Highly efficient fluorescent probe and preparation method and application thereof

Abstract

The application discloses a high-efficiency fluorescein probe and a preparation method and application thereof, and relates to the field of fluorescein probes. The high-efficiency fluorescein probe prepared by the application has an extremely low detection limit for cathepsin B, has excellent fluorescence performance and good biocompatibility, is suitable for detection of tumor-related cathepsin B, and has important application significance in early diagnosis of tumors.

Description

Technical Field

[0001] This invention relates to the field of fluorophore probes, and more specifically to a high-efficiency fluorophore probe, its preparation method, and its application. Background Technology

[0002] Fluorescein probes, due to their advantages such as high sensitivity and real-time response, have been widely used in biomedical imaging, environmental monitoring, and food safety. An ideal fluorophore probe must meet core requirements such as high sensitivity, excellent fluorescence performance (e.g., long-wavelength emission, high quantum yield, large Stokes shift), and good biocompatibility. However, some traditional fluorophores suffer from low quantum yields due to insufficient conjugated systems or quenching effects such as photoinduced electron transfer and distorted intramolecular charge transfer. Furthermore, small Stokes shifts can easily cause spectral overlap between excitation and emission light, leading to self-absorption and reducing the signal-to-noise ratio. They are also susceptible to background fluorescence interference during detection. Traditional probes mostly emit visible light, making them vulnerable to autofluorescence interference from biological tissues. Moreover, some probes contain toxic groups (such as heavy metal complexes) that may damage cells or organisms, making long-term dynamic monitoring difficult.

[0003] Chinese invention patent CN112679543A discloses a lipophilic fluorescein probe and its preparation method. This lipophilic fluorescein probe has high stability and is not easily self-hydrolyzed. Under the action of lipases, it can rapidly hydrolyze to release fluorescein, thus exhibiting very high affinity for lipases and a very low detection limit. However, its fluorescence performance is still insufficient. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing a high-efficiency fluorophore probe and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A high-efficiency luciferin probe has the structural formula shown in formula (I):

[0007] (I).

[0008] A method for preparing a high-efficiency fluorophore probe, the process route is as follows:

[0009] Compound (II) was mixed with perylene-3-carboxylic acid in a solvent, and then dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added to react and give the highly efficient fluorophore probe of formula (I); the reaction equation is as follows:

[0010]

[0011] The molar ratio of the compound of formula (II) to perylene-3-carboxylic acid is 1:(1.1-1.2).

[0012] The solvent is DMF.

[0013] The reaction was carried out at room temperature.

[0014] The reaction time is controlled to be 10-14 hours.

[0015] The compound of formula (II) is prepared by the following method:

[0016] (1) 3,5-Diiodo-L-tyrosine reacts with paraformaldehyde to generate intermediate 1;

[0017] (2) Intermediate 1 reacts with di-tert-butyl dicarbonate to generate intermediate 2;

[0018] (3) Intermediate 2 is converted into intermediate 3 under the action of palladium on carbon catalyst;

[0019] (4) Intermediate 3 reacts with (R)-(-)-1,2,3,4-tetrahydro-1-naphthylamine to generate intermediate 4;

[0020] (5) Intermediate 4 is reacted with hydrochloric acid to form intermediate 5;

[0021] (6) Intermediate 5 reacts with N-Boc-L-tert-leucine to generate intermediate 6;

[0022] (7) Intermediate 6 is converted into intermediate 7 under the action of trifluoroacetic acid;

[0023] (8) Intermediate 7 reacts with BOC-N-methyl-L-alanine to generate compound (II).

[0024] In step (1), the molar ratio of 3,5-diiodo-L-tyrosine to paraformaldehyde is 1:6.

[0025] In step (2), the molar ratio of intermediate 1 to ditert-butyl dicarbonate is 1:1.6;

[0026] In step (3), the mass ratio of intermediate 2 to palladium on carbon catalyst is 20:1;

[0027] In step (4), the molar ratio of intermediate 3 to (R)-(-)-1,2,3,4-tetrahydro-1-naphthylamine is 1:1.15.

[0028] In step (6), the molar ratio of intermediate 5 to N-Boc-L-tert-leucine is 1:(1.15);

[0029] In step (7), the reaction temperature is 25°C and the reaction time is 12h.

[0030] Application of a high-efficiency fluorescent probe in the detection of tumor-associated cathepsin B.

[0031] Due to the adoption of the above technical solutions, the beneficial effects of the present invention include:

[0032] The high-efficiency fluorophore probe prepared by this invention has an extremely low detection limit for cathepsin B, excellent fluorescence properties, and good biocompatibility. It is suitable for the detection of tumor-associated cathepsin B and has important application significance in the early diagnosis of tumors. Attached Figure Description

[0033] Figure 1 The image shows the 1H NMR spectrum of the high-efficiency fluorophore probe prepared in Example 2.

[0034] Figure 2 The high-resolution mass spectrum of the high-efficiency fluorophore probe prepared in Example 2 is shown. Detailed Implementation

[0035] The following description, in conjunction with specific embodiments, provides further details, but the present invention is not limited to these embodiments.

[0036] Example 1 Preparation of compound (II):

[0037] (1) Under nitrogen protection, 14.7 L of 0.1 M hydrochloric acid solution was mixed with 2.98 mol of 3,5-diiodo-L-tyrosine. At 0 °C, a mixture of 1.5 L of DME (ethylene glycol dimethyl ether) and 17.88 mol of paraformaldehyde was added in 5 equal batches (each batch 20 min apart). The mixture was heated to 70 °C and stirred for 12 h. TLC (methanol / dichloromethane = 1 / 5 (V / V), product Rf = 0.3) showed that the raw materials were completely consumed. After filtration of the reaction solution, the filter cake was washed with DME (3 × 600 ml) to obtain intermediate 1 (yield 76.8%, liquid phase purity 98.3%). The reaction equation is shown below:

[0038]

[0039] Its 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d6) δ 14.15 (s, 1H), 10.10 (s, 2H), 9.67 (s, 1H), 7.72 (s, 1H), 4.31 (dd, J = 11.0, 4.6 Hz, 1H), 4.15-3.96 (m, 2H), 3.28-3.16 (m, 1H), 3.14-3.00 (m, 1H); LCMS (m / z):445.9 [M+H] + .

[0040] (2) Under nitrogen protection, at 0°C, 6.5 L of 1M NaOH solution was mixed with 2.20 mol of intermediate 1, and 2.6 L of THF solution containing 3.52 mol of di-tert-butyl dicarbonate was added. The mixture was stirred at room temperature for 6 h. TLC (methanol / dichloromethane = 1 / 5 (V / V), product Rf = 0.7) showed that the raw materials were completely consumed. The pH of the reaction solution was adjusted to 4 with 0.1M hydrochloric acid aqueous solution, 2 L of water was added to quench the reaction, and the mixture was extracted with ethyl acetate (3 × 2 L). The organic phase was dried with 600 g of anhydrous sodium sulfate for 1 h, filtered, and concentrated under reduced pressure at 40°C for 2 h to obtain intermediate 2 (yield 83.7%, liquid phase purity 93.9%). The reaction equation is shown below:

[0041]

[0042] Its 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 12.62 (s, 1H), 9.49 (s,1H), 7.32 (t, J = 1.0 Hz, 1H), 4.62 (s, 1H), 4.45 (s, 1H), 4.26 (s, 1H),3.20-2.85 (m, 2H), 1.41 (s, 9H); LCMS (m / z):445.9 [M-100+H] + .

[0043] (3) Mix 2.5 L of methanol with 985.7 g of intermediate 2, and add 5.79 mol of triethylamine and 49.3 g of palladium on carbon catalyst (palladium content 5 wt%) sequentially at 25 °C. Stir for 12 h at 25 °C under a hydrogen atmosphere. TLC detection (methanol / dichloromethane = 1 / 10 (V / V), product Rf = 0.6) showed that the raw materials were completely converted. Filter the reaction solution, wash the filter cake with methanol (3 × 50 ml), combine the filtrates, and concentrate under reduced pressure at 40 °C for 2 h to obtain intermediate 3 (yield 91.3%, liquid phase purity 97.5%). The reaction equation is shown below:

[0044]

[0045] Its 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 12.62 (s, 1H), 8.59 (s,1H), 6.90 (dt, J = 7.4, 1.0 Hz, 1H), 6.58 (dd, J = 7.5, 2.0 Hz, 1H), 6.48(dt, J = 2.0, 1.0 Hz, 1H), 4.72 (d, J = 1.0 Hz, 1H), 4.46 (s, 1H), 4.32 (d, J= 1.0 Hz, 1H), 3.24-2.76 (m, 2H), 1.41 (s, 9H); LCMS (m / z): 194.1 [M-100+H] + .

[0046] (4) Under nitrogen protection, 2.8 L of DMF was mixed with 954.59 mmol of intermediate 3 and 1.10 mol of (R)-(-)-1,2,3,4-tetrahydro-1-naphthylamine. The mixture was then added in 5 equal batches at 0 °C with a mixed solution of 2.86 mol of N,N-diisopropylethylamine and 1.00 mol of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (each batch was 20 min apart). The mixture was stirred at room temperature for 12 h. TLC detection (methanol / dichloromethane = 1 / 10 (V / V), product Rf = 0.5) showed that the raw materials were completely converted. The reaction solution was quenched with 5.6 L of water and extracted with ethyl acetate (3 × 2.0 L). The combined organic phases were washed with saturated brine (3 × 3.0 L), dried over 200 g of anhydrous sodium sulfate for 1 h, filtered, and concentrated under reduced pressure at 40 °C for 3 h to obtain intermediate 4 (yield 86.7%, liquid phase purity 99.2%). The reaction equation is shown below:

[0047]

[0048] Its 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d6) δ 8.59 (s, 1H), 8.15 (d,J = 0.6 Hz, 1H), 7.24-7.12 (m, 4H), 6.95 (dt, J = 7.7, 1.1 Hz, 1H), 6.58 (dd,J = 7.5, 2.0 Hz, 1H), 6.48 (dt, J = 2.1, 1.0 Hz, 1H), 4.97 (t, J = 0.6 Hz,1H), 4.69 (d, J = 1.0 Hz, 1H), 4.54 (s, 1H), 4.38 (d, J = 1.0 Hz, 1H), 3.33-2.97 (m, 2H), 2.80 (dd, J = 1.7, 0.6 Hz, 2H), 1.98-1.75 (m, 4H), 1.41 (s,9H); LCMS (m / z):323.2 [M-100+H] + .

[0049] (5) Under nitrogen protection, at 0°C, 946.69 mmol of intermediate 4 was added in three equal portions to a 2.0 L solution of 1M hydrochloric acid and dioxane (hydrochloric acid to dioxane volume ratio of 1:2, with a 15 min interval between each batch), and the mixture was heated to 25°C and stirred for 12 h. TLC detection (methanol / dichloromethane = 1 / 20 (V / V), product Rf = 0.6) showed complete conversion of the starting material. The reaction solution was concentrated under reduced pressure at 40°C for 3 h to obtain intermediate 5 (yield 98.9%, liquid phase purity 88.1%). The reaction equation is shown below:

[0050]

[0051] Its 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d6) δ 11.06 (s, 1H), 8.73 (s,1H), 8.01 (d, J = 0.6 Hz, 1H), 7.24-7.12 (m, 4H), 6.98 (dt, J = 7.4, 1.0 Hz,1H), 6.58 (dd, J = 7.6, 2.0 Hz, 1H), 6.43 (dt, J = 2.1, 1.0 Hz, 1H), 4.96 (t,J = 0.6 Hz, 1H), 3.93-3.88 (m, 1H), 3.83 (d, J = 0.5 Hz, 1H), 3.75-3.71 (m,1H), 3.39 (d, J = 0.6 Hz, 1H), 3.02 (dd, J = 85.6, 1.0 Hz, 2H), 2.80 (dd, J =1.7, 0.6 Hz, 2H), 1.93 (d, J = 67.8 Hz, 4H); LCMS (m / z): 323.2 [M+H] + .

[0052] (6) Under nitrogen protection, 2.0 L of DMF was mixed with 809.74 mmol of intermediate 5 and 931.20 mmol of N-Boc-L-tert-leucine. The mixture was then added in 5 equal batches at 0 °C with a mixed solution of 1.78 mol N,N-diisopropylethylamine and 1.37 mol 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (each batch was 20 min apart). The mixture was stirred at room temperature for 12 h. TLC detection (methanol / dichloromethane = 1 / 20 (V / V), product Rf = 0.5) showed that the raw materials were completely converted. The reaction solution was quenched with 4.0 L of water and extracted with ethyl acetate (3 × 1.5 L). The combined organic phases were washed with saturated brine (3 × 2.0 L), dried over 150 g of anhydrous sodium sulfate for 1 h, filtered, and concentrated under reduced pressure at 40 °C for 3 h to obtain intermediate 6 (yield 70.4%, liquid phase purity 98.5%). The reaction equation is shown below:

[0053]

[0054] Its 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d6) δ 8.59 (s, 1H), 8.13 (d,J = 0.6 Hz, 1H), 7.24-7.12 (m, 4H), 6.98 (dt, J = 7.5, 1.0 Hz, 1H), 6.58 (dd,J = 7.5, 2.0 Hz, 1H), 6.48 (dt, J = 2.0, 1.0 Hz, 1H), 6.33 (d, J = 0.5 Hz,1H), 4.98 (t, J = 0.7 Hz, 1H), 4.85-4.54 (m, 3H), 4.11 (d, J = 0.5 Hz, 1H),3.32-2.99 (m, 2H), 2.83-2.78 (m, 2H), 1.98-1.73 (m, 4H), 1.40 (s, 9H), 0.97(s, 9H); LCMS (m / z):536.3[M+H] + .

[0055] (7) Under nitrogen protection, 3.5 L of dichloromethane was mixed with 705.64 mmol of intermediate 6, and 500 mL of trifluoroacetic acid was added in five equal batches at 0 °C (15 min interval between each batch). The mixture was heated to 25 °C and stirred for 12 h. TLC analysis (petroleum ether / ethyl acetate = 1 / 1 (V / V), product Rf = 0.3) showed complete conversion of the starting material. The reaction solution was concentrated under reduced pressure at 40 °C for 3 h to obtain intermediate 7 (yield 98.1%, liquid phase purity 95.9%). The reaction equation is shown below:

[0056]

[0057] Its 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d6) δ 8.91 (s, 1H), 8.59 (s,1H), 8.13 (d, J = 0.6 Hz, 1H), 7.24-7.12 (m, 4H), 6.98 (dt, J = 7.5, 1.0 Hz,1H), 6.58 (dd, J = 7.5, 2.0 Hz, 1H), 6.48 (dt, J = 2.0, 1.0 Hz, 1H), 5.02 (t,J = 0.6 Hz, 1H), 4.88-4.43 (m, 3H), 3.30-3.02 (m, 2H), 2.94 (d, J = 0.6 Hz,1H), 2.80 (dd, J = 1.7, 0.6 Hz, 2H), 2.60 (d, J = 0.5 Hz, 1H), 2.04-1.81 (m,5H), 0.99 (s, 9H); LCMS (m / z):436.3[M+H] + .

[0058] (8) Under nitrogen protection, 4.0 L of DMF was mixed with 941.30 mmol of intermediate 7 and 1016.60 mmol of BOC-N-methyl-L-alanine. The mixture of 5.65 mol of N-methylimidazole and 753.01 mmol of N,N,N',N'-tetramethylchloromethanesulfonate hexafluorophosphate was added in 5 equal batches at 0 °C (15 min interval between each batch). The mixture was stirred at room temperature for 12 h. TLC detection (petroleum ether / ethyl acetate = 2 / 1 (V / V), product Rf = 0.7) showed that the raw materials were completely converted. The reaction solution was quenched with 8.0 L of water and extracted with ethyl acetate (3 × 3.0 L). The organic phases were combined, washed with saturated brine (3 × 3.0 L), dried over 900 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 40 °C for 3 h, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate V:V = 2:5). The crude product was then distilled under reduced pressure at 40 °C for 3 h to give compound (II) (yield 78.6%, HPLC purity 99.6%). The reaction equation is shown below:

[0059]

[0060] Its 1H NMR data are as follows: 1 H NMR (400 MHz, DMSO- d6) δ 9.28 (d, J = 30.1 Hz,1H), 8.25-8.08 (m, 1H), 7.44 (d, J = 96.2 Hz, 1H), 7.16-6.83 (m, 5H), 6.71-6.53 (m, 2H), 5.01 (t, J = 48.7 Hz, 1H), 4.87 (dd, J = 13.8, 8.5 Hz, 1H), 4.81-4.73 (m, 1H), 4.73-4.59 (m, 1H), 4.59 4.49 (m, 2H), 3.03-2.83 (m, 2H),2.81-2.64 (m, 5H), 1.92-1.75 (m, 2H), 1.75-1.63 (m, 1H), 1.62-1.52 (m, 1H), 1.40 (s, 9H), 1.14 (dd, J = 23.1, 7.1 Hz, 3H), 0.98 (d, J = 31.9 Hz, 9H); LCMS(m / z):521.4[M-100+H] + .

[0061] Example 2 Preparation of compound (I):

[0062] 4.0 L of DMF was mixed with 0.6 mol of compound (II) and 0.66 mol of perylene-3-carboxylic acid. Then, 0.6 mol of dicyclohexylcarbodiimide and 2.2 g of 4-dimethylaminopyridine were added, and the mixture was stirred at room temperature for 10 h. TLC analysis (methanol / dichloromethane = 1 / 10 (V / V), product Rf = 0.6) showed complete conversion of the starting material. The reaction solution was quenched with 8.0 L of water and extracted with ethyl acetate (3 × 3.0 L). The organic phases were combined, washed with saturated brine (3 × 3.0 L), dried over 200 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 40 °C for 3 h, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate V / V = 1:2). The product was then distilled under reduced pressure at 40 °C for 3 h to obtain compound (I) (yield 74.9%, HPLC purity 99.8%), which is the high-efficiency fluorophore probe. The reaction equation is shown below:

[0063]

[0064] Its proton NMR spectrum is as follows Figure 1 The 1H NMR data are shown below:

[0065] 1 H NMR (400 MHz, DMSO- d6) δ 8.26 (d, J = 0.5 Hz, 1H), 8.19 (d, J = 0.5Hz, 2H), 8.16 (d, J = 0.6 Hz, 1H), 8.13 (d, J = 0.6 Hz, 1H), 8.08-7.93 (m,4H), 7.73 (d, J = 0.5 Hz, 1H), 7.63-7.57 (m, 3H), 7.23-7.12 (m, 5H), 7.09(dd, J = 7.5, 2.0 Hz, 1H), 6.88 (dt, J = 2.0, 1.0 Hz, 1H), 5.01-4.96 (m, 1H),4.81-4.50 (m, 3H), 4.41 (d, J = 0.5 Hz, 1H), 4.15 (s, 1H), 3.17 (dd, J =101.6, 1.0 Hz, 2H), 2.87 (s, 3H), 2.83-2.78 (m, 2H), 1.98-1.72 (m, 4H), 1.39 (d, J = 14.9 Hz, 12H), 0.97 (s, 9H);

[0066] Its high-resolution mass spectrum is as follows Figure 2 The high-resolution mass spectrometry data are as follows: HRMS (m / z): 799.4340 [M-100+H] + .

[0067] Example 3 Preparation of compound (I):

[0068] 4.0 L of DMF was mixed with 0.6 mol of compound (II) and 0.69 mol of perylene-3-carboxylic acid, and 0.6 mol of dicyclohexylcarbodiimide and 2.2 g of 4-dimethylaminopyridine were added. The mixture was stirred at room temperature for 12 h. TLC analysis (methanol / dichloromethane = 1 / 10, product Rf = 0.6) showed complete conversion of the starting material. The reaction solution was quenched with 8.0 L of water and extracted with ethyl acetate (3 × 3.0 L). The organic phases were combined, washed with saturated brine (3 × 3.0 L), dried over 900 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 40 °C for 3 h, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate V / V = 1:2). The crude product was then distilled under reduced pressure at 40 °C for 3 h to obtain compound (I) (yield 75.3%, HPLC purity 99.9%).

[0069] Example 4 Preparation of compound (I):

[0070] 4.0 L of DMF was mixed with 0.6 mol of compound (II) and 0.72 mol of perylene-3-carboxylic acid, and 0.6 mol of dicyclohexylcarbodiimide and 2.2 g of 4-dimethylaminopyridine were added. The mixture was stirred at room temperature for 14 h. TLC analysis (methanol / dichloromethane = 1 / 10, product Rf = 0.6) showed complete conversion of the starting material. The reaction solution was quenched with 8.0 L of water and extracted with ethyl acetate (3 × 3.0 L). The organic phases were combined, washed with saturated brine (3 × 3.0 L), dried over 900 g of anhydrous sodium sulfate for 1 h, filtered, concentrated under reduced pressure at 40 °C for 3 h, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate V / V = 1:2). The crude product was then distilled under reduced pressure at 40 °C for 3 h to obtain compound (I) (yield 78.4%, HPLC purity 99.8%).

[0071] Comparative Example 1

[0072] The preparation method of the high-efficiency fluorescent probe is basically the same as that in Example 3, except that perylene-3-carboxylic acid in the preparation step is replaced with an equimolar amount of benzoic acid.

[0073] Comparative Example 2

[0074] The preparation method of the high-efficiency fluorescent probe is basically the same as that in Example 3, except that perylene-3-carboxylic acid in the preparation step is replaced with an equimolar amount of 1-naphthoic acid.

[0075] Comparative Example 3

[0076] The preparation method of the high-efficiency fluorescent probe is basically the same as that in Example 3, except that perylene-3-carboxylic acid in the preparation step is replaced with an equimolar amount of 1-pyrenecarboxylic acid.

[0077] The palladium-on-carbon catalyst used in the embodiments and comparative examples of this application is model Pd 101, manufactured by Xi'an Kaili New Materials Co., Ltd.

[0078] The high-efficiency fluorophore probes prepared in Examples 1, 2 and Comparative Examples 1-3 were tested for fluorescence performance, specificity and cathepsin B detection limit. The test results are shown in Table 1.

[0079] Weigh 10 mg of the fluorescein probe prepared in Examples 1, 2 and Comparative Examples 1-3 respectively, mix thoroughly with 2 ml of dichloromethane, pour into a fluorescence cuvette, and measure its fluorescence spectrum using an LF-1804004 fluorescence spectrophotometer (fluorescence spectral scanning conditions: excitation wavelength 365 nm, slit width 5.0 nm, scanning range 380 nm-1000 nm), and record the fluorescence emission wavelength;

[0080] Take 2 ml of the prepared fluorescein probe solution and mix it with 1 ml of 0.1 mol / L cathepsin B, cathepsin A, and cathepsin D respectively. Add 1 ml of phosphate buffer (0.1 M, pH=6), pour it into a fluorescence cuvette, and use an LF-1804004 fluorescence spectrophotometer to measure its fluorescence spectrum (fluorescence spectral scanning conditions: excitation wavelength 365 nm, slit width 5.0 nm, scanning range 380 nm-1000 nm), and record the fluorescence intensity.

[0081] Take 2 ml of the prepared fluorescein probe solution and mix it with 1 ml of cathepsin B at concentrations of 0.1, 0.5, 1, and 5 mol / L, respectively. Add 1 ml of phosphate buffer (0.1 M, pH=6) and pour it into a fluorescence cuvette. Measure the fluorescence spectrum using an LF-1804004 fluorescence spectrophotometer (fluorescence spectral scanning conditions: excitation wavelength 365 nm, slit width 5.0 nm, scanning range 380 nm-1000 nm). Record the fluorescence intensity. Use the fluorescein probe aqueous solution without protease as a blank control. Calculate the detection limit for cathepsin B according to the formula LOD=3σ / K (σ represents the standard deviation obtained from ten measurements of the blank sample, and k represents the slope of the calibration curve).

[0082] Table 1

[0083]

[0084] As can be seen from Table 1, the high-efficiency fluorescent probe prepared in Example 3 of this application has excellent fluorescence performance and can detect cathepsin B with a low detection limit.

[0085] The high-efficiency luciferin probe prepared in Example 2 exhibits a longer fluorescence emission wavelength, primarily due to the presence of a large conjugated system within the probe. This system reduces the energy difference between the highest occupied orbital and the lowest unoccupied orbital, enabling long-wavelength luminescence and enhancing cell penetration depth. In contrast, the luciferin probes prepared in Comparative Examples 1, 2, and 3 have smaller conjugated systems than those in the Example 2, resulting in a larger energy difference between the highest occupied and lowest unoccupied orbitals and relatively shorter emission wavelengths.

[0086] The high-efficiency fluorophore probe prepared in Example 2 can detect cathepsin B with a low detection limit, mainly due to the presence of two peptide bonds in the probe. These two peptide bonds serve as substrates for cathepsin B. When the probe comes into contact with cathepsin, the protease hydrolyzes the peptide bonds, causing a conformational change in the probe molecule, thereby significantly enhancing fluorescence intensity. Furthermore, the peptide bonds can increase the Stokes shift through a twisted intramolecular charge transfer effect, reducing interference from excitation light to emission light and improving the detection sensitivity of the fluorophore probe.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. However, any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention based on the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A high-efficiency fluorophore probe, characterized in that, Its structural formula is shown in equation (I): （I）。 2. A method for preparing a high-efficiency fluorophore probe, characterized in that, The process route is as follows: Compound (II) is mixed with perylene-3-carboxylic acid in a solvent, and then dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added to react and give the highly efficient fluorophore probe of formula (I); the reaction equation is as follows: 。 3. The method for preparing a high-efficiency fluorophore probe according to claim 2, characterized in that, The molar ratio of the compound of formula (II) to perylene-3-carboxylic acid is 1:(1.1-1.2).

4. The method for preparing a high-efficiency fluorophore probe according to claim 2, characterized in that, The solvent is DMF.

5. The method for preparing a high-efficiency fluorophore probe according to claim 2, characterized in that, The reaction was carried out at room temperature.

6. The method for preparing a high-efficiency fluorophore probe according to claim 2, characterized in that, The reaction time is controlled to be 10-14 hours.

7. The method for preparing a high-efficiency fluorophore probe according to claim 2, characterized in that, The compound of formula (II) is prepared by the following method: (1) 3,5-Diiodo-L-tyrosine reacts with paraformaldehyde to generate intermediate 1; (2) Intermediate 1 reacts with di-tert-butyl dicarbonate to generate intermediate 2; (3) Intermediate 2 is converted into intermediate 3 under the action of palladium on carbon catalyst; (4) Intermediate 3 reacts with (R)-(-)-1,2,3,4-tetrahydro-1-naphthylamine to generate intermediate 4; (5) Intermediate 4 is converted into intermediate 5 under the action of hydrochloric acid; (6) Intermediate 5 reacts with N-Boc-L-tert-leucine to generate intermediate 6; (7) Intermediate 6 is converted into intermediate 7 under the action of trifluoroacetic acid; (8) Intermediate 7 reacts with BOC-N-methyl-L-alanine to generate compound (II); The chemical structural formulas of intermediates 1-7 are as follows: 。 8. The method for preparing a high-efficiency fluorophore probe according to claim 7, characterized in that, In step (1), the molar ratio of 3,5-diiodo-L-tyrosine to paraformaldehyde is 1:

6. In step (2), the molar ratio of intermediate 1 to ditert-butyl dicarbonate is 1:1.6; In step (3), the mass ratio of intermediate 2 to palladium on carbon catalyst is 20:1; In step (4), the molar ratio of intermediate 3 to (R)-(-)-1,2,3,4-tetrahydro-1-naphthylamine is 1:1.

15.

9. The method for preparing a high-efficiency fluorophore probe according to claim 7, characterized in that, In step (6), the molar ratio of intermediate 5 to N-Boc-L-tert-leucine is 1:(1.15); In step (7), the reaction temperature of intermediate 6 with trifluoroacetic acid is 25°C and the reaction time is 12h.

10. The use of the high-efficiency fluorescent probe of claim 1 in the preparation of a tumor-associated cathepsin B detection reagent.

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