Fluorescent probe for preparing reagent for detecting elastase as well as synthesis method and application of fluorescent probe

Abstract

The invention belongs to the field of chemical analysis and testing, and particularly relates to a fluorescent probe for preparing a reagent for detecting elastase as well as a synthesis method and application of the fluorescent probe. The preparation method comprises the following steps: dissolving 2-dicyanomethylene-3-cyano-4, 5, 5-trimethyl-2, 5-dihydrofuran and 6-aminopyridine-3-formaldehyde in ethanol, and heating and refluxing a reaction solution at 50 DEG C under the acidic condition of ammonium acetate to obtain an intermediate TCF-N-NH2; and dissolving the intermediate TCF-N-NH2 and pentafluoropropionic anhydride in dichloromethane, stirring the reaction solution at the temperature of 0 DEG C to room temperature under the alkaline condition of pyridine, and separating and purifying the obtained crude product through thin layer chromatography to obtain the final orange product TCF-NF. The fluorescent probe obtained by the invention can quickly recognize elastase in deionized water.

Description

Technical Field

[0001] The present invention belongs to the field of chemical analysis and testing, and particularly relates to a fluorescent probe for preparing an elastase detection reagent, a synthesis method thereof, and an application thereof. Background Art

[0002] Neutrophil elastase (NE) is a 29 - 33 kDa serine protease, mainly secreted by neutrophils and released from the bone marrow. NE can degrade a variety of extracellular matrix proteins, such as elastin, fibronectin, laminin, and collagen, and it plays an important role in the immune defense of the inflammatory response. In addition, overexpression of NE may lead to various diseases, such as rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis, or delayed wound healing. Developing a simple and rapid detection technology for elastase is of great significance for human life and health.

[0003] Currently, the methods for measuring elastase activity include: indirect separation methods, namely high - performance liquid chromatography (HPLC) or liquid chromatography - mass spectrometry (LC - MS), the combination of direct electrochemistry and ultraviolet - visible spectrophotometry, and enzyme - linked immunosorbent assay, etc. However, due to the use of peptide - based substrates, these methods have complex operation procedures, long detection cycles, and high costs. Summary of the Invention

[0004] The present invention provides a fluorescent probe TCF - NF for preparing an elastase detection reagent, and the structural formula of the fluorescent probe is:

[0005]

[0006] The present invention also provides a synthesis method of a fluorescent probe for preparing an elastase detection reagent, and the chemical reaction formula for synthesizing the fluorescent probe is:

[0007]

[0008] The specific synthesis method steps are as follows:

[0009] (1) Dissolve 2 - dicyanomethylene - 3 - cyano - 4,5,5 - trimethyl - 2,5 - dihydrofuran and 6 - aminopyridine - 3 - carbaldehyde in ethanol according to a molar equivalent of 1:1.1 - 2, then add 1.1 - 2 molar equivalents of ammonium acetate, and heat under reflux at 50 °C for 10 hours under nitrogen protection. After the reaction is completed and cooled to room temperature, place it in an ice - water bath for 30 minutes, and a solid will precipitate. Filter the solid by suction and wash it three times with ethanol to obtain a purple solid intermediate TCF - N - NH2.

[0010] (2) Dissolve the intermediate TCF-NH2 and pyridine in dichloromethane at a molar equivalent ratio of 1:2 to 3, and slowly add 9 to 10 molar equivalents of pentafluoropropionic anhydride under stirring. React at 0 °C to room temperature for 10 hours. After the reaction is completed, remove the solvent. The crude product is extracted with dichloromethane and water, and the solvent is removed under reduced pressure. Finally, it is purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 to obtain the orange solid product TCF-NF.

[0011] The present invention also provides the application of the above-mentioned fluorescent probe: the prepared fluorescent probe is used to prepare a reagent that can specifically recognize elastase in deionized water.

[0012] The probe of the present invention uses 6-aminopyridine as the donor of intramolecular electrons, which increases the conjugation of the molecule, is beneficial to the conduction of electrons within the molecule, and causes the fluorescence wavelength to redshift to about 580 nm. The compound intermediate TCF-N-NH2 is a conjugated fluorescent group, which is linked to the pentafluoropropionyl group that specifically recognizes elastase through an amide bond to form the final fluorescent probe TCF-NF. After the probe of the present invention recognizes elastase, the fluorescence intensity is significantly enhanced, thereby achieving the purpose of specifically detecting elastase.

[0013] Beneficial effects

[0014] The raw materials of the present invention are easily obtained, the synthesis method is simple, the reaction conditions are easy to control, and a pure product can be obtained through simple post-treatment. The probe TCF-NF reported in the present invention solves the problem of rapid recognition of elastase in deionized water. From the fluorescence phenomenon, after adding elastase, the fluorescence intensity of the probe TCF-NF at 580 nm is significantly enhanced, thereby showing the recognition effect of the probe TCF-NF on elastase in this system. Description of the drawings

[0015] Figure 1 Fluorescence intensity diagram of the fluorescent probe prepared in Example 1 at a concentration of 5×10 -5 mol / L in deionized water after reacting with different proteins, amino acids or metal ions.

[0016] Figure 2 Absorption spectrum diagram of the fluorescent probe prepared in Example 1 at a concentration of 5×10 -5 mol / L in deionized water after reacting with different proteins, amino acids or metal ions.

[0017] Figure 3 Fluorescence spectrum diagram of the fluorescent probe prepared in Example 1 at a concentration of 5×10 -5 mol / L in deionized water after reacting with different concentrations of elastase.

[0018] Figure 4 The fluorescence probe prepared in Example 1 was at a concentration of 5×10 -5 mol / L in deionized water and the fluorescence intensity graph at 580 nm after adding five-fold interferents with elastase.

[0019] Figure 5 1H NMR spectrum of the probe prepared in Example 1. Detailed implementation manners

[0020] The present invention will be further described in detail below in conjunction with specific implementation manners:

[0021] Example 1

[0022] (1) Dissolve 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (59.8 mg, 0.3 mmol) and 6-aminopyridine-3-carbaldehyde (40.3 mg, 0.33 mmol) in 1.5 mL of ethanol, then add ammonium acetate (25.5 mg, 0.33 mmol), and heat under reflux at 50 °C for 10 hours under nitrogen protection. After the reaction is completed, let the solution cool to room temperature, then place it in an ice-water bath for 30 minutes. Solids will precipitate. Filter by suction and wash three times with cold ethanol to obtain 84.2 mg of purple solid intermediate TCF-N-NH2 with a yield of 92.5%.

[0023] (2) Dissolve the intermediate obtained in step (1) (38.4 mg, 0.13 mmol) and pyridine (21 mg, 0.26 mmol) in 1 mL of dichloromethane. Slowly add pentafluoropropionic anhydride (362.8 mg, 1.17 mmol) with stirring, and react at 0 °C to room temperature for 10 hours. After the reaction is completed, remove the solvent. The crude product is extracted with dichloromethane and water, and the organic solvent is removed under reduced pressure. Finally, it is purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 as the developing agent to obtain 25.6 mg of orange solid final product with a yield of 42.9%.

[0024] The specific application method is as follows: Add 2 μL of common proteins (elastase, pepsin, trypsin, bromelain, hemoglobin, casein, human serum albumin, heparin sodium, human transferrin, concentration 1×10 - 4 g / mL), amino acids (phenylalanine, serine, valine, arginine, tyrosine, tryptophan, cysteine, concentration 1×10 -4 mol / L)) or metal ions (Pd 2+ 、Co 3+ 、Cu 2+ 、Sn 4+ , concentration 1×10-4 mol / L)), 196 μL of deionized water and 2 μL of the fluorescent probe TCF-NF (concentration 5×10 -5 mol / L). At the same time, a fluorescent probe TCF-NF solution without enzyme (Blank) was used as a control. The solution in each well was mixed evenly, and the fluorescence intensity of the solution in each well was detected by a microplate reader. The results showed that after adding elastase, the fluorescence intensity of the probe at 580 nm increased significantly; and there was no obvious change in the fluorescence intensity of the fluorescent probe for other interfering substances, thus showing the selective recognition effect of the fluorescent probe on elastase in deionized water.

[0025] Figure 1 For the fluorescent probe TCF-NF (concentration 5×10 -5 mol / L) prepared in Example 1 in deionized water with different analytes (proteins (elastase, pepsin, trypsin, bromelain, hemoglobin, casein, human serum albumin, heparin sodium, human transferrin, concentration 1×10 -4 g / mL), amino acids (phenylalanine, serine, valine, arginine, tyrosine, tryptophan, cysteine, concentration 1×10 -4 mol / L) or metal ions (Pd 2+ 、Co 3+ 、Cu 2+ 、Sn 4+ ,concentration 1×10 - 4 mol / L)) acting on the fluorescence spectra. The figure shows the changes in the fluorescence intensity of the probe after adding different analyte solutions. When the elastase solution was added, the fluorescence intensity of the fluorescent probe solution at 580 nm increased significantly (the curve pointed by the arrow); while in deionized water, the probe had no particularly obvious fluorescence change for other analytes except elastase, thus showing the selective recognition effect of the probe on elastase in this system.

[0026] Figure 2 For the fluorescent probe TCF-NF (concentration 5×10 -5 mol / L) prepared in Example 1 in deionized water with different analytes (proteins (elastase, pepsin, trypsin, bromelain, hemoglobin, casein, human serum albumin, heparin sodium, human transferrin, concentration 1×10 -4 g / mL), amino acids (phenylalanine, serine, valine, arginine, tyrosine, tryptophan, cysteine, concentration 1×10 -4 mol / L) or metal ions (Pd 2+ 、Co 3+ 、Cu 2+, Sn 4+ , concentration 1×10 - 4 Absorption spectra after the action of mol / L)). The figure shows the changes in the maximum absorption wavelength of the probe after adding different analyte solutions. In deionized water, the maximum absorption wavelength of this probe for analytes other than elastase is 405 nm. When an elastase solution is added, the maximum absorption wavelength redshifts to 435 nm, and the detection system changes from yellow to orange, showing an obvious visual change, thus demonstrating the selective recognition of elastase by the probe in this system.

[0027] Figure 3 The fluorescence spectra of the fluorescent probe prepared in Example 1 after reacting with different concentrations of elastase in deionized water at a concentration of 5×10 -5 mol / L. The figure shows that as the concentration of elastase increases, the fluorescence intensity of the TCF-NF probe solution gradually rises. When its concentration is 10 μg / mL, the fluorescence intensity of TCF-NF levels off. When its lowest concentration is 0.1 μg / mL, the fluorescence peak of the probe at 580 nm can still be distinguished from the background fluorescence curve without adding elastase, indicating that the probe has a low detection limit and high sensitivity for elastase.

[0028] Figure 4 The fluorescence intensity diagram at 580 nm of the fluorescent probe prepared in Example 1 after reacting with elastase (concentration 1×10 -5 mol / L) in deionized water at a concentration of 5×10 -4 mol / L after adding five-fold interferents. The figure shows that when only elastase is added, the fluorescence intensity of the probe solution at 580 nm increases significantly relative to the blank. However, for other interferents (pepsin, trypsin, bromelain, hemoglobin, casein, human serum albumin, heparin sodium, human transferrin, phenylalanine, serine, valine, arginine, tyrosine, tryptophan, cysteine, Pd 2+ , Co 3+ , Cu 2+ , Sn 4+ , blank (Blank)), there is no particularly obvious fluorescence change; then the change in the fluorescence intensity of the probe at 580 nm after adding 5-fold other interferents to the detection system shows that the addition of other proteins, amino acids, and metal ions does not cause a change in the detection result of elastase by this probe, indicating that this fluorescent probe has strong anti-interference ability during the detection process.

[0029] Figure 5 1H NMR spectrum of the fluorescent probe for detecting elastase prepared in Example 1. 11H NMR (400 MHz, DMSO-d6) δ 8.94 (d, J = 2.3 Hz, 1H), 8.53 (dd, J = 8.9, 2.3 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 16.5 Hz, 1H), 7.32 (d, J = 16.7 Hz, 1H), 4.11 (q, J = 5.3 Hz, 1H), 1.82 (s, 6H).

[0030] Example 2

[0031] (1) 2-Dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (99.6 mg, 0.5 mmol) and 6-aminopyridine-3-carbaldehyde (122.2 mg, 1.0 mmol) were dissolved in 2 mL of ethanol solution. Subsequently, ammonium acetate (77.1 mg, 1.0 mmol) was added, and the mixture was heated under reflux at 50 °C for 10 h under nitrogen protection. After the reaction was completed, the solution was cooled to room temperature and then placed in an ice-water bath for 30 min. A solid precipitated out. The solid was filtered by suction and washed three times with cold ethanol to obtain 139.1 mg of purple solid intermediate TCF-N-NH2 with a yield of 91.7%.

[0032] (2) The intermediate obtained in step (1) (91.0 mg, 0.3 mmol) and pyridine (71.2 mg, 0.9 mmol) were dissolved in 2 mL of dichloromethane solution. Pentafluoropropionic anhydride (930.2 mg, 3.0 mmol) was slowly added with stirring, and the reaction was carried out at 0 °C to room temperature for 10 h. After the reaction was completed, the solvent was removed. The crude product was extracted with dichloromethane and water, and the organic solvent was removed under reduced pressure. Finally, it was purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 as the developing agent to obtain 56.8 mg of orange solid final product with a yield of 42.1%.

[0033] Example 3

[0034] (1) 2-Dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (159.4 mg, 0.8 mmol) and 6-aminopyridine-3-carbaldehyde (146.6 mg, 1.2 mmol) were dissolved in 2.5 mL of ethanol solution. Subsequently, ammonium acetate (92.5 mg, 1.2 mmol) was added, and the mixture was heated under reflux at 50 °C for 10 h under nitrogen protection. After the reaction was completed, the solution was cooled to room temperature and then placed in an ice-water bath for 30 min. A solid precipitated out. The solid was filtered by suction and washed three times with cold ethanol to obtain 218.2 mg of purple solid intermediate TCF-N-NH2 with a yield of 89.9%.

[0035] (2) Dissolve the intermediate obtained in step (1) (60.7 mg, 0.2 mmol) and pyridine (39.6 mg, 0.5 mmol) in 1.5 mL of dichloromethane solution, and slowly add pentafluoropropionic anhydride (589.1 mg, 1.9 mmol) under stirring. React at 0 °C to room temperature for 10 hours. After the reaction is completed, remove the solvent. The crude product is extracted with dichloromethane and water, and the organic solvent is removed under reduced pressure. Finally, it is purified by thin-layer chromatography using a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 as the developing agent to obtain 47.7 mg of the orange solid final product, with a yield of 53.1%.

Claims

1. A fluorescent probe, characterized in that: The structural formula of the fluorescent probe is shown as follows:

2. A method for synthesizing the fluorescent probe according to claim 1, characterized in that: The steps of the synthesis method are as follows: (1) Using ethanol as the solvent, dissolve 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran and 6-aminopyridine-3-carbaldehyde in a round-bottom flask, then add ammonium acetate, and heat under reflux at 50 °C for 10 hours under nitrogen protection; after the reaction is completed, place it in an ice-water bath for 30 minutes, and a solid will precipitate. After suction filtration, wash it three times with ethanol to obtain a purple solid intermediate TCF-N-NH2; (2) Dissolve the intermediate TCF-N-NH2 obtained in step (1) and pyridine in dichloromethane, add pentafluoropropionic anhydride under stirring conditions, and react at 0 °C to room temperature for 10 hours. After the reaction is completed, remove the solvent. The crude product is extracted with dichloromethane and water, and the organic solvent is removed under reduced pressure. Finally, it is purified by column chromatography using petroleum ether and ethyl acetate as the eluent to obtain an orange fluorescent probe.

3. The synthesis method of the fluorescent probe according to claim 2, characterized in that: In step (1), the molar ratio of 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran, 6-aminopyridine-3-carbaldehyde, and ammonium acetate is: 1:1.1 - 2:1.1 - 2.

4. The synthesis method of the fluorescent probe according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate TCF-N-NH2, pentafluoropropionic anhydride, and pyridine is: 1:9 - 10:2 - 3, and the eluent is a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:

1.

5. Use of a fluorescent probe as described in claim 1, characterized in that: The fluorescent probe is used to prepare a reagent for detecting elastase.

6. The application of the fluorescent probe according to claim 5, wherein: The prepared reagent detects elastase by fluorescence enhancement in deionized water.

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