Synthesis method and application of a fluorescent probe for lipase detection

Abstract

The application discloses a synthesis method and application of a fluorescent probe for lipase detection, and the fluorescent probe has the following structural formula; the synthesis method comprises the following steps: under the action of inert gas protection, a catalyst and alkali, triphenylol compound and a fatty chain or an aromatic ring organic acid are reacted in an organic solvent at 25-100 DEG C for 8-24h; after the reaction is stopped, dichloromethane and water are added to carry out extraction; organic phases are combined, dried, filtered, and the solvent is removed under reduced pressure; and the product is separated and purified; the fluorescent probe is basically non-fluorescent by itself, releases bright fluorescence after reacting with lipase, and can realize rapid detection of bacteria by using the characteristics that most bacteria secrete lipase in the growth and reproduction process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a synthesis method and application of a fluorescent probe for lipase detection. BACKGROUND

[0002] Bacterial infection, especially infection caused by multi-drug resistant bacteria, has become a global public health problem. With the large-scale and irrational use of antibiotics, superbugs and multi-drug resistant bacteria have become commonplace. Previous studies have found that when the bacterial concentration in the wound tissue reaches 10 5 cm 2 / g, it is the threshold of bacterial infection. When the wound is infected, the body will have an immune response to pain, swelling, inflammation and tissue damage. Therefore, it is particularly important to quickly and sensitively detect bacterial infection. Rapid detection of bacteria is essential for diagnostic tools in medical settings, which helps to prevent bacterial infection and improve patient survival rate. The main clinical detection methods for tissue wounds are tissue biopsy, plate counting, scraping and wound swabbing. These methods increase the pain and cost of patients because they involve removing dressings and bacterial culture, which are both cumbersome and time-consuming, and may cause secondary damage to the wound. Therefore, researchers have been trying to develop new methods for rapid bacterial detection. An attractive alternative is to use small molecule fluorescent and colorimetric probes because they are simple to use, highly sensitive, low cost and fast detection time. Based on the contact between amphiphilic carbon quantum dots (CDs) and bacterial cell membranes, carbon-hydrogen chain functionalized CDs can easily bind to bacterial cells after short-term culture. Different bacteria can be detected by fluorescence emission spectrum and microscope.

[0003] Organic fluorescent probes are important carriers of optical imaging technology. In addition, due to the strong modifiability of organic fluorescent probes, the fluorescence is easy to control, and the cost is relatively low, etc. Organic fluorescent probes have attracted widespread attention from researchers. Although a large number of fluorescent probes have been reported at present, fluorescent probes with extremely fast response to lipase secreted by bacteria still need to be further developed, especially fluorescent probes that can detect bacteria while killing bacteria. SUMMARY

[0004] The purpose of the present application is to provide a synthesis method and application of a fluorescent probe for lipase detection, which utilizes the characteristics of lipase secreted by bacteria during growth and reproduction to achieve rapid detection of bacteria.

[0005] The technical scheme for solving the above technical problems of the present application is as follows:

[0006] A fluorescent probe having a response to lipase has the following structural formula:

[0007]

[0008] R1 is, but not limited to, H,

[0009] R2 is, but not limited to,

[0010] The method for preparing the above-mentioned fluorescent probe responsive to fat, characterized by comprising the following steps:

[0011]

[0012] Under the protection of nitrogen, organic acid compound, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) are taken, dry acetonitrile (10 mL-50 mL) is injected into the above-mentioned system, and stirring is carried out at 25-100 DEG C for 30 min to completely dissolve the compound and activate the carboxyl group. Then, 3,5-diphenyl-trithiophenol is dissolved in anhydrous acetonitrile (the molar ratio of trithiophenol to organic acid compound is 0.1-10:1), and the above-mentioned system is added by using a syringe, and stirring is carried out at 25-100 DEG C for 6-48 h. After the reaction is completed, extraction is carried out with an organic solvent and water, and the organic phase is combined. The organic phase is dried with anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the product. Other esters are synthesized by the same method, and the yield is between 61.6% and 85.0%. All the compounds are characterized by NMR and LC-MS.

[0013] The fluorescent probe is prepared by connecting acids containing different substituents and corresponding alcohols in the form of ester bond, PET effect is utilized in the process, the fluorescence of the ester is quenched, when the ester bond is hydrolyzed after the action of lipase, the fluorescence of the corresponding alcohol is quickly recovered, the fluorescence of the system is significantly enhanced, and rapid and sensitive detection of lipase is realized. By utilizing the characteristics that bacteria secrete lipase in the process of growth and reproduction, rapid detection of bacteria is realized.

[0014] The condensing agent in the application is at least one of dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDCI).

[0015] The base in the application is at least one of p-dimethylaminopyridine (DMAP), pyridine (Pyridine), triethylamine (TEA), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium acetate (CH3COONa).

[0016] The solvent in the application is at least one of acetonitrile, dichloromethane, trichloromethane, toluene, acetone, tetrahydrofuran and 1,4-dioxane.

[0017] The molar ratio of the terthiophene alcohol (R1-TSF-OH) to the acid (R2-COOH) organic compound described in this invention is 0.5-5:1.

[0018] The reaction temperature and time described in this invention are 6-48 hours at 25-100℃.

[0019] The inert gas described in this invention is nitrogen or argon.

[0020] The beneficial effects of this invention are:

[0021] 1) In this invention, by introducing acids with different substituent units into the molecule, a molecular PET system is constructed to achieve fluorescence quenching of the probe. When it reacts with lipase, the ester bond is hydrolyzed, and fluorescence is released, thereby realizing the detection of lipase.

[0022] 2) The fluorescent probe of the present invention detects bacteria based on the characteristics of bacteria secreting lipase during their growth and reproduction. Attached Figure Description

[0023] Figure 1 This is the structural formula of the fluorescent probe of the present invention.

[0024] Figure 2 Ester bond compound structure.

[0025] Figure 3 Comparison of fluorescence changes in ester-bonded compounds before and after hydrolysis.

[0026] Figure 4 The response of Ester-J compounds to different lipase concentrations.

[0027] Figure 5 Fluorescence changes of Ester-J compound in response to lipase response time.

[0028] Figure 6 Fluorescence changes of Ester-J compounds in bacterial cultures of different concentrations.

[0029] Figure 7 The hydrogen NMR spectrum of the Ester-J compound.

[0030] Figure 8 Ester-J compound LC-MS image. Detailed Implementation

[0031] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be given to the present application in combination with the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application, i.e., the described examples are only a part of the examples of the present application, but not all the examples. The components of the embodiments of the present application generally described and shown in the accompanying drawings can be arranged and designed in various different configurations.

[0032] Therefore, the detailed description of the embodiments of the present application provided in the accompanying drawings below is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0033] Example 1

[0034] A method for synthesizing a fluorescent probe responsive to lipase, comprising the following steps:

[0035]

[0036] (1) Preparation of Ester-A

[0037] Hexanoic acid (30 mg, 0.25 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 47.9 mg, 0.25 mmol), 4-dimethylaminopyridine (DMAP, 30.5 mg, 0.25 mmol) were taken under nitrogen protection, dry dichloromethane (10 mL) was injected into them, and they were stirred at room temperature for 30 min to completely dissolve the compounds and activate the carboxyl group. Then, terthiophene alcohol (55.68 mg, 0.20 mmol) was dissolved in anhydrous dichloromethane and added to the above system with a syringe, and stirred at room temperature for 6 h. After the reaction was completed, extraction was performed with dichloromethane and water, and the organic phases were combined. The organic phase was dried with anhydrous sodium sulfate, concentrated, purified by column chromatography (PE / EA = 5 / 1) to obtain Ester-A (yellow powder, 63.9 mg, yield 85%). 1H NMR (500 MHz, Chloroform-d) δ 7.25 (dd, J = 5.1, 1.2 Hz, 1H), 7.20 (dd, J = 3.6, 1.2 Hz, 1H), 7.09 (q, J = 3.8 Hz, 2H), 7.05 (dd, J = 4.9, 3.6 Hz, 2H), 7.02 - 6.99 (m, 1H), 5.25 (d, J = 0.8 Hz, 2H), 2.37 (t, J = 7.5 Hz, 2H), 1.67 (dq, J = 9.5, 7.4 Hz, 2H), 1.35 - 1.28 (m, 4H), 0.93 - 0.89 (m, 3H). 13 C NMR (126 MHz, CDC13) δ 173.58, 138.54, 137.21, 137.02, 136.56, 135.86, 128.91, 127.91, 125.19, 124.51, 124.34, 123.80, 123.14, 60.44, 34.22, 31.27, 24.58, 22.31, 13.92. ESI HRMS: calcd. for C19H20O2S3 [M] + : 376.0625, found: 376.0651.

[0038] Example 2

[0039] Preparation of Ester-B

[0040]

[0041] Benzoic acid (30.5 mg, 0.25 mmol), l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 47.9 mg, 0.25 mmol), 4-dimethylaminopyridine (DMAP, 30.5 mg, 0.25 mmol) were taken under nitrogen protection, dry dichloromethane (10 mL) was injected into them, and stirred at room temperature for 30 min to completely dissolve the compound and activate the carboxyl group. Then, TSF-OH (55.68 mg, 0.20 mmol) was dissolved in anhydrous dichloromethane, added to the above system with a syringe, and stirred at room temperature for 12 h. After the reaction was completed, extraction was performed with dichloromethane and water, and the organic phase was combined. The organic phase was dried with anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE / EA = 5 / 1) to obtain Ester-B (yellow powder, 54.4 mg, yield 85.0%). 1H NMR (500 MHz, Chloroform-d) δ 8.13 - 8.09 (m, 2H), 7.62 - 7.57 (m, 1H), 7.50 - 7.45 (m, 2H), 7.26 - 7.19 (m, 2H), 7.12 - 7.03 (m, 5H), 5.50 (d, J = 0.7 Hz, 2H). 13 C NMR (126 MHz, CDC13) δ 166.32, 138.70, 137.05, 137.03, 136.60, 135.83, 133.22, 129.84, 129.80, 129.08, 128.44, 127.92, 124.62, 124.57, 124.35, 123.81, 123.19, 61.20. ESI HRMS: calcd. for C 20 H 14 O2S3[M] + : 382.0156, found: 382.0173.

[0042] Example 3

[0043] Preparation of Ester-H

[0044]

[0045] (1) Preparation of Intermediate TPA-TSF-OH

[0046] To a 2-necked flask, 4-(diphenylamino)phenylboronic acid (92.52 mg, 0.32 mmol), bromotriphene methanol (Br-TSF-OH) (107.19 mg, 0.3 mmol) and Pd(dppf)Cl2(21.95 mg, 0.03 mmol) were added, toluene (15 mL) was added under nitrogen protection, then 2M sodium carbonate solution (2 mL) was added. The reaction was refluxed at 110 °C for 8 h. After the reaction was completed, it was cooled to room temperature, and the reaction system was extracted with ethyl acetate and water. The organic phase was combined, dried with anhydrous sodium sulfate, and purified by column chromatography (PE / EA = 3 / 1) to obtain a yellow powder (106.5 mg, yield 68.1%).1H NMR (500 MHz, CDC13-d) δ 7.81 (d, J = 1.6 Hz, 2H), 7.75 - 7.70 (m, 5H), 7.52 (dd, J = 8.5, 6.9 Hz, 4H), 7.46 - 7.41 (m, 2H), 7.38 (d, J = 3.7 Hz, 1H), 7.21 (d, J = 3.7 Hz, 1H), 7.15 (d, J = 3.7 Hz, 1H), 7.11 (d, J = 3.8 Hz, 1H), 7.08 (d, J = 3.5 Hz, 1H), 6.96 (dd, J = 3.6, 0.9 Hz, 1H), 4.85 (d, J = 0.8 Hz, 2H). ESI HRMS: Caclued for: C 31 H 23 NOS3[M] + : 521.0942, found: 521.0961.

[0047] (2) Synthesis of Ester-H

[0048] Under nitrogen protection, 4-(2,2-dicyanovinyl)benzoic acid (39.6 mg, 0.2 mmol), l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 47.9 mg, 0.25 mmol), 4-dimethylaminopyridine (DMAP, 30.5 mg, 0.25 mmol) were taken, dry and anhydrous acetonitrile (10 mL) was injected into it, stirred at room temperature for 30 min to completely dissolve the compound and activate the carboxyl group. Then, TPA-TSF-OH (130 mg, 0.25 mmol) was dissolved in anhydrous dichloromethane, added to the above system with a syringe, stirred at room temperature for 24 h. After the reaction was completed, it was extracted with dichloromethane and water, and the organic phase was combined. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE / EA = 5 / 1) to obtain Ester-H (yellow brown powder, 83.9 mg, yield 62.6%). 1H NMR (500 MHz, Chloroform-d) δ 8.33 - 8.30 (m, 2H), 8.28 - 8.25 (m, 2H), 7.49 - 7.46 (m, 2H), 7.32 - 7.29 (m, 3H), 7.18 - 7.05 (m, 15H), 5.55 (s, 2H). ESI HRMS: calcd. for C38H26N2O4S3[M]+: 670.1055, found: 670.1087.

[0049] Example 4

[0050] Preparation of Ester-J

[0051]

[0052] Preparation of intermediate Tph-TSF-OH

[0053] 3,5-Diphenylboronic acid (87.72 mg, 0.32 mmol), bromo-trithiophenylmethanol (Br-TSF-OH) (107.19 mg, 0.3 mmol) and Pd(dppf)Cl2(21.95 mg, 0.03 mmol) were added to a two-necked flask, to which toluene (15 mL) was added under nitrogen protection, and then 2M sodium carbonate solution (2 mL) was added. The reaction was refluxed at 110°C for 8 h. After the reaction was completed, it was cooled to room temperature, and the reaction system was extracted with ethyl acetate and water. The organic phase was combined, dried over anhydrous sodium sulfate, and purified by column chromatography (PE / EA = 5 / 1) to obtain a yellow powder (109.5 mg, yield 72.0%). 1H-NMR (500 MHz, CDC13-d) δ 7.81 (d, J = 1.6 Hz, 2H), 7.75-7.70 (m, 5H), 7.52 (dd, J = 8.5, 6.9 Hz, 4H), 7.46-7.41 (m, 2H), 7.38 (d, J = 3.7 Hz, 1H), 7.21 (d, J = 3.7 Hz, 1H), 7.15 (d, J = 3.7 Hz, 1H), 7.11 (d, J = 3.8 Hz, 1H), 7.08 (d, J = 3.5 Hz, 1H), 6.96 (dd, J = 3.6, 0.9 Hz, 1H), 4.85 (d, J = 0.8 Hz, 2H).13C NMR (126 MHz, CDCL3) δ 142.92, 142.63, 142.13, 140.80, 136.78, 136.44, 136.44, 136.18, 135.92, 134.96, 134.96, 128.90, 76.77, 66.04. ESI HRMS: cacl. for: C 31 H 22 OS3[M] + : 506.0833, found: 506.0862.

[0054] (2) Preparation of product Ester-J

[0055] To p-nitrobenzoic acid (208.9 mg, 1.0 mmol), l-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 191.6 mg, 1.0 mmol), 4-dimethylaminopyridine (DMAP, 122.0 mg, 1.0 mmol), inject dry anhydrous acetonitrile (50 mL) into it, stir at room temperature for 30 min to completely dissolve the compound. Then, dissolve TPh-TSF-OH (126.7 mg, 0.25 mmol) in anhydrous acetonitrile, add it into the above system with a syringe, stir at room temperature for 48 h. After the reaction is completed, extract with dichloromethane and water, combine the organic phase. Dry the organic phase with anhydrous sodium sulfate, concentrate, purify by column chromatography (PE / EA = 3 / 1) to obtain Ester-J. In this reaction, since p-nitrobenzoic acid is not soluble in anhydrous dichloromethane, the solvent is changed to anhydrous acetonitrile, and the general procedure is consistent. Orange yellow powder, 81.7 mg, yield 62.4%. 1H NMR (500 MHz, DMSO-d6) δ 8.35 - 8.31 (m, 2H), 8.21 - 8.17 (m, 2H), 7.89 - 7.84 (m, 3H), 7.65 - 7.59 (m, 4H), 7.47 - 7.41 (m, 4H), 7.41 - 7.36 (m, 3H), 7.32 (d, J = 6.3 Hz, 1H), 7.27 (d, J = 6.1 Hz, 1H), 7.12 - 7.08 (m, 3H), 5.37 (s, 2H). ESI HRMS: calcd. for C 38 H 25 NO4S3[M] + : 655.0946, found: 655.1197. NMR and mass spectra are shown in Figure 6 and Figure 7 .

[0056] Example 5

[0057] When Ester-J reacts with lipase, Tph-TSF-OH is released, emitting bright fluorescence. With the increase of lipase concentration, the fluorescence intensity at 450 nm and 475 nm gradually increases within 30 min, as shown in Figure 3 . By controlling the lipase concentration at 100 pg / mL, the fluorescence of Ester-J at different reaction times was determined. The results show that when the action time is greater than 10 min, the change of fluorescence intensity is no longer obvious, indicating that Ester-J reacts quickly.

[0058] The lipase in the bacterial culture solution was detected with Ester-J. The results show that Ester-J also has a rapid reaction to the bacterial culture medium, and the fluorescence intensity at 475 nm is positively correlated with the concentration of the bacterial culture medium. Ester-J has a significant reaction to the culture medium of 10 5 CFU / mL of Pseudomonas aeruginosa, while the reaction to Staphylococcus aureus is relatively slow, as shown in Figure 5 .

Claims

1. A fluorescent probe for lipase detection, characterized in that, Its structure is shown in one of the following ways: 、 、 、 。 2. The method for synthesizing the lipase fluorescent probe according to claim 1, characterized in that, Includes the following steps: ， Under inert gas protection, p-nitrobenzoic acid, a condensing agent, and a base were added, followed by 10-50 mL of dry acetonitrile to completely dissolve the compound and activate the carboxyl group. Then, 3,5-diphenylphenyl-terthiophenol was dissolved in anhydrous acetonitrile and added to the above system using a syringe. After the reaction was completed, the mixture was extracted with an organic solvent and water, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the product.

3. The method for synthesizing the lipase fluorescent probe according to claim 2, characterized in that, The condensing agent is at least one of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI).

4. The method for synthesizing the lipase fluorescent probe according to claim 2, characterized in that, The base is at least one of p-dimethylaminopyridine (DMAP), pyridine, triethylamine (TEA), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), and sodium acetate (CH3COONa).

5. The method for synthesizing the lipase fluorescent probe according to claim 2, characterized in that, The organic solvent is at least one selected from acetonitrile, dichloromethane, chloroform, toluene, acetone, tetrahydrofuran, and 1,4-dioxane.

6. The method for synthesizing the lipase fluorescent probe according to claim 2, characterized in that, The molar ratio of 3,5-diphenylphenyl-terthiophenol to p-nitrobenzoic acid is 0.5-5:

1.

7. The method for synthesizing the lipase fluorescent probe according to claim 2, characterized in that, The reaction conditions are 6-48 h at a temperature of 25-100℃.

8. The method for synthesizing the lipase fluorescent probe according to claim 2, characterized in that, The inert gas is nitrogen or argon.

9. The method for synthesizing the lipase fluorescent probe according to claim 2, characterized in that, The activation conditions for the carboxyl groups are stirring at 25-100℃ for 30 minutes.

10. The application of the lipase detection fluorescent probe according to claim 1 in the preparation of lipase detection and bacterial detection products.

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