Synthesis and application of pentamethylcyanine dye fluorescent probe for dynamic super-resolution imaging of cell membranes

Abstract

The present invention has developed a pentamethine cyanine dye fluorescent probe for long-term super-resolution imaging of cell membrane dynamics. This dye has the advantages of low-cost synthetic raw materials and mild synthesis conditions. Research has found that this type of dye forms a buffer pool by linking a sulfonamide structure via an aliphatic chain at the 3-position of the indole ring of the cyanine dye, leveraging the weak binding ability of sulfonamide with carbonic anhydrase. This buffering strategy circumvents the susceptibility of cyanine dyes to photobleaching, allowing long-term super-resolution dynamic imaging of cell membranes expressing carbonic anhydrase 9 to observe morphological changes in the cell membrane without changing the dye's original spectral properties.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence imaging, and in particular relates to a method for synthesizing a pentamethine cyanine dye fluorescent probe suitable for long-term dynamic super-resolution imaging of cell membranes and its application in the field of fluorescence imaging. Background Art

[0002] The cell membrane is a dynamic structure composed of a phospholipid bilayer and a variety of proteins anchored within it. It plays an important role in the transmission of information between cells and in regulating the exchange of substances between cells and between cells and the external environment. Monitoring the dynamic processes of the cell membrane helps us understand the biological activities of cells and appreciate their physiological significance. Fluorescence imaging technology is one of the most powerful tools in the field of modern life sciences. The emergence of super-resolution imaging technology has increased the resolution of fluorescence imaging to the nanoscale, allowing us to clearly observe the fine structure of cells. Dyes with higher brightness and photostability are necessary to achieve high-resolution imaging. Unfortunately, most existing fluorescent dyes lack sufficient brightness for super-resolution dynamic imaging.

[0003] Pentamethine cyanine dyes are widely used in biology due to their high molar extinction coefficient, wide range of absorption and emission wavelengths, low background fluorescence, and excellent biocompatibility. However, the positive charge of the cyanine dye matrix makes it easier to target mitochondria. Furthermore, due to the high activity of the methine chain, they are easily attacked by singlet oxygen in the environment, leading to photobleaching, which limits their application in super-resolution imaging. Therefore, the rational modification of pentamethine cyanine dyes to regulate their targeting ability and enable long-term super-resolution dynamic imaging is a key research focus. Summary of the Invention

[0004] This study has developed a pentamethine cyanine dye fluorescent probe suitable for long-term super-resolution imaging of cell membrane dynamics. This probe incorporates a sulfonamide structure into the pentamethine cyanine dye matrix, utilizing a buffering strategy to enhance the imaging stability of the dye without altering its luminescent properties. Research has shown that this type of dye exhibits high brightness and enables long-term super-resolution dynamic imaging of cell membranes, enabling observation of the growth and fusion of pseudopodia on the cell membrane.

[0005] The pentamethine cyanine dye fluorescent probe suitable for long-term cell membrane dynamic super-resolution imaging of the present invention has the following structural formula:

[0006]

[0007] At the same time, the present invention also provides a general synthesis method of the pentamethine cyanine dye, the synthesis steps are as follows:

[0008]

[0009] Weigh 2.0-5.0 g of 2,3,3-trimethyl-3H-indole and 5.0-10.0 g of methyl iodide into a sealed tube, heat to 70-100°C, and react for 10-20 hours. Transfer the solid to a Buchner funnel and wash with ether to obtain 1,2,3,3-tetramethyl-3H-indolium iodide.

[0010] Step 2: Synthesis of intermediate 2-acetyl-2-methylcyclohexanone

[0011] Weigh 2.0-5.0g of 2-acetylcyclohexanone and 3.0-7.0g of iodomethane into a round-bottom flask and dissolve in methanol. Slowly add 1.0-3.0g of an aqueous solution of sodium hydroxide at low temperature. Raise the temperature to 70-100°C and react for 10-20 hours. Dissolve the reaction mixture in 60-100ml of water and extract with dichloromethane. Remove the solvent under reduced pressure and perform silica gel column chromatography to obtain the intermediate 2-acetyl-2-methylcyclohexanone.

[0012] Step 3: Synthesis of intermediate 6-methyl-7-oxooctanoic acid methyl ester

[0013] A mixture of 1.0-4.0 g of the intermediate 2-acetyl-2-methylcyclohexanone and 0.4-1.0 g of lithium methoxide was dissolved in 10-30 ml of methanol, added to a dry round-bottom flask, and stirred at room temperature overnight. The mixture was dissolved in water and adjusted to a pH of 5-6 with HCl. The product was then extracted with dichloromethane and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue was purified by column chromatography to yield the intermediate methyl 6-methyl-7-oxooctanoate.

[0014] Step 4: Synthesis of intermediate 6-methyl-7-oxooctanoic acid

[0015] 2.0-4.0 g of the intermediate methyl 6-methyl-7-oxooctanoate was dissolved in 10-20 ml of 12-18% HCl, and the mixture was stirred under reflux overnight. The reaction solution was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the intermediate 6-methyl-7-oxooctanoic acid.

[0016] Step 5: Synthesis of intermediate 2,3-dimethyl-3H-indole-3-pentanoic acid

[0017] Weigh 0.4-1.0g of phenylhydrazine and 0.4-1.0g of the intermediate 6-methyl-7-oxooctanoic acid into a round-bottom flask, dissolve in 4-10ml of acetic acid, and stir under reflux at 100-120°C for 2-4 hours. Remove the solvent under reduced pressure, and perform silica gel column chromatography to obtain the intermediate 2,3-dimethyl-3H-indole-3-pentanoic acid. Step 6: Synthesis of the intermediate 1,2,3-trimethyl-3H-indole-3-pentanoic acid iodide (1:1). Weigh 0.1-0.3g of the intermediate 2,3-dimethyl-3H-indole-3-pentanoic acid, 0.3-0.5g of iodomethane, and 1-3ml of acetonitrile in a sealed tube. Reaction mixture at 80-100°C for 12-18 hours. Remove the solvent under reduced pressure, and perform silica gel column chromatography to obtain the intermediate 1,2,3-trimethyl-3H-indole-3-pentanoic acid iodide (1:1). (The ratio of 1:1 means that the molar ratio of 1,2,3-trimethyl-3H-indole-3-pentanoic acid to iodine in the compound is 1:1)

[0018] Step 7: Synthesis of intermediate N-[3-(phenylamino)-2-propylene-1-ylidene]aniline

[0019] Weigh 6.0-9.0 g of 1,1,3,3-tetraethoxypropane and 3-8 ml of hydrochloric acid into 60-80 ml of distilled water and stir at 30-60°C. Then, add a solution of 4.0-8.0 ml of aniline, 10-20 ml of hydrochloric acid, and 60-80 ml of distilled water dropwise to the reaction mixture and continue stirring at 50-70°C for 1-3 hours. After cooling, filtering, and drying, the intermediate N-[3-(phenylamino)-2-propen-1-ylidene]aniline is obtained.

[0020] Step 8: Synthesis of intermediate carboxyl-containing pentamethine cyanine dye

[0021] 0.2-0.4 g of the intermediate 1,2,3,3-tetramethyl-3H-indolium iodide and 0.3-0.6 g of the intermediate N-[3-(phenylamino)-2-propylene-1-ylidene]aniline were weighed and added to 5-10 ml of acetic anhydride, and the mixture was stirred at 30-60 ° C for 3-5 hours. The solution was cooled to room temperature and the solvent was evaporated under reduced pressure. Then, 5-10 mL of anhydrous ethanol, 0.3-0.5 g of the intermediate 1,2,3-trimethyl-3H-indole-3-pentanoic acid-iodide (1:1) and 0.2-0.5 g of sodium acetate were added; the mixture was stirred at 30-60 ° C for 1-3 hours. After the reaction was completed, the product was purified by silica gel column chromatography to obtain the intermediate carboxyl-containing pentamethine cyanine dye.

[0022] Step 9: Synthesis of Pentamethine Cyanine Dye for Super-Resolution Dynamic Imaging of Cell Membranes

[0023] A mixture of 40-70 mg of the intermediate carboxyl-containing pentamethicone cyanine dye and 60-90 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate was weighed and added to a round-bottom flask. 80-100 μL of diisopropylethylamine was dissolved in anhydrous N,N-dimethylformamide and added to the flask. The mixture was stirred at room temperature for 30-60 minutes. Then, 60-90 mg of 4-aminomethylbenzenesulfonamide hydrochloride was dissolved in anhydrous N,N-dimethylformamide and added to the flask. Silica gel column chromatography was used to obtain pentamethicone cyanine dye suitable for super-resolution dynamic imaging of cell membranes.

[0024] In step 1, 2.0-5.0 g of 2,3,3-trimethyl-3-H-indole and 5.0-10.0 g of iodomethane.

[0025] In step 2, 2.0-5.0 g of 2-acetylcyclohexanone, 3.0-7.0 g of iodomethane, and 1.0-3.0 g of sodium hydroxide in water

[0026] In step 3, 1.0-4.0 g of the intermediate 2-acetyl-2-methylcyclohexanone and 0.4-1.0 g of lithium methoxide are used.

[0027] In step 4, 2.0-4.0 g of the intermediate methyl 6-methyl-7-oxooctanoate and 10-20 ml of 12-18% HCl are used.

[0028] In step 5, 0.4-1.0 g of phenylhydrazine, 0.4-1.0 g of the intermediate 6-methyl-7-oxooctanoic acid, and 4-10 ml of acetic acid.

[0029] In step six, 0.1-0.3 g of the intermediate 2,3-dimethyl-3H-indole-3-pentanoic acid and 0.3-0.5 g of iodomethane.

[0030] In step 7, 6.0-9.0 g of 1,1,3,3-tetraethoxypropane, 3-8 ml of hydrochloric acid, 4.0-8.0 ml of aniline, and 10-20 ml of hydrochloric acid are added. In step 8, 0.2-0.4 g of the intermediate 1,2,3,3-tetramethyl-3H-indolium iodide, 0.3-0.6 g of the intermediate N-[3-(phenylamino)-2-propen-1-ylidene]aniline, 5-10 ml of anhydrous ethanol, 0.3-0.5 g of the intermediate 1,2,3-trimethyl-3H-indole-3-pentanoic acid iodide (1:1 ratio), and 0.2-0.5 g of sodium acetate are added.

[0031] In step nine, 40-70 mg of the intermediate carboxyl-containing pentamethine cyanine dye, 60-90 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 80-100 μL of diisopropylethylamine, 60-90 mg of benzenesulfonamide, and 4-(aminomethyl)-hydrochloride (1:1) are added. The above-mentioned cell membrane fluorescent probe can label the cell membrane in living cells and achieve long-term super-resolution dynamic imaging.

[0032] The present invention has the following features:

[0033] This type of probe has the advantages of low-cost synthetic raw materials and mild synthesis conditions.

[0034] This type of probe enables specific binding to carbonic anhydrase 9 without changing the dye's original spectral properties. It can rapidly label and stain cell membranes transfected with carbonic anhydrase membrane protein, and perform long-term super-resolution dynamic imaging to observe morphological changes in the cell membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : is the H NMR spectrum of the product in Example 1;

[0036] Figure 2 : is the H NMR spectrum of the product in Example 3;

[0037] Figure 3 : is the H NMR spectrum of the product in Example 4;

[0038] Figure 4 : is the H NMR spectrum of the product in Example 5;

[0039] Figure 5 : is the H NMR spectrum of the product in Example 8;

[0040] Figure 6 : is the H NMR spectrum of the product in Example 9;

[0041] Figure 7 : Normalized fluorescence excitation and emission spectra of the cell membrane probe S-Cy5 prepared in Example 9 in water, the abscissa is the wavelength, the ordinate is the normalized fluorescence intensity and absorption intensity, and the concentration of the fluorescent probe is 2 μM;

[0042] Figure 8 : Long-term super-resolution dynamic imaging of HeLa cells using the cell membrane probe S-Cy5 prepared in Example 9. DETAILED DESCRIPTION

[0043] The present invention provides a pentamethine cyanine dye fluorescent probe suitable for long-term dynamic super-resolution imaging of cell membranes and its application in the field of fluorescence imaging.

[0044] Example 1

[0045] The synthetic route and product structure of compound 1 are as follows:

[0046]

[0047] 2,3,3-Trimethyl-3-H-indole (3.50 ml, 21.80 mmol) and iodomethane (6.77 ml, 0.11 mol) were weighed into a sealed tube and heated at 100°C under reflux overnight. The solid was transferred to a Buchner funnel and washed with 30 ml of ether to obtain Compound 1 (5.92 g, 19.67 mmol, 90%) as a light pink solid.

[0048] 1 H NMR (400MHz, DMSO) δ7.94–7.88(m,1H),7.85–7.80(m,1H),7.66–7.59(m,2H),3.97(d,J=0.4Hz,3H),2.76(s,3H),1.53(s,6H).

[0049] Example 2

[0050] The synthetic route and product structure of compound 2 are as follows:

[0051]

[0052] 2-Acetylcyclohexanone (3 ml, 23.1 mol) and iodomethane (4.32 ml, 69.3 mol) were weighed into a round-bottom flask and dissolved in methanol (9 ml). A 6 ml aqueous solution of sodium hydroxide (924 mg, 23.1 mol) was slowly added under an ice bath. The reaction temperature was raised to 70°C and refluxed for 12 h. The reaction solution was dissolved in 100 ml of water, extracted with dichloromethane, washed with 2 mol / L NaOH (100 ml), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography (volume ratio, petroleum ether:ethyl acetate = 20:1). Compound 2 (1.50 ml, 12.0 mmol, 52%) was obtained.

[0053] Example 3

[0054] The synthetic route and product structure of compound 3 are as follows:

[0055]

[0056] A mixture of compound 2 (2 g, 13.0 mmol) and lithium methoxide (543 mg, 14.3 mmol) was weighed and dissolved in 10 ml of methanol, added to a dry round-bottom flask, and stirred at room temperature for 12 h. The mixture was dissolved in water and adjusted to pH 5-6 with concentrated hydrochloric acid. The product was then extracted with dichloromethane and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue was purified by column chromatography (volume ratio, petroleum ether: ethyl acetate = 10:1). Compound 3 was a colorless liquid (1.30 g, 7.0 mmol, 54%).

[0057] 1 H NMR (400MHz, CDCl3) δ3.71–3.65(m,3H),2.52(dd,J=13.6,6.8Hz,1H),2.32(t,J=7.4Hz ,1H),2.15(s,1H),1.65(dd,J=14.7,7.1Hz,1H),1.43–1.22(m,1H),1.14–1.06(m,1H).

[0058] Example 4

[0059] The synthetic route and product structure of compound 4 are as follows:

[0060]

[0061] Compound 3 (1.32 g, 7.0 mmol) was weighed and dissolved in 6 ml of 12% aqueous HCl solution. The mixture was stirred under reflux for 12 h. The reaction mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. This gave compound 4 (0.87 g, 5.1 mmol, 73%) as a colorless, transparent liquid. 1 H NMR (400MHz, CDCl3) δ10.08 (s, 1H), 2.50–2.39 (m, 1H), 2.29 (t, J = 7.4Hz, 2H), 2.07 (s ,3H),1.59(ddt,J=22.3,14.8,7.3Hz,3H),1.35–1.16(m,3H),1.02(d,J=7.0Hz,3H).

[0062] Example 5

[0063] The synthetic route and product structure of compound 5 are as follows:

[0064]

[0065] Phenylhydrazine (432.6 mg, 4 mmol) and compound 4 (688.4 mg, 4 mmol) were weighed into a round-bottom flask and dissolved in 4 ml of acetic acid. The mixture was stirred and refluxed at 120°C for 3.5 hours. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (volume ratio, dichloromethane:ethyl acetate:methanol = 100:50:5). The resulting product was recrystallized from ethyl acetate-n-hexane (0.6-2). 001-09 was obtained as yellow crystals (324.9 mg, 1.33 mmol, 33%).

[0066] 1 H NMR (400MHz, CDCl3) δ12.50(s,1H),7.48(d,J=7.6Hz,1H),7.21(td,J=7.4,1.3Hz,1H),7.17–7.08(m,2H),2.18(s,3H),2.11(td,J=7.4,2. 0Hz,2H),1.88–1.77(m,1H),1.77–1.67(m,1H),1.48–1.35(m,2H),1.21(s,3H),0.71(dd,J=12.5,7.3Hz,1H),0.58(dd,J=12.6,7.4Hz,1H).

[0067] Example 6

[0068] The synthetic route and product structure of compound 6 are as follows:

[0069]

[0070] Compound 5 (245.1 mg, 0.66 mmol), iodomethane (311 μL, 5 mmol), and 1 mL of acetonitrile were dissolved in a sealed tube. The mixture was reacted at 100°C overnight. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (ethyl acetate:methanol = 100:10 by volume) to afford Compound 6 (184.7 mg, 0.48 mmol, 72%) as a yellow solid.

[0071] Example 7

[0072] The synthetic route and product structure of compound 7 are as follows:

[0073]

[0074] 1,1,3,3-Tetraethoxypropane (7.19 mL, 30 mmol) and 4.3 mL of concentrated hydrochloric acid were added to 70 mL of distilled water and stirred at 50°C. A solution of 5.47 mL of aniline, 10 mL of hydrochloric acid, and 70 mL of distilled water was then added dropwise to the reaction mixture and stirred at 50°C for 1 hour. After cooling, filtration, and drying, compound 7 was obtained, which was carried on to the next step without further purification.

[0075] Example 8

[0076] The synthetic route and product structure of compound 8 are as follows:

[0077]

[0078] Compound 1 (361.2 mg, 1.2 mmol) and compound 7 (455.9 mg, 1.2 mmol) were weighed into (Ac)2O (5 mL), and the mixture was stirred at 60°C for 5 hours. The solution was cooled to room temperature, and the solvent was evaporated under reduced pressure. EtOH (5 mL), compound 6 (387.1 mg, 1.0 mmol), and NaOAc (246.1 mg, 3.0 mmol) were then added; the mixture was stirred at 50°C for 1.5 hours. After completion of the reaction, the product was purified by silica gel column chromatography (volume ratio, dichloromethane:methanol = 10:1) to obtain compound 8 (90.3 mg, 0.15 mmol, 15%).

[0079] 1 H NMR(400MHz,MeOD)δ8.25(dd,J=25.4,12.5Hz,2H),7.55–7.38(m,4H),7.28(dd,J=17.9,9.2Hz ,4H),6.64(t,J=12.4Hz,1H),6.30(dd,J=13.7,2.2Hz,2H),3.63(s,3H),3.61(s,3H),2.50–2. 39(m,1H),2.21(td,J=13.0,4.3Hz,1H),2.11(t,J=7.2Hz,2H),1.73(d,J=1.6Hz,6H),1.70(s, 3H), 1.46 (td, J=13.8, 7.3Hz, 2H), 0.91 (dd, J=28.6, 12.9Hz, 1H), 0.62 (dd, J=12.3, 6.4Hz, 1H).

[0080] Example 9

[0081] The synthetic route and product structure of compound 9 are as follows:

[0082]

[0083] A mixture of compound 8 (59.6 mg, 0.1 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (76 mg, 0.3 mmol) was weighed and added to a round-bottom flask. Diisopropylethylamine (87 μl, 0.5 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 ml) and added to the flask. The mixture was stirred at room temperature for 30 minutes. 4-aminomethylbenzenesulfonamide hydrochloride (66.8 mg, 0.3 mmol) was then dissolved in anhydrous N,N-dimethylformamide (2 ml) and added to the flask. Silica gel column chromatography obtained pentamethyl cyanine dye S-Cy5 suitable for super-resolution dynamic imaging of cell membranes.

[0084] 1 H NMR (700MHz, MeOD) δ8.23(t,J=13.1Hz,1H),8.18(t,J=13.0Hz,1H),7.82(d,J=8.1Hz,2H),7.49(d,J=7.4Hz,1H),7.42(dd,J= 17.5,7.7Hz,3H),7.33(d,J=8.1Hz,2H),7.30(d,J=7.9Hz,1H),7.26(dd,J=18.8,8.6Hz,3H),6.62(t,J=12.4Hz,1H),6.29(d, J=13.7Hz,1H),6.24(d,J=13.6Hz,1H),4.31(q,J=15.4Hz,2H),3.64(s,3H),3.55(s,3H),2.44(td,J=13.4,4.4Hz,1H),2.19( td,J=13.7,4.1Hz,1H),2.14–2.04(m,2H),1.71(s,6H),1.68(s,3H),1.57–1.42(m,2H),0.94–0.87(m,1H),0.62–0.53(m,1H).

[0085] The dye S-Cy5 to be tested was dissolved in dimethyl sulfoxide solution to prepare a 2 mM stock solution. Test solutions of different concentrations were prepared as needed to detect the changes in fluorescence spectrum and cell membrane fluorescence imaging.

[0086] Example 10

[0087] Spectral test of S-Cy5 in water: Take 3 μL of S-Cy5 stock solution and add it to 3 mL of water to prepare a 2 μM fluorescent probe test solution. Then perform UV-visible absorption and fluorescence emission spectroscopy (excitation light is 610 nm).

[0088] Figure 7The concentration of the fluorescent probe is 2μM, which is the absorption spectrum and fluorescence spectrum of S-Cy5 in water. The molar extinction coefficient of S-Cy5 in water is 180000M -1 cm -1 , the quantum yield reaches 0.14, and the probe has high brightness.

[0089] Example 11

[0090] Super-resolution fluorescence imaging was performed after staining HeLa cells expressing carbonic anhydrase 9 on the cell membrane with S-Cy5. 0.5 μL of 0.2 mmol / L S-Cy5 stock solution was dissolved in 1 mL of HeLa culture medium and incubated at room temperature for 15 minutes before long-term super-resolution dynamic imaging.

[0091] Figure 8 In the experiment, S-Cy5 was able to precisely locate the cell membrane of HeLa cells, image the cell membrane for up to 60 minutes, and observe the growth changes of pseudopodia on the cell membrane.

Claims

1. Pentamethine cyanine dye suitable for long-term dynamic super-resolution imaging of cell membranes, characterized by: Its structural formula is shown below, 。 2. A method for synthesizing the pentamethine cyanine dye fluorescent probe according to claim 1, characterized in that: The specific method of this synthesis is as follows: (1): Intermediate 1, 2, 3, 3-tetramethyl-3 H -Synthesis of indolium iodides Weigh 2.0-5.0 g of 2, 3, 3-trimethyl-3-H-indole and 5.0-10.0 g of iodomethane into a sealed tube and heat to 70-100 °C for 10-20 hours. Transfer the solid and wash it with ether to obtain 1, 2, 3, 3-tetramethyl-3-H-indole. H -Indolium iodide; (2): Synthesis of intermediate 2-acetyl-2-methylcyclohexanone Weigh 2.0-5.0 g of 2-acetylcyclohexanone and 3.0-7.0 g of iodomethane into a container and dissolve them in 5-15 ml of methanol. Add 0.8-3.0 g of sodium hydroxide in 4-8 ml of aqueous solution. Heat the mixture to 70-100°C and react for 10-20 hours. Dissolve the reaction mixture in 60-100 ml of water and extract with dichloromethane. Remove the solvent under reduced pressure and perform silica gel column chromatography to obtain the intermediate 2-acetyl-2-methylcyclohexanone. (3): Synthesis of intermediate 6-methyl-7-oxooctanoic acid methyl ester A mixture of 1.0-4.0 g of the intermediate 2-acetyl-2-methylcyclohexanone and 0.4-1.0 g of lithium methoxide was dissolved in 10-30 ml of methanol, added to a container, and stirred at room temperature for 10-15 hours. The mixture was dissolved in water and adjusted to pH 5-6 with concentrated hydrochloric acid. The product was then extracted with dichloromethane and dried over anhydrous sodium sulfate. The solvent was removed and purified to obtain the intermediate methyl 6-methyl-7-oxooctanoate. (4): Synthesis of the intermediate 6-methyl-7-oxooctanoic acid Weigh 2.0-4.0 g of the intermediate methyl 6-methyl-7-oxooctanoate and dissolve it in 10-20 ml of 12-18% HCl. Stir and reflux for 12-15 hours. Extract the reaction solution with dichloromethane, dry it over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain the intermediate 6-methyl-7-oxooctanoic acid. (5): Synthesis of the intermediate 2,3-dimethyl-3H-indole-3-pentanoic acid Weigh 0.4-1.0 g of phenylhydrazine and 0.4-1.0 g of the intermediate 6-methyl-7-oxooctanoic acid into a container, add 4-10 ml of acetic acid, and stir and reflux at 100-120°C for 2-4 hours. Remove the solvent under reduced pressure, and obtain the intermediate 2,3-dimethyl-3H-indole-3-pentanoic acid by silica gel column chromatography. (6): Synthesis of intermediate 1:1 1,2,3-trimethyl-3H-indole-3-pentanoic acid-iodide Weigh 0.1-0.3 g of the intermediate 2,3-dimethyl-3H-indole-3-pentanoic acid, 0.3-0.5 g of iodomethane, and 1-3 ml of acetonitrile and dissolve them in a sealed tube; react at 80-100°C for 12-18 hours; remove the solvent under reduced pressure, and perform silica gel column chromatography to obtain the intermediate 1:1 1,2,3-trimethyl-3H-indole-3-pentanoic acid iodide; (7): Synthesis of intermediate N-[3-(phenylamino)-2-propylene-1-ylidene]aniline Weigh 6.0-9.0 g of 1,1,3,3-tetraethoxypropane and 3-8 ml of concentrated hydrochloric acid into 60-80 ml of distilled water and stir at 30-60°C. Then, add 4.0-8.0 ml of aniline, 10-20 ml of concentrated hydrochloric acid, and 60-80 ml of distilled water dropwise to the reaction mixture and continue stirring at 50-70°C for 1-3 hours. After cooling, filtering, and drying, the intermediate N-[3-(phenylamino)-2-propylene-1-ylidene]aniline is obtained. (8): Synthesis of intermediate carboxyl-containing pentamethine cyanine dye Weigh 0.2-0.4 g of the intermediate 1,2,3,3-tetramethyl-3 H -indolium iodide and 0.3-0.6 g of the intermediate N-[3-(phenylamino)-2-propen-1-ylidene]aniline are added to 5-10 ml of acetic anhydride, and the mixture is stirred at 30-60°C for 3-5 hours; the solution is cooled to room temperature, and the solvent is evaporated under reduced pressure; then 5-10 mL of anhydrous ethanol, 0.3-0.5 g of the intermediate 1:1 1,2,3-trimethyl-3H-indole-3-pentanoic acid-iodide and 0.2-0.5 g of sodium acetate are added; the mixture is stirred at 30-60°C for 1-3 hours; after the reaction is completed, the product is purified by silica gel column chromatography to obtain the intermediate carboxyl-containing pentamethicone cyanine dye; (9): Synthesis of Pentamethine Cyanine Dye A mixture of 40-70 mg of the intermediate carboxyl-containing pentamethicone cyanine dye and 60-90 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is weighed and added to a container; 80-100 μL of diisopropylethylamine is dissolved in anhydrous N,N-dimethylformamide and added to the container; the mixture is stirred at room temperature for 30-60 minutes; then 60-90 mg of 4-aminomethylbenzenesulfonamide hydrochloride is dissolved in anhydrous N,N-dimethylformamide and added to the container; and silica gel column chromatography is performed to obtain a suitable pentamethicone cyanine dye.

3. Use of the pentamethine cyanine dye according to claim 1 in fluorescence imaging.

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