Fluoro-substituted pentamethine cyanine dye and preparation method and application thereof

Abstract

The invention discloses a fluorine-substituted pentamethine cyanine dye and a preparation method and application thereof, the fluorine-substituted pentamethine cyanine dye has a structure shown in a general formula I: # imgabs0 #, and by introducing fluorine atoms to an indole benzene ring and introducing different groups to an N site, accurate regulation and control of light stability of dye molecules are realized. The fluorine-substituted pentamethine cyanine dye prepared by the invention shows remarkable advantages in spectral characteristics, has a molar extinction coefficient higher than that of the traditional cyanine dye and has strong light energy absorption capacity, and is beneficial to improving the light energy utilization rate and reducing the dye dosage; the absolute fluorescence quantum yield reaches a high level, the intensity and sensitivity of imaging signals are remarkably enhanced, and a more sensitive and reliable tool is provided for biological imaging and molecular detection.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent dyes, and particularly to a fluorine-substituted pentamethine cyanine dye, a preparation method thereof, and an application thereof. Background Art

[0002] With the in-depth expansion of the scientific research field, the biofluorescent staining technology has become an indispensable tool for exploring the mysteries of life and analyzing the structure and function of organisms. This technology cleverly bonds fluorescent dye molecules with target biomolecules (such as proteins, antibodies, cell membranes, etc.) through covalent or non-covalent bonds, realizing the precise labeling and efficient detection of specific components in biological samples, and providing strong support for the visualization analysis of intracellular dynamic processes. As a new cancer treatment strategy, photodynamic therapy takes a unique approach. Through the energy conversion of dye molecules under light excitation, a series of active factors with a killing effect on cancer cells are induced, thereby inhibiting tumor growth and showing unique advantages that traditional radiotherapy and chemotherapy do not have.

[0003] In the research field of traditional pentamethine cyanine dyes, fluorine-substituted pentamethine cyanine dyes can be effectively used in in vivo and in vitro bio-detection applications because they have a high molar extinction coefficient and extremely strong light stability, which can effectively reduce the interference of dye photobleaching during long-term imaging.

[0004] However, the current development of pentamethine cyanine dyes also faces many challenges, specifically manifested as: solubility problems, poor solubility in water, which hinders the wide application of dyes in biological systems; low fluorescence quantum yield, which limits the application of dyes; and a single synthesis method, resulting in fewer sites where the dyes can be modified. Therefore, it is necessary to develop near-infrared fluorescent dyes to address these problems, overcome the existing defects, and enable them to have better imaging, labeling, and detection capabilities. Summary of the Invention

[0005] Aiming at the technical problem of insufficient light stability of dyes existing in the prior art, the present invention provides a fluorine-substituted pentamethine cyanine dye, a preparation method thereof, and an application thereof.

[0006] To achieve the above object, the present invention provides a fluorine-substituted pentamethine cyanine dye having a structure of general formula I:

[0007]

[0008] In general formula I,

[0009] R1 is one of hydrogen, an alkyl group having 1-18 carbons, an aryl group, an aryl cyanide group, an aryl carboxyl group, an aryl nitro group, and an aryl sulfonate group;

[0010] R2 is one of hydrogen, an alkyl group having 1 to 18 carbons, an aryl group, an aryl cyanide group, an aryl carboxyl group, an aryl nitro group, and an aryl sulfonate group;

[0011] Y - is a halogen ion.

[0012] To achieve the above object, the present invention also provides a preparation method of a fluorine-substituted pentamethine cyanine dye, comprising the following steps:

[0013]

[0014] S1. Dissolve Y1 and 3-methyl-2-butanone in an organic acid, heat under reflux in an inert atmosphere for 6 to 12 h to obtain reaction solution 1; extract reaction solution 1 with a first organic solvent, adjust the pH of the organic phase to alkaline with a base, then extract with the first organic solvent again, evaporate the organic phase to dryness and purify to obtain compound Y2; wherein, the molar ratio of Y1 to 3-methyl-2-butanone is 1:1 to 5;

[0015] The N-alkylation reagent is selected from one of a halogen-substituted alkyl group, an aryl group, an aryl halogen group, an aryl carboxyl group, an aryl sulfonate group, an aryl nitro group, and an aryl cyanide group;

[0016] S2. Dissolve compound Y2 and the N-alkylation reagent in a second organic solvent, reflux and react under the protection of an inert gas for 12 to 24 h and then purify to obtain compounds Y3-R1 and Y3-R2 respectively; wherein, the molar ratio of Y2 to the N-alkylation reagent is 1:2 to 10;

[0017] S3. Dissolve compound Y3-R1 and compound S1 in an organic acid and acetic anhydride solvent, heat to 40 to 120 °C in an inert atmosphere and react for 2 to 6 h; cool to room temperature and recrystallize, separate the solid and liquid phases, take the solid phase and purify to obtain compound Y4; wherein, the molar ratio of Y3-R1 to compound S1 is 1:0.8 to 1.2, and the volume ratio of the organic acid to the acetic anhydride solvent is 1:0.5 to 2;

[0018] S4. Dissolve compound Y4, Y3-R2, a base catalyst and an acetic anhydride solvent, heat to 40 to 120 °C in an inert atmosphere and react for 2 to 6 h, cool to room temperature and recrystallize, separate the solid and liquid phases, take the solid phase and dry it, and use dichloromethane and methanol as elution solvents to purify to obtain the fluorine-substituted pentamethine cyanine dye; wherein the molar ratio of Y4, Y3-R2 to the base catalyst is 1:0.8 to 1.2:2 to 10.

[0019] Further, in S1, the base is selected from at least one of sodium hydroxide, potassium hydroxide, ammonia water, and sodium bicarbonate.

[0020] Further, in S2, the second organic solvent is selected from at least one of toluene, o-dichlorobenzene, ethanol, methanol, isopropanol, acetonitrile, tetrahydrofuran, and sulfolane.

[0021] Further, in S1 and S3, the organic acid is selected from at least one of acetic acid, hydrochloric acid, sulfuric acid, and polyphosphoric acid.

[0022] Further, in S4, the base catalyst is selected from at least one of sodium carbonate, sodium bicarbonate, sodium acetate, triethylamine, sodium hydroxide, and potassium hydroxide.

[0023] Further, in S1, the first organic solvent is selected from at least one of dichloromethane, ethyl acetate, toluene, and n-hexane.

[0024] Further, in S2, S3, and S4, the solvents used for recrystallization are all selected from at least one of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone, propanol, and isopropanol.

[0025] To achieve the above object, the present invention also provides an application of the fluorine-substituted pentamethine cyanine dye, and the fluorine-substituted pentamethine cyanine dye is applied to cell imaging, protein labeling, specific recognition of antibodies, nucleic acid labeling, DNA sequencing, and preparation of reagents for specific recognition and photodynamic therapy of tumors.

[0026] Further, the fluorescence imaging emission wavelength of the fluorine-substituted pentamethine cyanine dye during application is 600 - 800 nm.

[0027] The present invention has the following beneficial effects:

[0028] 1. The fluorine-substituted pentamethine cyanine dye provided by the present invention shows significant advantages in spectral characteristics. The molar extinction coefficients of different fluorine-substituted pentamethine cyanine dyes in PBS buffer are higher than those of traditional pentamethine cyanine dyes, reaching 2.42×10 5 L / mol -1 ·cm -1 , indicating that it has a strong ability to absorb light energy, which helps to improve the light energy utilization rate and reduce the amount of dye used;

[0029] 2. The absolute fluorescence quantum yield of the fluorine-substituted pentamethine cyanine dye provided by the present invention reaches a relatively high level, up to 0.080. The high fluorescence quantum yield means that the dye molecules can more effectively convert light energy into fluorescence emission after absorbing light energy, thereby significantly enhancing the intensity and sensitivity of the imaging signal, providing a more sensitive and reliable tool for biological imaging and molecular detection;

[0030] 3. The absolute fluorescence quantum yield of the fluorine-substituted pentamethine cyanine dye provided by the present invention reaches a relatively high level, up to 0.080. The fluorine-substituted pentamethine cyanine dye with high fluorescence has high photostability. By introducing strong electron-withdrawing group fluorine atoms at the indole benzene ring position of the dye molecule, the redox potential of the dye is adjusted, further greatly improving the photostability of the dye molecule. The photostability of different fluorine-substituted pentamethine cyanine dyes in the mixed solvent of PBS and methanol is significantly improved compared with that of traditional cyanine dyes.

[0031] 4. The fluorine-substituted pentamethine cyanine dye provided by the present invention has high structural diversity and modifiability. By introducing groups such as benzyl to regulate the structure of the indole N position of the cyanine dye, the formation of π-π stacking within the dye molecule is successfully achieved, increasing the steric hindrance, thereby hindering the attack of reactive oxygen species on the dye molecule, further inhibiting the photobleaching of the dye molecule, and thus realizing the regulation of the photostability of the dye molecule. This structural diversity and modifiability not only enrich the types of dyes but also provide a broad space for further optimizing their spectral properties and application performance. Brief Description of the Drawings

[0032] 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, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0033] Figure 1 It is the high-resolution mass spectrum of Dye 1 prepared in Example 1 disclosed by the present invention;

[0034] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of Dye 1 prepared in Example 1 disclosed by the present invention;

[0035] Figure 3 It is the high-resolution mass spectrum of Dye 3 prepared in Example 3 disclosed by the present invention;

[0036] Figure 4 It is the nuclear magnetic resonance hydrogen spectrum of Dye 3 prepared in Example 3 disclosed by the present invention;

[0037] Figure 5 It is the high-resolution mass spectrum of Dye 4 prepared in Example 4 disclosed by the present invention;

[0038] Figure 6 It is the nuclear magnetic resonance hydrogen spectrum of Dye 4 prepared in Example 4 disclosed by the present invention;

[0039] Figure 7Absorption spectra of Dye 1, Dye 2, Dye 3, and Dye 4 prepared in Examples 1-4 of the present invention in PBS buffer solution;

[0040] Figure 8 Fluorescence emission spectra of Dye 1, Dye 2, Dye 3, and Dye 4 prepared in Examples 1-4 of the present invention in PBS buffer solution;

[0041] Figure 9 Photostability test chart of Dye 1 prepared in Example 1 of the present invention;

[0042] Figure 10 Photostability test chart of Dye 2 prepared in Example 2 of the present invention;

[0043] Figure 11 Photostability test chart of Dye 3 prepared in Example 3 of the present invention;

[0044] Figure 12 Photostability test chart of Dye 4 prepared in Example 4 of the present invention;

[0045] Figure 13 Molecular structure and photostability test chart of the traditional pentamethine cyanine dye as a comparative molecule in the present invention. Detailed implementation manners

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0047] Unless otherwise specified, the terms used herein have the following meanings. The term "halogen" used in the present invention includes fluorine, chlorine, bromine, and iodine; "alkyl" includes straight-chain alkyl and branched-chain alkyl; "room temperature" refers to the temperature range of 20-25°C.

[0048] Embodiment

[0049] Embodiment 1:

[0050] Preparation of fluorine-substituted pentamethine cyanine dye 1:

[0051] S1 Preparation of compound Y2

[0052]

[0053] 2,4-Difluorophenylhydrazine hydrochloride (5.00 g, 27.69 mmol) and 3-methyl-2-butanone (2.62 g, 30.46 mmol) were added to a 250 mL two-necked round-bottom flask, followed by 50 mL of acetic acid. The mixture was heated to 100 °C under a nitrogen atmosphere and reacted for 12 h. After the reaction, the reaction mixture was extracted with dichloromethane and water. After liquid separation, sodium carbonate was added to the organic phase until no more bubbles emerged, and then anhydrous sodium sulfate was added for drying. The mixture was filtered, and the filtrate was rotary evaporated. Using petroleum ether / ethyl acetate (8:1, v / v) as the elution solvent, the crude product was purified by silica gel chromatography to obtain Y2 (4.15 g, 21.26 mmol), with a yield of 76.8%.

[0054] Preparation of Compound Y3.1

[0055]

[0056] Y2 (2.00 g, 10.25 mmol) and iodoethane (3.20 g, 20.49 mmol) were added to a 100 mL two-necked round-bottom flask containing 10 mL of acetonitrile. The mixture was refluxed under a nitrogen atmosphere for 12 h. After cooling to room temperature, the remaining solid in the reaction mixture was dissolved in a small amount of methanol and slowly dropped into 150 mL of ether, and at this time, the solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Y3.1 as a light pink solid powder (2.40 g, 6.83 mmol), with a yield of 66.7%.

[0057] Preparation of Fluoro-Substituted Pentamethine Cyanine Dye 1

[0058]

[0059] Y3.1 (300 mg, 0.85 mmol), malondialdehyde diphenylamine hydrochloride (111 mg, 0.43 mmol), and sodium acetate (175 mg, 2.14 mmol) were added to a 50 mL two-necked round-bottom flask, followed by acetic anhydride as the solvent. The mixture was heated to 50 °C under a nitrogen atmosphere and reacted for 4 h. After cooling to room temperature, the reaction mixture was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. Using dichloromethane / methanol (40 / 1 - 10 / 1, v / v) as the elution solvent, the crude product was purified by silica gel chromatography to obtain fluoro-substituted pentamethine cyanine dye 1 (110 mg, 0.18 mmol), with a yield of 42.2%. 11H NMR (600 MHz, Methanol-d4) δ 8.31 (dd, J = 13.7, 12.4 Hz, 2H), 7.24 (dd, J = 7.5, 2.3 Hz, 2H), 7.09 (ddd, J = 11.8, 9.1, 2.3 Hz, 2H), 6.72–6.64 (m, 1H), 6.33 (d, J = 13.7 Hz, 2H), 4.25 (q, J = 7.2 Hz, 4H), 1.75 (s, 12H), 1.43 (t, J = 7.2 Hz, 6H).

[0060] Example 2:

[0061] Preparation of Fluoro-Substituted Pentamethine Cyanine Dye 2:

[0062] S1: The same as Example 1;

[0063] S2: Preparation of Compound Y3.2

[0064]

[0065] Y2 (2.00 g, 10.25 mmol) and benzyl bromide (3.50 g, 20.49 mmol) were added to a 100 mL two-necked round-bottom flask containing 10 mL of o-dichlorobenzene, and the mixture was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly dropped into 150 mL of ether, and at this time, the solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Y3.2 as a light pink solid powder (2.10 g, 5.73 mmol), with a yield of 56.0%.

[0066] S3: Preparation of Fluoro-Substituted Pentamethine Cyanine Dye 2:

[0067]

[0068] Y3.2 (300 mg, 0.82 mmol), malondialdehyde diphenylamine hydrochloride (106 mg, 0.41 mmol), and sodium acetate (168 mg, 2.05 mmol) were added to a 50 mL two-necked round-bottom flask, and then acetic anhydride solvent was added. The mixture was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. Using dichloromethane / methanol (v / v) with a ratio of 40 / 1 - 10 / 1 as the elution solvent, the crude product was purified by silica gel chromatography to obtain fluoro-substituted pentamethine cyanine dye 2 (52 mg, 0.076 mmol), with a yield of 18.5%. 11H NMR (600 MHz, Methanol-d4) δ 8.30 (t, J = 13.0 Hz, 2H), 7.37 (t, J = 7.6 Hz, 4H), 7.30 (dd, J = 20.3, 7.4 Hz, 4H), 7.23 (d, J = 7.7 Hz, 4H), 7.05 (dd, J = 11.6, 9.0 Hz, 2H), 6.49 (s, 1H), 6.30 (d, J = 13.6 Hz, 2H), 5.43 (s, 4H), 1.80 (d, J = 1.9 Hz, 12H).

[0069] Example 3:

[0070] Preparation of Fluoro-Substituted Pentamethine Cyanine Dye 3:

[0071] S1: The same as Example 1;

[0072] S2: Preparation of Compound Y3.3

[0073]

[0074] Y2 (2.00 g, 10.25 mmol) and p-cyanobenzyl bromide (4.02 g, 20.49 mmol) were added to a 100 mL two-necked round-bottom flask containing 20 mL of o-dichlorobenzene, and the mixture was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly added dropwise to 150 mL of ether, and at this time, a solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Y3.3 as a light pink solid powder (2.50 g, 6.39 mmol), with a yield of 62.4%.

[0075] S3: Preparation of Fluoro-Substituted Pentamethine Cyanine Dye 3

[0076]

[0077] Y3.3 (300 mg, 0.77 mmol), malondialdehyde diphenylamine hydrochloride (99 mg, 0.38 mmol), and sodium acetate (157 mg, 1.92 mmol) were added to a 50 mL two-necked round-bottom flask, and then acetic anhydride solvent was added. The mixture was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. Using dichloromethane / methanol (v / v) with a ratio of 40 / 1 - 10 / 1 as the elution solvent, the crude product was purified by silica gel chromatography to obtain fluoro-substituted pentamethine cyanine dye 3 (82 mg, 0.11 mmol), with a yield of 29.0%. 11H NMR (600 MHz, Methanol-d4) δ 8.37 (t, J = 13.0 Hz, 2H), 7.78–7.73 (m, 4H), 7.45–7.40 (m, 4H), 7.31 (dd, J = 7.5, 2.3 Hz, 2H), 7.05 (ddd, J = 11.6, 9.0, 2.3 Hz, 2H), 6.51 (t, J = 12.4 Hz, 1H), 6.29 (d, J = 13.6 Hz, 2H), 5.53 (s, 4H), 1.83 (s, 12H).

[0078] Example 4:

[0079] The preparation of fluorine-substituted pentamethine cyanine dye 4 includes the following steps:

[0080] S1: The same as Example 1;

[0081] S2: Preparation of compound Y3.4

[0082]

[0083] Y2 (2.00 g, 10.25 mmol) and p-bromomethylbenzoic acid (4.41 g, 20.49 mmol) were added to a 100 mL two-necked round-bottom flask containing 20 mL of o-dichlorobenzene, and the reaction was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly dropped into 150 mL of ether, and at this time, the solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Y3.4 as a light pink solid powder (2.00 g, 6.39 mmol), with a yield of 47.6%.

[0084] S3: Preparation of fluorine-substituted pentamethine cyanine dye 4

[0085]

[0086] Y3.4 (300 mg, 0.73 mmol), malondialdehyde diphenylamine hydrochloride (95 mg, 0.37 mmol), and sodium acetate (150 mg, 1.83 mmol) were added to a 50 mL two-necked round-bottom flask, and then acetic anhydride solvent was added. The reaction was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. Using dichloromethane / methanol (v / v) with a ratio of 40 / 1 - 10 / 1 as the elution solvent, the crude product was purified by silica gel chromatography to obtain fluorine-substituted pentamethine cyanine dye 4 (50 mg, 0.064 mmol), with a yield of 17.6%. 11H NMR (600 MHz, Methanol-d4) δ 8.29 (t, J = 13.0 Hz, 2H), 7.97 (d, J = 8.0 Hz, 4H), 7.28 (td, J = 5.7, 2.8 Hz, 5H), 7.05 (ddd, J = 11.6, 8.9, 2.3 Hz, 2H), 6.48 (t, J = 12.5 Hz, 1H), 6.28 (d, J = 13.6 Hz, 2H), 5.48 (s, 4H), 1.80 (s, 12H).

[0087] Example 5:

[0088] Preparation of the fluorine-substituted structure near-infrared fluorescent dye compound 5, comprising the following steps:

[0089] S1: The same as Example 1;

[0090] S2: Preparation of compound Y3.5

[0091]

[0092] Add Y2 (2.00 g, 10.25 mmol) and p-nitrobenzyl bromide (4.43 g, 20.49 mmol) to a 100 mL two-necked round-bottom flask containing 20 mL of o-dichlorobenzene, and reflux the reaction for 12 h under a nitrogen atmosphere. After cooling to room temperature, dissolve the remaining solid in the reaction solution with a small amount of methanol and slowly drop it into 150 mL of ether, at which time the solid precipitates. Then filter, wash, and dry to obtain Y3.5 as a light pink solid powder (2.30 g, 5.59 mmol), with a yield of 54.6%.

[0093] S3: Preparation of the fluorine-substituted pentamethine cyanine dye 5

[0094]

[0095] Add Y3.5 (300 mg, 0.73 mmol), malondialdehyde diphenylamine hydrochloride (94 mg, 0.36 mmol), and sodium acetate (150 mg, 1.82 mmol) to a 50 mL two-necked round-bottom flask, then add acetic anhydride as the solvent, and heat the reaction to 50 °C for 4 h under a nitrogen atmosphere. After cooling to room temperature, add the reaction solution to 50 mL of ether, filter, wash the filter cake with ethyl acetate, and dry. Use silica gel column chromatography to purify the crude product with 40 / 1 - 10 / 1 dichloromethane / methanol (v / v) as the elution solvent to obtain the fluorine-substituted pentamethine cyanine dye 5 (45 mg, 0.058 mmol), with a yield of 15.9%. 11H NMR (600 MHz, Methanol-d4) δ 8.43 (t, J = 13.0 Hz, 2H), 7.76–7.71 (m, 4H), 7.45–7.40 (m, 4H), 7.31 (dd, J = 7.5, 2.3 Hz, 2H), 7.05 (ddd, J = 11.6, 9.0, 2.3 Hz, 2H), 6.51 (t, J = 12.4 Hz, 1H), 6.29 (d, J = 13.6 Hz, 2H), 5.54 (s, 4H), 1.78 (s, 12H).

[0096] Example 6:

[0097] Preparation of fluorine-substituted pentamethine cyanine dye 6, comprising the following steps:

[0098] S1: The same as Example 1;

[0099] S2: Preparation of compound Y3.6

[0100]

[0101] Y2 (2.00 g, 10.25 mmol) and p-bromomethylbenzenesulfonic acid (5.15 g, 20.49 mmol) were added to a 100 mL two-necked round-bottom flask containing 20 mL of o-dichlorobenzene, and the reaction was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly dropped into 150 mL of ether, and at this time, the solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Y3.6 as a light pink solid powder (1.80 g, 4.93 mmol), with a yield of 48.1%.

[0102] S3: Preparation of fluorine-substituted pentamethine cyanine dye 6

[0103]

[0104] Y3.6 (300 mg, 0.82 mmol), malondialdehyde diphenylamine hydrochloride (106 mg, 0.41 mmol), and sodium acetate (168 mg, 1.82 mmol) were added to a 50 mL two-necked round-bottom flask, and then acetic anhydride solvent was added. The reaction was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. Using dichloromethane / methanol (v / v) with a ratio of 40 / 1 - 10 / 1 as the elution solvent, the crude product was purified by silica gel chromatography to obtain fluorine-substituted pentamethine cyanine dye 6 (45 mg, 0.057 mmol), with a yield of 13.9%. 11H NMR (600 MHz, Methanol-d4) δ 8.45 (t, J = 13.0 Hz, 2H), 7.76–7.71 (m, 4H), 7.45–7.40 (m, 4H), 7.31 (dd, J = 7.5, 2.3 Hz, 2H), 7.08 (ddd, J = 11.6, 9.0, 2.3 Hz, 2H), 6.44 (t, J = 12.4 Hz, 1H), 6.33 (d, J = 13.6 Hz, 2H), 5.48 (s, 4H), 1.72 (s, 12H).

[0105] Example 7:

[0106] The preparation of fluorine-substituted pentamethine cyanine dye 7 comprises the following steps:

[0107] S1: The same as Example 1;

[0108] S2: Preparation of compound Y3.7

[0109]

[0110] Y2 (2.00 g, 10.25 mmol) and p-bromomethylbenzenesulfonic acid (3.79 g, 20.49 mmol) were added to a 100 mL two-necked round-bottom flask containing 20 mL of o-dichlorobenzene, and the mixture was refluxed for 12 h under a nitrogen atmosphere. After cooling to room temperature, the remaining solid in the reaction solution was dissolved in a small amount of methanol and slowly added dropwise to 150 mL of ether, and at this time, the solid precipitated. Subsequently, it was filtered, washed, and dried to obtain Y3.7 as a light pink solid powder (2.20 g, 4.93 mmol), with a yield of 56.5%.

[0111] S3: Preparation of fluorine-substituted pentamethine cyanine dye 7

[0112]

[0113] Y3.7 (300 mg, 0.79 mmol), malondialdehyde diphenylamine hydrochloride (102 mg, 0.39 mmol), and sodium acetate (162 mg, 1.97 mmol) were added to a 50 mL two-necked round-bottom flask, and then acetic anhydride solvent was added. The mixture was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. Using 40 / 1 - 10 / 1 dichloromethane / methanol (v / v) as the elution solvent, the crude product was purified by silica gel chromatography to obtain fluorine-substituted pentamethine cyanine dye 7 (40 mg, 0.056 mmol), with a yield of 14.17%. 11H NMR (600 MHz, Methanol-d4) δ 8.38 (t, J = 13.0 Hz, 2H), 7.77–7.72 (m, 4H), 7.47–7.42 (m, 4H), 7.28 (dd, J = 7.5, 2.3 Hz, 2H), 7.06 (ddd, J = 11.6, 9.0, 2.3 Hz, 2H), 6.45 (t, J = 12.4 Hz, 1H), 6.33 (d, J = 13.6 Hz, 2H), 5.48 (s, 4H), 2.29 (s, 6H), 1.70 (s, 12H).

[0114] Example 8:

[0115] Preparation of fluorine-substituted pentamethine cyanine dye 8, comprising the following steps:

[0116] Preparation of compound Y4.8:

[0117]

[0118] Y3.4 (0.500 g, 1.22 mmol) prepared in Example 4 and malondialdehyde diphenylamine hydrochloride (0.315 g, 1.22 mmol) were added to a 50 mL two-necked round-bottom flask, and then a mixed solvent of acetic acid:acetic anhydride = 1:1 was added. The reaction was heated to 100 °C under a nitrogen atmosphere for 4 h. After cooling to room temperature, the reaction solution was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by silica gel chromatography to obtain crude Y4.8 (0.423 g, 0.073 mmol), with a yield of 59.7%;

[0119] Preparation of fluorine-substituted pentamethine cyanine dye 8:

[0120]

[0121] Y4.8 (0.300 g, 0.516 mmol), Y3.2 (0.189 mg, 0.516 mmol) and sodium acetate (0.212 mg, 2.58 mmol) were added to a 50 mL two-necked round-bottom flask, and then acetic anhydride solvent was added. The reaction was heated to 50 °C under a nitrogen atmosphere for 4 h. After cooling to room temperature, the reaction solution was added to 50 mL of ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by silica gel chromatography to obtain fluorine-substituted pentamethine cyanine dye 8 (55 mg, 0.075 mmol), with a yield of 14.6%. 1HNMR(600MHz, Methanol-d4) δ 8.33 (t, J=13.0Hz, 2H), 7.97 (d, J=8.0Hz, 5H), 7.28 (td, J=5.7, 2.8Hz, 5H), 7.05 (ddd, J=11.6, 8.9, 2.3Hz, 2H), 6.48 (t, J=12.5Hz, 1H), 6.28 (d, J=13.6Hz, 2H), 5.48 (s, 4H), 1.80 (s, 12H).

[0122] Example 9:

[0123] Preparation of Fluoro-Substituted Pentamethine Cyanine Dye 9:

[0124]

[0125] Y4.8 (0.300 g, 0.516 mmol) prepared in Example 8, Y3.3 (0.202 mg, 0.516 mmol) prepared in Example 3, and sodium acetate (0.212 mg, 2.58 mmol) were added to a 50 mL two-necked round-bottom flask. Subsequently, acetic anhydride solvent was added, and the mixture was heated to 50 °C under a nitrogen atmosphere and reacted for 4 h. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by silica gel column chromatography to obtain fluoro-substituted pentamethine cyanine dye 9 (60 mg, 0.079 mmol), with a yield of 15.4%. 1 H NMR(600MHz, Methanol-d4) δ 8.46 (t, J=13.0Hz, 2H), 7.78–7.72 (m, 4H), 7.41–7.36 (m, 4H), 7.34 (dd, J=7.5, 2.3Hz, 2H), 7.10 (ddd, J=11.6, 9.0, 2.3Hz, 2H), 6.44 (t, J=12.4Hz, 1H), 6.33 (d, J=13.6Hz, 2H), 5.53 (s, 4H), 1.68 (s, 12H).

[0126] Example 10:

[0127] Preparation of Fluoro-Substituted Pentamethine Cyanine Dye 10:

[0128]

[0129] Y4.8 prepared in Example 8 (0.300 g, 0.516 mmol), Y3.5 prepared in Example 5 (0.202 mg, 0.516 mmol) and sodium acetate (0.212 mg, 2.58 mmol) were added to a 50 mL two-necked round-bottom flask. Subsequently, acetic anhydride solvent was added, and the mixture was heated to 50 °C under a nitrogen atmosphere and reacted for 4 h. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by silica gel chromatography to obtain fluorine-substituted pentamethine cyanine dye 10 (40 mg, 0.052 mmol) with a yield of 10.0%. 1 H NMR (600 MHz, Methanol-d4) δ 8.44 (t, J = 13.0 Hz, 2H), 7.78–7.72 (m, 4H), 7.43–7.38 (m, 4H), 7.31 (dd, J = 7.5, 2.3 Hz, 2H), 7.13 (ddd, J = 11.6, 9.0, 2.3 Hz, 2H), 6.46 (t, J = 12.4 Hz, 1H), 6.34 (d, J = 13.6 Hz, 2H), 5.55 (s, 4H), 1.70 (s, 12H).

[0130] Example 11:

[0131] Preparation of fluorine-substituted pentamethine cyanine dye 11:

[0132]

[0133] Y4.8 prepared in Example 8 (0.300 g, 0.516 mmol), Y3.6 prepared in Example 6 (0.189 mg, 0.516 mmol) and sodium acetate (0.212 mg, 2.58 mmol) were added to a 50 mL two-necked round-bottom flask. Subsequently, acetic anhydride solvent was added, and the mixture was heated to 50 °C under a nitrogen atmosphere and reacted for 4 h. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by silica gel chromatography to obtain fluorine-substituted pentamethine cyanine dye 11 (35 mg, 0.048 mmol) with a yield of 9.3%. 1 H NMR (600 MHz, Methanol-d4) δ 8.48 (t, J = 13.0 Hz, 2H), 7.73–7.68 (m, 4H), 7.43–7.38 (m, 4H), 7.33 (dd, J = 7.5, 2.3 Hz, 2H), 7.10 (ddd, J = 11.6, 9.0, 2.3 Hz, 2H), 6.45 (t, J = 12.4 Hz, 1H), 6.33 (d, J = 13.6 Hz, 2H), 5.52 (s, 4H), 1.73 (s, 12H).

[0134] Example 12:

[0135] Preparation of fluorine-substituted pentamethine cyanine dye 12:

[0136]

[0137] Y4.8 (0.300 g, 0.516 mmol) prepared in Example 8, Y3.7 (0.196 mg, 0.516 mmol) prepared in Example 7 and sodium acetate (0.212 mg, 2.58 mmol) were added into a 50 mL two-necked round-bottom flask. Subsequently, acetic anhydride solvent was added, and the mixture was heated to 50 °C under a nitrogen atmosphere and reacted for 4 h. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate and dried. The crude product was purified by silica gel chromatography to obtain fluorine-substituted pentamethine cyanine dye 12 (38 mg, 0.051 mmol), with a yield of 9.9%. 1 H NMR (600 MHz, Methanol-d4) δ 8.48 (t, J = 13.0 Hz, 2H), 7.73–7.68 (m, 4H), 7.43–7.38 (m, 4H), 7.33 (dd, J = 7.5, 2.3 Hz, 2H), 7.10 (ddd, J = 11.6, 9.0, 2.3 Hz, 2H), 6.45 (t, J = 12.4 Hz, 1H), 6.33 (d, J = 13.6 Hz, 2H), 5.52 (s, 4H), 2.28 (s, 3H), 1.73 (s, 12H).

[0138] Comparative Example 1:

[0139] Preparation of traditional pentamethine cyanine dye:

[0140]

[0141] Preparation of compound Y3.8:

[0142] 2,3,3-Trimethyl-3H-indole (1.00 g, 6.28 mmol) and iodoethane (2.94 g, 18.84 mmol) were added into a two-necked round-bottom flask containing 20 mL of acetonitrile, and the mixture was refluxed under a nitrogen atmosphere for 16 h. After cooling to room temperature, the reaction solution was slowly dropped into 150 ml of diethyl ether, and a solid precipitated. Subsequently, it was filtered, washed and dried to obtain a light pink solid powder Y3.8 (1.60 g, 5.08 mmol), with a yield of 80.8%, and directly used for the next step of reaction

[0143] Preparation of traditional pentamethine cyanine dye:

[0144] Y3.8 (300 mg, 0.952 mmol), malondialdehyde diphenylamine hydrochloride (123 mg, 0.476 mmol), and sodium acetate (195 mg, 2.38 mmol) were added to a 50 mL two-necked round-bottom flask. Subsequently, acetic anhydride solvent was added, and the mixture was heated to 50 °C and reacted for 4 h under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was added to 50 mL of diethyl ether, filtered, and the filter cake was washed with ethyl acetate and dried. Using dichloromethane / methanol (v / v) with a ratio of 30 / 1 - 10 / 1 as the elution solvent, the crude product was purified by silica gel chromatography to obtain Comparative Example 1 as a blue-violet solid (160 mg, 0.297 mmol), with a yield of 62.4%. 1 H NMR (600 MHz, Methanol-d4) δ 8.26 (dd, J = 13.7, 12.4 Hz, 2H), 7.50 (dd, J = 7.5, 1.2 Hz, 2H), 7.41 (td, J = 7.7, 1.2 Hz, 2H), 7.33–7.22 (m, 4H), 6.64 (t, J = 12.4 Hz, 1H), 6.30 (d, J = 13.7 Hz, 2H), 4.16 (q, J = 7.3 Hz, 4H), 1.73 (s, 12H), 1.39 (t, J = 7.3 Hz, 6H).

[0145] Experiment and result analysis:

[0146] (1) Identification of the molecular structure of fluorine-substituted pentamethine cyanine dyes:

[0147] High-precision and high-sensitivity high-resolution mass spectrometry analysis technology was used to identify fluorine-substituted pentamethine cyanine dye 1, fluorine-substituted pentamethine cyanine dye 3, and fluorine-substituted pentamethine cyanine dye 4. The results are as shown in Figure 1 , Figure 3 and Figure 5 . The high-resolution mass spectrometry analysis results of fluorine-substituted pentamethine cyanine dye 1 are as shown in Figure 1 . A clear and sharp main peak is shown in the figure. The position of this peak highly coincides with the molecular weight calculated theoretically for fluorine-substituted pentamethine cyanine dye 1, and no obvious impurity peaks or fragment peaks are observed. This fully proves the purity of fluorine-substituted pentamethine cyanine dye 1 and the correctness of its structure, marking the successful preparation of compound 1. At the same time, the distribution of characteristic peaks in the mass spectrometry pattern coincides with the functional groups and connection modes in the expected structure, further verifying the specified structure of fluorine-substituted pentamethine cyanine dye 1. Similarly, Figure 3 and Figure 5The high-resolution mass spectrometry analysis results of fluorine-substituted pentamethine cyanine dye 3 and fluorine-substituted pentamethine cyanine dye 4 are respectively shown. Similar to fluorine-substituted pentamethine cyanine dye 1, the mass spectrometry graphs of fluorine-substituted pentamethine cyanine dye 3 and fluorine-substituted pentamethine cyanine dye 4 also present a main peak, whose position precisely corresponds to the theoretical molecular weight of fluorine-substituted pentamethine cyanine dye 3, and the graphs are clear and interference-free, reflecting the high purity and structural accuracy of fluorine-substituted pentamethine cyanine dye 3.

[0148] Fluorine-substituted pentamethine cyanine dye 1, fluorine-substituted pentamethine cyanine dye 3 and fluorine-substituted pentamethine cyanine dye 4 were identified using nuclear magnetic resonance spectrometer analysis technology, and the results are as Figure 2 , Figure 4 and Figure 6 shown. The nuclear magnetic resonance hydrogen spectrum analysis results for fluorine-substituted pentamethine cyanine dye 1 are as Figure 2 shown. Each peak in the nuclear magnetic spectrum can find its attribution in the molecule, and the total number of hydrogen atoms can correspond, and no obvious impurity peaks are observed, which further proves the purity of fluorine-substituted pentamethine cyanine dye 1 and the correctness of its structure. Similarly, Figure 4 and Figure 6 respectively show the nuclear magnetic resonance hydrogen spectrum analysis results of fluorine-substituted pentamethine cyanine dye 3 and fluorine-substituted pentamethine cyanine dye 4, proving the purity and structural correctness of fluorine-substituted pentamethine cyanine dye 3 and fluorine-substituted pentamethine cyanine dye 4.

[0149] (2) UV-Vis absorption spectrum, fluorescence spectrum and photophysical property test of fluorescent dye compounds:

[0150] (2.1) Dye preparation method:

[0151] In this experiment, a balance with a precision of one ten-thousandth was used to accurately weigh the dye that had been strictly vacuum-dried. Subsequently, the weighed dye was dissolved in dimethyl sulfoxide (DMSO) to prepare a dye stock solution with a concentration of 3 mmol / L. This stock solution was carefully aliquoted into brown sample bottles to prevent light from interfering with its stability and stored in a 4 °C refrigerator to ensure chemical stability and activity during long-term storage. Before performing UV-Vis absorption spectrum and fluorescence spectrum tests, test solutions need to be prepared. The specific steps are as follows: Use a high-precision micropipette to accurately measure 3 μL of the dye stock solution, and slowly inject it into a quartz cuvette containing 3 mL of a predetermined solvent (such as water). Gently shake to ensure uniform mixing of the solution. This operation dilutes the dye concentration to 3 μmol / L, which is suitable for subsequent spectral analysis.

[0152] (2.2) Broad-spectrum conditions and photophysical property test conditions:

[0153] All spectroscopic tests were performed in a strictly controlled environment at a constant temperature of 25 °C to eliminate the potential influence of temperature fluctuations on the experimental results and ensure the reproducibility and accuracy of the obtained data. During the photophysical property tests, a precisely prepared dye stock solution was used as the test solution. According to the principle of Beer's law, by measuring the absorbance at a specific wavelength and combining with the molar extinction coefficient calculation formula, the molar extinction coefficient of each dye was calculated; a high-precision absolute fluorescence quantum yield meter (Hamamatsu, model C11347) was used to measure the absolute fluorescence quantum yield of each dye sample under the same conditions to comprehensively evaluate its fluorescence emission efficiency.

[0154] (2.3) Result analysis:

[0155] Figure 7 and Figure 8 respectively show the absorption spectra and fluorescence emission spectra of fluorine-substituted pentamethine cyanine dye 1, fluorine-substituted pentamethine cyanine dye 2, fluorine-substituted pentamethine cyanine dye 3, and fluorine-substituted pentamethine cyanine dye 4 in PBS buffer. The results show that these dyes have similar maximum absorption peaks and fluorescence emission peaks, which are located at about 644 nm and 668 nm respectively.

[0156] It should be noted that even after specific activation treatment, the maximum emission wavelength of the fluorine-substituted pentamethine cyanine dye did not change significantly, indicating its good structural stability. Through targeted chemical modification, the spectral properties of such dyes, including absorption and emission wavelengths, can be finely tuned, providing broad possibilities for their applications in cutting-edge scientific and technological fields such as near-infrared biofluorescence imaging and protein labeling detection.

[0157] The photophysical property test results of fluorine-substituted pentamethine cyanine dyes 1-12 are shown in Table 1.

[0158] Table 1 Photophysical property parameter table of fluorine-substituted pentamethine cyanine dyes 1-12

[0159]

[0160]

[0161] Table 1 details the key photophysical property parameters of the fluorine-substituted pentamethine cyanine dyes prepared in Examples 1-12 and the traditional pentamethine cyanine dye prepared in Comparative Example 1, including absorption wavelength, emission wavelength, molar extinction coefficient (ε), and absolute fluorescence quantum yield

[0162] As can be seen from Table 1, the molar extinction coefficients of the fluorine-substituted pentamethine cyanine dyes prepared in the examples of this application are significantly higher than those of the traditional pentamethine cyanine dyes, and can reach 2.42×10 5 L / mol -1 ·cm-1 (Fluorine-substituted pentamethine cyanine dye 1). A high molar extinction coefficient means that the dye molecules have a stronger ability to absorb light, enabling effective light energy capture and conversion at lower concentrations, thereby improving the light energy utilization rate, reducing the amount of dye used, and lowering potential biological toxicity. All compounds exhibit a relatively high fluorescence quantum yield, with a value reaching 0.178 (for Fluorine-substituted pentamethine cyanine dye 6). A high fluorescence quantum yield means that the dye molecules can more effectively convert the absorbed light energy into fluorescence emission after absorbing light energy, thereby enhancing the intensity and sensitivity of the imaging signal and improving the imaging quality. The above data not only reveal the diversity and superiority of the spectral characteristics of this dye but also deeply reflect the remarkable effectiveness of the present invention in solving the problems of the prior art.

[0163] (3) Photostability test of fluorine-substituted pentamethine cyanine dyes

[0164] (3.1) Test method for the photostability of dyes:

[0165] Use a high-precision micropipette to accurately measure 4 μL of the dye stock solution and slowly inject it into a quartz cuvette containing a 3 mL mixed solvent of PBS buffer and methanol with an equal volume ratio. Gently shake to ensure uniform mixing of the solution. This operation dilutes the dye concentration to 3 μmol / L, irradiate it under a 660 nm LED lamp, and adjust the distance between the quartz cuvette and the lamp so that the light power is 20 mW / cm 2 , and measure the absorption spectrum of the dye every 10 minutes.

[0166] (3.2) Results of the photostability of dyes:

[0167] Table 2: Photostability results of the fluorine-substituted pentamethine cyanine dyes prepared in Examples 1-4 and the traditional pentamethine cyanine dye

[0168]

[0169]

[0170] Figures 9 - 12 respectively reflect the photostability test results of Fluorine-substituted pentamethine cyanine dye 1, Fluorine-substituted pentamethine cyanine dye 2, Fluorine-substituted pentamethine cyanine dye 3, and Fluorine-substituted pentamethine cyanine dye 4. Figure 13 reflects the photostability test result of the traditional pentamethine cyanine dye in Comparative Example 1. Combining the results in Table 2, it can be seen that the fluorine-substituted pentamethine cyanine dyes at a light power of 20 mW / cm at 660 nm 2After 1 h of illumination, its maximum absorption only attenuated by about 7.0%, while the maximum absorption of the traditional pentamethine cyanine dye attenuated by 22.7% under the same conditions, indicating that the introduction of fluorine atoms can greatly improve the light stability of the dye and enhance the imaging effect of the dye; after introducing a benzyl group at the N position of the indole of the dye molecule, the light stability of the molecule was further improved. Among them, when the substituent group is p-carboxybenzyl, that is, the fluorine-substituted pentamethine cyanine dye 4 prepared, its light stability is the best, and the maximum absorption only decreases by 1.2% under the same test conditions.

[0171] In summary, by introducing fluorine atoms on the indole benzene ring and different groups at the N position, precise regulation of the light stability of the dye molecule was achieved. The pentamethine cyanine dye prepared in the present invention exhibits significant advantages in spectral characteristics. The molar extinction coefficient is higher than that of traditional cyanine dyes, and its ability to absorb light energy is strong, which helps to improve the light energy utilization rate and reduce the amount of dye used; the absolute fluorescence quantum yield reaches a relatively high level, significantly enhancing the intensity and sensitivity of the imaging signal, providing a more sensitive and reliable tool for biological imaging and molecular detection.

[0172] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorine-substituted pentamethine cyanine dye, characterized in that, It has a structure of General Formula I: In General Formula I, R1 is one of hydrogen, an alkyl group having 1 - 18 carbons, an aryl group, an aryl cyano group, an aryl carboxyl group, an aryl nitro group, and an aryl sulfonate group; R2 is one of hydrogen, an alkyl group having 1 - 18 carbons, an aryl group, an aryl cyano group, an aryl carboxyl group, an aryl nitro group, and an aryl sulfonate group; Y - is a halogen ion.

2. The preparation method of the fluorine-substituted pentamethine cyanine dye described in claim 1, characterized in that, It includes the following steps: S1. Dissolve Y1 and 3 - methyl - 2 - butanone in an organic acid, heat and reflux for 6 - 12 h under an inert atmosphere to obtain a reaction solution; extract the reaction solution with a first organic solvent, adjust the pH of the organic phase to alkaline with a base, then extract with the first organic solvent again, and after evaporating the organic phase to dryness and purification, obtain compound Y2; wherein, the molar ratio of Y1 to 3 - methyl - 2 - butanone is 1:1 - 5; The N - alkylating agent is selected from one of a halogen - substituted alkyl group, an aryl group, an aryl halogen group, an aryl carboxyl group, an aryl sulfonate group, an aryl nitro group, and an aryl cyano group; S2. Dissolve compound Y2 and the N - alkylating agent in a second organic solvent, reflux and react for 12 - 24 h under the protection of an inert gas and then through purification, respectively obtain compounds Y3 - R1 and Y3 - R2; wherein, the molar ratio of Y2 to the N - alkylating agent is 1:2 - 10; S3. Dissolve compound Y3 - R1 and compound S1 in an organic acid and acetic anhydride solvent, heat to 40 - 120 °C and react for 2 - 6 h under an inert atmosphere; after cooling to room temperature, perform recrystallization, separate the solid and liquid phases, take the solid phase, and after purification, obtain compound Y4; wherein, the molar ratio of Y3 - R1 to compound S1 is 1:0.8 - 1.2, and the volume ratio of the organic acid to the acetic anhydride solvent is 1:0.5 - 2; S4. Dissolve compound Y4, Y3 - R2, a base catalyst and an acetic anhydride solvent, heat to 40 - 120 °C and react for 2 - 6 h under an inert atmosphere, after cooling to room temperature, perform recrystallization, separate the solid and liquid phases, take the solid phase, dry it, and use dichloromethane and methanol as elution solvents, and after purification, obtain a fluorine - substituted pentamethine cyanine dye; wherein the molar ratio of Y4, Y3 - R2 to the base catalyst is 1:0.8 - 1.2:2 - 10.

3. The preparation method of the fluorine-substituted pentamethine cyanine dye according to claim 2, wherein In S1, the base is selected from at least one of sodium hydroxide, potassium hydroxide, ammonia water, and sodium bicarbonate.

4. The preparation method of the fluorine-substituted pentamethine cyanine dye according to claim 2, characterized in that, In S2, the second organic solvent is selected from at least one of toluene, o - dichlorobenzene, ethanol, methanol, isopropanol, acetonitrile, tetrahydrofuran, and sulfolane.

5. The preparation method of the fluorine-substituted pentamethine cyanine dye according to claim 2, characterized in that, In S1 and S3, the organic acid is selected from at least one of acetic acid, hydrochloric acid, sulfuric acid, and polyphosphoric acid.

6. The preparation method of the fluorine-substituted pentamethine cyanine dye according to claim 2, wherein, In S4, the base catalyst is selected from at least one of sodium carbonate, sodium bicarbonate, sodium acetate, triethylamine, sodium hydroxide, and potassium hydroxide.

7. The preparation method of the fluorine-substituted pentamethine cyanine dye according to claim 2, characterized in that, In S1, the first organic solvent is selected from at least one of dichloromethane, ethyl acetate, toluene, and n - hexane.

8. The preparation method of the fluorine-substituted pentamethine cyanine dye according to claim 2, characterized in that, In S2, S3, and S4, the solvents used for recrystallization are all selected from at least one of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone, propanol, and isopropanol.

9. Use of the fluorine-substituted pentamethine cyanine dye according to claim 1, characterized in that, The fluorine - substituted pentamethine cyanine dye is applied to cell imaging, protein labeling, specific recognition of antibodies, nucleic acid labeling, DNA sequencing, and preparation of reagents for specific recognition and photodynamic therapy of tumors.

10. The application of the fluorine-substituted pentamethine cyanine dye according to claim 9, characterized in that, The fluorescence imaging emission wavelength of the described fluorine-substituted pentamethine cyanine dye during application is 600 - 800 nm.

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