CN116836565B - A type of water-soluble squarylium cyanine dye and its synthesis method and application - Google Patents

Abstract

The invention discloses a water-soluble squaraine dye, a synthesis method and application thereof. One type of water-soluble squaraine dye has the following structural general formula: the invention provides a water-soluble squaraine dye, a synthesis method and application thereof, wherein an amphiphilic squaraine dye is synthesized through design, hydrophilic diglycolamine is introduced into a rigid azaindole squaraine dye, and hydrophilic and hydrophobic properties are regulated, so that the amphiphilic squaraine dye can be accurately positioned on a cell membrane. Meanwhile, the change of aggregation form further improves the photo-thermal performance in organisms, and realizes real-time biological imaging and treatment.

Description

A type of water-soluble squarylium cyanine dye and its synthesis method and application

Technical Field

The invention relates to the technical field of organic dyes, in particular to a class of water-soluble squarylium cyanine dyes and a synthesis method and application thereof.

Background Art

The cell membrane is an elastic semipermeable membrane composed of a phospholipid bilayer located outside the cell. Due to its selectivity and permeability, it is particularly important in regulating material exchange and maintaining the stability of the intracellular environment. Abnormalities in the cell membrane are also often used as reference indicators for some related diseases. Fluorescence imaging, as a high-sensitivity and high-resolution technology, has been widely used in cell membrane imaging and plays an important role in real-time imaging and detection of cell membranes.

Photothermal therapy uses photothermal materials to convert external light sources into local heat energy, denaturing proteins, nucleic acids or other intracellular biomacromolecules, affecting cell membrane permeability, and ultimately causing irreversible damage. It can be used to ablate microorganisms or cancer. As a minimally invasive local cancer treatment method, it has the advantages of being non-invasive and remotely controllable, and has attracted widespread attention from researchers in recent years.

However, the cell membrane fluorescent dyes currently studied still have some disadvantages, such as low signal-to-noise ratio, high cytotoxicity, and easy photobleaching, which hinder their application. In addition, there are relatively few reports on dyes that can target cell membranes for photothermal therapy. Therefore, it is very necessary to synthesize a cell membrane-targeted photothermal agent with high signal-to-noise ratio and good biocompatibility for real-time imaging and treatment of cancer.

Summary of the invention

In view of the problems of low signal-to-noise ratio, high cytotoxicity, and easy photobleaching in existing cell membrane fluorescent dyes, the purpose of the present invention is to provide a class of water-soluble squaryl cyanine dyes and their synthesis methods and applications, by designing and synthesizing an amphiphilic squaryl cyanine dye, introducing hydrophilic diglycolamine into the rigid azaindole squaryl cyanine dye, adjusting the hydrophilic and hydrophobic properties, so that it can be accurately positioned on the cell membrane. At the same time, the change in its aggregation morphology also further improves the photothermal performance in the organism, realizing real-time biological imaging and treatment.

In order to achieve the above object, the technical solution of the present invention is: a water-soluble cyanine dye has a structure of general formula I:

In the general formula I,

_R1 and _R4 are each independently selected from at least one of a carboxyalkyl group, a hydroxyl group, and an alkyl sulfonate having 1 to 18 carbon atoms;

_R2 and _R3 are each independently selected from at least one of hydrogen, aryl, and alkyl having 1 to 4 carbon atoms.

Furthermore, _R1 and _R4 are each independently selected from any one of the following structural groups:

Further,

The _R2 and _R3 are independently selected from any one of the following structural groups:

A method for synthesizing a water-soluble cyanine dye comprises the following steps:

(1) Add the carboxyl-substituted 7-azaindole J-1 to a methanol solution, slowly add SOCl ₂ at 0°C, react for 3-5 hours, reflux at 60-80°C for 3-6 hours, then add an amine with R ₁ substitution, and react at 40-70°C for 3-5 hours to obtain the intermediate J-2;

(2) dissolving the intermediate J-2 obtained in step (1) in a first organic solvent at 50-120° C., adding a halogenated alkane substituted with R ₂ to react, and obtaining an intermediate J-3;

(3) dissolving the intermediate J-3 obtained in step (2) in a second organic solvent at 70-120° C., adding squaric acid, and condensing under the catalysis of the first organic base to obtain the intermediate J-4 after purification;

(4) Dissolving the intermediate J-4 obtained in step (3) in a third organic solvent at 100-120° C., adding 5-amide-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine quaternary ammonium salt S-1 substituted with R ₃ and R ₄ , and causing a condensation reaction under the catalysis of a second organic base to obtain a fluorescent dye.

Furthermore, in step (2), the first organic solvent is selected from at least one of benzene, toluene, o-dichlorobenzene, acetonitrile and acetone.

Further, in step (3), the second organic solvent is selected from at least one of ethanol, acetic acid, acetic anhydride, DMF, n-butanol, trimethyl orthoformate, and triethyl orthoformate;

The first organic base is selected from at least one of triethylamine, pyridine and DIPEA.

Further, in step (4), the third organic solvent is selected from at least one of ethanol, acetic acid, acetic anhydride, DMF, trimethyl orthoformate, and triethyl orthoformate;

The second organic base is selected from at least one of triethylamine, pyridine and DIPEA.

Furthermore, in step (2), an amine substituted with R ₁ is added, and the reaction is carried out at 40-70° C. for 3-5 hours, followed by purification by recrystallization to obtain an intermediate J-3, wherein the solvent used for recrystallization is selected from at least one of methanol, ethanol, ethyl acetate, and diethyl ether.

Application of a class of water-soluble squarylium cyanine dyes in biological and medical fields.

Furthermore, it can be used for cell imaging and tumor photothermal therapy.

Furthermore, when applied, the excitation wavelength is 600-950nm, and the fluorescence detection wavelength is 650-1000nm.

In summary, the present invention has the following beneficial effects:

1. The dye of the present invention changes the hydrophilic and hydrophobic properties of the molecule due to the introduction of hydrophilic groups. The lipophilic structure in the dye matrix allows it to enter the cell, while the hydrophilic flexible chains connected at both ends repel the lipid layer, allowing the dye to be accurately positioned on the cell membrane, thereby enabling real-time cell imaging.

2. The present invention significantly enhances the water solubility of the azaindole cyanine dye by modifying the azaindole cyanine dye, thereby solving the problem that the azaindole cyanine dye is easy to aggregate in aqueous solution and making the dye molecules more likely to produce molecular vibration, thereby improving the photothermal performance in vivo. Both in vitro and cell experiments show good photothermal effects, and the dye has good biocompatibility and can be used in aspects such as photothermal therapy of tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

FIG1 is a high-resolution mass spectrum of compound 1 prepared in Example 1 of the present invention;

FIG2 is a normalized emission spectra of Compound 1, Compound 2 and Compound 3 prepared in Example 1 of the present invention in dichloromethane;

FIG3 is an MTT test graph of compound 1 prepared in Example 1 of the present invention;

FIG4 is a graph showing the MTT test of compound 2 prepared in Example 2 of the present invention;

FIG5 is an MTT test graph of compound 3 prepared in Example 3 of the present invention;

FIG6 is a confocal imaging image of compound 1, compound 2, compound 3 prepared in the examples of the present invention and the comparative example in cells.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in further detail.

Unless otherwise specified, the terms used herein have the following meanings.

The term "MTT" as used in the present invention refers to a method for detecting cell survival and growth.

The instruments and equipment used in the examples are:

In the column chromatography process of the present invention, column chromatography silica gel with 200-300 mesh and 100-200 mesh purchased from Qingdao Meigao Group Co., Ltd. and analytical pure quartz sand with 20-40 mesh purchased from Tianda Chemical Reagent Factory are used.

In the process of detecting compounds, the mass spectrometer used was the Synapt G2-Si HDMS high-resolution mass spectrometer from Waters, USA, which adopted a double-needle electrospray ion source to detect the compounds in positive and negative modes.

The absorption and emission spectra of the dyes were measured using a Cary 60 UV-visible spectrophotometer and a Cary Eclipse fluorescence spectrophotometer (Agilent).

The cytotoxicity test was measured using Varioskan LUX Multimode Microplate Reader from Thermofisher, USA.

Cellular uptake experiments were measured using a single-photon confocal microscope from Olympus, Japan.

The cell membrane-targeted squaraine dyes shown in Formula I are described in detail below in conjunction with the examples.

In the general formula I,

_R1 and _R4 are each independently selected from any one of the following structural groups:

_R2 and _R3 are each independently selected from any one of the following structural groups:

Specific examples of the compound represented by the general formula I are given below, but the present invention is not limited to these specific examples.

The synthesis mechanism of the compound shown in the general formula I of the present invention is:

The compound represented by the general formula I of the present invention can be synthesized by the method described below.

Example

Example 1

Preparation of compound 1 wherein _R1 and _R4 are both hydroxyl groups, and _R2 and _R3 are both methyl groups

(1) Manufacturing intermediate 1.1

Carboxyl-substituted 7-azaindole (1 g, 4.90 mmol) was added to 20 mL of methanol solution, and 4 mL of SOCl ₂ was slowly added dropwise at 0°C. After reacting for 3 h, the mixture was transferred to an oil bath and the temperature was raised to 60°C. The mixture was refluxed for 6 h, and then 2.6 mL of diglycolamine was added. The mixture was reacted at 50°C for 3-5 h. After concentration, intermediate 1.1 (0.42 g, 1.44 mmol, yield was 29%) was obtained.

HRMS-ESI:m/z calcd.M ⁺ for C ₁₅ H ₂₂ N ₃ O ₃ ⁺ ,292.1661; found,292.1658.

¹ HNMR(400MHz,Chloroform-d)δ8.79(d,J＝1.6Hz,1H),8.53(t,J＝6.8Hz,1H),8.30(d,J＝1.6Hz,1H),4.24(t ,J＝7.4Hz,1H),3.70–3.51(m,8H),2.66(s,3H),1.36(s,6H).

(2) Manufacturing intermediate 1.2

At 65°C, the intermediate 1.1 obtained in step (1) was dissolved in 20 mL of acetonitrile, and iodomethane (0.82 g, 5.77 mmol) was added for reaction. After recrystallization and purification, intermediate 1.2 (0.34 g, 1.11 mmol, yield 77%) was obtained.

HRMS-ESI:m/z calcd.M ⁺ for C ₁₆ H ₂₄ N ₃ O ₃ ⁺ ,306.1812; found,306.1813.

(3) Preparation of Compound 1

At 115°C, the intermediate 1.2 obtained in step (2) was dissolved in 10 mL of n-butanol, and squaric acid (0.07 g, 0.57 mmol) was added for condensation reaction. After purification by column chromatography, compound 1 (0.025 g, 0.04 mmol, yield 6%) was obtained.

HRMS-ESI:m/z calcd.M ⁺ for C ₃₆ H ₄₅ N ₆ O ₈ ⁺ ,689.3283; found,689.3299.

¹ HNMR(400MHz,Chloroform-d)δ8.86(d,J＝1.4Hz,1H),8.74(d,J＝1.4Hz,1H),8.37–8.26(m,2H),8.12–8.02(m, 2H),7.98(d,J＝1.4Hz,1H),7.24(s,1H),4.08–3.98(m,5H),3.68–3.50(m,16H),3.39(s,3H),1.52(s ,6H),1.41(s,6H) (refer to Figure 1).

Example 2

Preparation of compound 2 wherein _R1 and _R4 are both carboxyl groups, and _R2 and _R3 are both methyl groups

(1) Manufacturing intermediate 2.1

Carboxyl-substituted 7-azaindole (1 g, 4.90 mmol) was added to 20 mL of methanol solution, and 4 mL of SOCl ₂ was slowly added dropwise at 0°C. After reacting for 5 h, the mixture was transferred to an oil bath and the temperature was raised to 60°C. The mixture was refluxed for 6 h, and then 2.8 mL of amino-monoethylene glycol-carboxylic acid was added. The mixture was reacted at 40°C for 5 h. After concentration, intermediate 2.1 (0.51 g, 1.60 mmol, yield 33%) was obtained.

HRMS-ESI:m/z calcd.M ⁺ for C ₁₆ H ₂₂ N ₃ O ₄ ⁺ ,320.1610; found,320.1601.

¹ HNMR(400MHz,Chloroform-d)δ8.79(d,J＝1.6Hz,1H),8.64(td,J＝6.6,0.9Hz,1H),8.30(d,J＝1.4Hz,1H),3.76 (t,J＝7.1Hz,2H),3.66–3.50(m,4H),2.63(s,3H),2.49(t,J＝7.0Hz,2H),1.36(s,6H).

(2) Manufacturing intermediates 2.2

At 65°C, the intermediate 2.1 was dissolved in 20 mL of acetonitrile, and iodomethane (0.85 g, 6.00 mmol) was added to react. After recrystallization and purification, the intermediate 2.2 (0.36 g, 1.08 mmol, yield 67%) was obtained.

HRMS-ESI:m/z calcd.M ⁺ for C ₁₇ H ₂₄ N ₃ O ₄ ⁺ ,334.1761; found,334.1750.

(3) Preparation of Compound 2

At 115°C, the intermediate 2 obtained in step (2) was dissolved in 10 mL of n-butanol, and squaric acid (0.07 g, 0.57 mmol) was added for condensation reaction. After purification by column chromatography, compound 2 (0.028 g, 0.04 mmol, yield 7%) was obtained.

¹ HNMR(400MHz,Chloroform-d)δ8.86(d,J＝1.4Hz,1H),8.40(d,J＝1.6Hz,1H),8.37–8.30(m,1H),8.27(s,1H) ,8.10–8.02(m,2H),7.98(d,J＝1.4Hz,1H),7.24(s,1H),4.06(s,3H),3.76(t,J＝7.1Hz,4H),3.66– 3.51(m,8H),3.39(s,3H),2.50(t,J＝7.1Hz,4H),1.60(s,6H),1.42(s,6H).

Example 3

Preparation of compound 1 wherein _R1 and _R4 are both alkyl sulfonates, and _R2 and _R3 are both methyl

(1) Manufacturing intermediate 3.1

Carboxyl-substituted 7-azaindole (1 g, 4.90 mmol) was added to 20 mL of methanol solution, and 4 mL of SOCl ₂ was slowly added dropwise at 0°C. After reacting for 5 h, the mixture was transferred to an oil bath and the temperature was raised to 60°C. The mixture was refluxed for 6 h, and then 2.4 mL of 3-aminopropanesulfonic acid was added. The mixture was reacted at 40°C for 3-5 h. After concentration, intermediate 3.1 (0.58 g, 1.64 mmol, yield 33%) was obtained.

HRMS-ESI:m/z calcd.M ^- for C ₁₅ H ₂₀ N ₃ O ₅ S ^- ,354.1129; found, 354.1140.

¹ HNMR(400MHz,Chloroform-d)δ8.79(d,J＝1.4Hz,1H),8.62(t,J＝6.8Hz,1H),8.30(d,J＝1.4Hz,1H),3.69(t ,J＝7.1Hz,2H),3.65–3.59(m,2H),3.59–3.50(m,2H),3.35(t,J＝7.1Hz,2H),2.52(s,3H),1.37(s, 6H) ^.

(2) Manufacturing intermediate 3.2

At 65°C, the intermediate 3.1 obtained in step (1) was dissolved in 20 mL of acetonitrile, and iodomethane (0.92 g, 6.56 mmol) was added for reaction. After recrystallization and purification, intermediate 3.2 (0.43 g, 1.16 mmol, yield 71%) was obtained.

HRMS-ESI:m/z calcd.M ⁺ for C ₁₆ H ₂₄ N ₃ O ₅ S ⁺ ,370.1437; found,370.1402.

(3) Preparation of Compound 3

At 115°C, the intermediate 3.2 obtained in step (2) was dissolved in 10 mL of n-butanol, and squaric acid (0.07 g, 0.57 mmol) was added for condensation reaction. After purification by column chromatography, compound 3 (0.027 g, 0.03 mmol, yield 6%) was obtained.

¹ HNMR(400MHz,Chloroform-d)δ8.86(d,J＝1.4Hz,1H),8.73(d,J＝1.6Hz,1H),8.34–8.26(m,1H),8.23(s,1H) ,8.10–8.02(m,2H),7.98(d,J＝1.4Hz,1H),7.24(s,1H),4.06(s,3H),3.70(t,J＝7.1Hz,4H),3.65– 3.51(m,8H),3.41–3.31(m,7H),1.60(s,6H),1.42(s,6H).

Comparative Example 1

Preparation of compound 4

(1) Manufacturing intermediate 4.1

7-Azaindole (1.00 g, 4 mmol) and iodomethane (1.77 g, 8 mmol) were added to a 100 mL double-necked round-bottom flask containing 20 mL of acetone and placed under nitrogen. The mixture was then heated to reflux overnight to terminate the reaction. After cooling to room temperature, 50 mL of ether was added for precipitation. The solid precipitate was filtered, washed with ether, and dried to obtain a brown solid intermediate 4.1 (1.64 g, 5.4 mmol, yield 87%).

(2) Preparation of Compound 4

Under nitrogen protection, square acid (300 mg, 2.6 mmol) and compound 1.2 (1.59 g, 5.3 mmol) were heated to react at 120°C in a mixed solvent of triethyl orthoformate and n-butanol. The reaction was stopped after stirring for 2 h. After the reaction solution cooled to room temperature, it was added dropwise into 150 mL of ether for recrystallization. The crude product was purified by silica gel column, and then purified by silica gel chromatography using 80:1 dichloromethane/methanol (v/v) as the elution solvent to obtain a blue solid compound 4 (0.067 g, 0.16 mmol, yield 6%)

HRMS-ESI: m/z calcd.M ⁺ for C ₂₆ H ₂₆ N ₄ O ₂ ⁺ ,427.2129; found, 427.2130.

¹ HNMR(400MHz,Chloroform-d)δ8.25(d,J＝4.7Hz,2H),7.60(d,J＝7.2Hz,2H),7.05(dd,J＝7.3,5.1Hz,2H),6.07 (s,2H),3.68(s,6H),1.80(d,J=6.1Hz,12H).

Performance Testing

The compounds prepared in the above Examples 1-3 and Comparative Example 1 were subjected to the following performance tests, and the test methods are as follows:

Test Example 1

The fluorescence spectra of compound 1, compound 2 and compound 3 obtained in the above examples were measured.

Use a 1/10,000 balance to accurately weigh the vacuum-dried dye, prepare a 2 mmol/L DMSO dye stock solution in a brown sample bottle, and store it in a 4°C refrigerator for later use.

When testing the UV-visible absorption spectrum and fluorescence spectrum, use a micropipette to measure 3 μL of the dye mother solution and dissolve it in a quartz cuvette containing 3 mL of the solvent to be tested, mix well, and obtain a dye concentration of 2.0 μmol/L for the absorption spectrum and fluorescence emission spectrum test. All tests were completed at 25°C.

FIG2 is a normalized emission spectra of compound 1, compound 2, and compound 3 in dichloromethane;

As shown in Figure 2, in dichloromethane solution, the emission of Compound 1, Compound 2, and Compound 3 is between 640-750nm, which is in the near-infrared region. The emission in the near-infrared region is beneficial to avoiding the influence of biological autofluorescence and can have better resolution and presentation effects. Therefore, this is more conducive to the application of dyes in biological imaging and treatment.

Test Example 2

Cytotoxicity test of compound 1, compound 2 and compound 3 prepared in Example

The toxicity of dye molecules to cells is evaluated by MTT assay. The principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in cells, and its light absorption value is measured at 490nm and 570nm wavelengths using a microplate reader, which can indirectly reflect the number of living cells.

MCF-7 cells were seeded in 96-well plates and incubated for 24 hours (1×10 ⁴ cells per well). DMEM culture medium containing different concentrations of compound 1, compound 2, and compound 3 was added to the wells, and light/dark treatment was given. After incubation for 24 hours, the cell activity was detected by MTT assay. The experimental data are shown in Figures 3, 4, and 5.

As can be seen from Figures 3, 4, and 5, Compound 1, Compound 2, and Compound 3 showed good biocompatibility in MCF-7 cells, which is conducive to the specific imaging of the dye in cells. At the same time, different concentrations of Compound 1, Compound 2, and Compound 3 were selected for phototoxicity experiments. The results showed that the dye had good phototoxicity and could effectively kill cells at 300mW, confirming that the dye can efficiently generate heat under light, thus having excellent photothermal therapy effects, and can therefore be applied to biological imaging and fields.

Test Example 3

Cellular uptake experiments were performed on Compound 1, Compound 2, Compound 3 obtained in Example 1 and Compound 4 obtained in Comparative Example 1.

MCF-7 cells were inoculated into the confocal culture dish one day in advance, and after the cells adhered to the wall, they were washed three times with PBS solution, and then 2 mL of culture medium and 1 μM of compound 1, compound 2, compound 3, and comparative example were added, and cell fluorescence images were taken with a confocal laser scanning microscope (CLSM) to observe the uptake of dyes and cells. The excitation wavelength was 640 nm, and the emission and receiving bands were 650-700 nm. The experimental data are shown in Figure 6.

As can be seen from Figure 6, compound 1, compound 2, compound 3 can be specifically positioned at cell membrane within 2h, and comparative example compound 4 has entered the cell, and has strong fluorescence in the cell, but is not positioned on the cell membrane. Therefore, it can be found that after introducing a water-soluble group, the water solubility of compound 1, compound 2, and compound 3 is enhanced, so that it has an amphipathic structure, and is more easily positioned at the cell membrane. The lipophilic structure in the dye matrix allows it to enter the cell, and the hydrophilic flexible chain connected at both ends repels from the lipid layer, so compound 1, compound 2, and compound 3 can be specifically positioned on the cell membrane, with the potential of an ideal fluorescent biological probe.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, 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 (9)

1. A water-soluble cyanine dye, characterized in that it has a structure of general formula I: In the general formula I, _R1 and _R4 are the same and are selected from at least one of carboxyl, hydroxyl, and alkyl sulfonate; _R2 and _R3 are the same and are alkyl groups having 1 to 4 carbons. 2. A water-soluble cyanine dye according to claim 1, characterized in that _R2 and _R3 are the same, and _R2 and _R3 are selected from any one of the following structural groups: 3. A method for synthesizing a water-soluble cyanine dye according to claim 1 or 2, characterized in that it comprises the following steps: (1) Add the carboxyl-substituted 7-azaindole J-1 to a methanol solution, slowly add SOCl ₂ at 0°C, react for 3-5 hours, reflux at 60-80°C for 3-6 hours, then add an amine with R ₁ substitution, and react at 40-70°C for 3-5 hours to obtain the intermediate J-2; (2) dissolving the intermediate J-2 obtained in step (1) in a first organic solvent at 50-120° C., adding an iodinated alkane substituted with R ₂ to react, and obtaining an intermediate J-3; (3) dissolving the intermediate J-3 obtained in step (2) in a second organic solvent at 70-120° C., adding squaric acid, and condensing under the catalysis of the first organic base to obtain the intermediate J-4 after purification; At 100-120° C., the prepared intermediate J-4 is dissolved in a third organic solvent, and 5-amide-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine quaternary ammonium salt S-1 with R ₃ and R ₄ substitutions is added, and a condensation reaction occurs under the catalysis of a second organic base to obtain a fluorescent dye. 4. The method for synthesizing a class of water-soluble cyanine dyes according to claim 3, characterized in that, in step (2), the first organic solvent is selected from at least one of benzene, toluene, o-dichlorobenzene, acetonitrile and acetone. 5. The method for synthesizing a water-soluble cyanine dye according to claim 3, characterized in that, in step (3), the second organic solvent is selected from at least one of ethanol, acetic acid, acetic anhydride, DMF, n-butanol, trimethyl orthoformate, and triethyl orthoformate; The first organic base is selected from at least one of triethylamine, pyridine and DIPEA. 6. The method for synthesizing a water-soluble cyanine dye according to claim 3, characterized in that, in step (3), the third organic solvent is selected from at least one of ethanol, acetic acid, acetic anhydride, DMF, trimethyl orthoformate, and triethyl orthoformate; The second organic base is selected from at least one of triethylamine, pyridine and DIPEA. 7. The method for synthesizing a water-soluble cyanine dye according to claim 3, characterized in that, in step (2), an amine substituted with R ₁ is added, reacted at 40-70° C. for 3-5 hours, and then purified by recrystallization to obtain an intermediate J-3, wherein the solvent used for recrystallization is selected from at least one of methanol, ethanol, ethyl acetate, and ether. 8. Application of the water-soluble squarylium cyanine dye according to claim 1 or 2 in the biological and medical fields. 9. The use according to claim 8, characterized in that the excitation wavelength is 600-950nm and the fluorescence detection wavelength is 650-1000nm.