A quinoline-malononitrile derivative fluorescent probe and its preparation method and application

Abstract

The invention discloses a quinoline-malononitrile derivative fluorescent probe with a structural formula of I. The preparation method comprises the following steps: (1) dissolving 2-methylquinoline and iodoalkane in an organic solvent for reaction to obtain compound 1; (2) dissolving compound 1 and malononitrile in an organic solvent, then adding sodium ethoxide for reaction to obtain compound 2; (3) mixing compound 2, 4-Boc aminobenzaldehyde and piperidine and dissolving them in an organic solvent for reaction at 85-90° C. to obtain compound 3; (4) dissolving compound 3 in an organic solvent, and adding trifluoroacetic acid to remove amino protection of Boc; the probe selectively detects sulfatase by hydrogen bonding, has a very rapid response, has high sensitivity, and achieves the purpose of rapid detection.

Description

Technical Field

[0001] The invention relates to a fluorescent probe, in particular to a quinoline-malononitrile derivative fluorescent probe and a preparation method and application thereof. Background Art

[0002] Sulfatase is a general term for highly conserved proteases that can hydrolyze various sulfates in organisms, including aromatic sulfatase, steroid sulfatase, and some other types of sulfatase. Sulfatase participates in various physiological processes in organisms, including hormone regulation, developmental cell signal transduction, and bacterial pathogenesis. In addition, sulfatase is also believed to be related to the development and growth of many hormone-dependent cancers, including breast cancer. Therefore, sulfatase has always been the focus of scientific researchers.

[0003] In the prior art, a variety of methods for detecting sulfatase have been developed. Compared with other detection methods such as immunoassay and gas chromatography-mass spectrometry, organic small molecule fluorescent probes have been widely used in the detection of enzymes in organisms due to their advantages of high spatiotemporal resolution, high sensitivity and high selectivity. Some organic small molecule fluorescent probes are prone to quenching at low concentrations. This phenomenon is called the ACQ effect, which is contrary to the concept of aggregation-induced emission (AIE). AIE probe molecules emit weak fluorescence at low concentrations and burst out strong fluorescence at high concentrations. When the molecules aggregate, the intramolecular motion that originally consumes the energy of the molecular excited state is suppressed, so that the non-radiative transition that quenches the molecular fluorescence is greatly suppressed, and the molecules mainly emit light by releasing energy through radiative transitions. The main factor for the solid-state luminescence of AIE molecules is that the intramolecular motion is restricted, including intramolecular rotation and vibration restrictions. Methods for restricting intramolecular motion also include electrostatic attraction, hydrogen bonding, hydrophobic interaction and solubility changes. The amino functional group and protein enzyme combine to form a hydrogen bonding force, which may affect the binding mode of the probe and the sulfatase, further hindering the intramolecular motion.

[0004] At present, AIE probe molecules show many advantages, including low background interference, high signal-to-noise ratio and excellent photostability. The fluorescent probes for selective detection of sulfatase that have been developed all use sulfate as the recognition group, which makes the design strategy of sulfatase fluorescent probes slightly simple and the traditional ACQ probe will produce fluorescence quenching at high concentrations, which is not conducive to monitoring. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a quinoline-malononitrile derivative fluorescent probe with short response time to sulfatase and high detection sensitivity; the second purpose of the present invention is to provide a method for preparing the quinoline-malononitrile derivative fluorescent probe; the third purpose of the present invention is to provide the application of the quinoline-malononitrile derivative fluorescent probe in detecting sulfatase.

[0006] Technical solution: The quinoline-malononitrile derivative fluorescent probe of the present invention has the following structural formula:

[0007]

[0008] Among them, the R group is a C1~C3 alkyl group.

[0009] Preferably, the R group is ethyl.

[0010] The method for preparing the quinoline-malononitrile derivative fluorescent probe of the present invention comprises the following steps:

[0011] (1) dissolving 2-methylquinoline and iodoalkane in an organic solvent and reacting them to obtain compound 1;

[0012] (2) dissolving compound 1 and malononitrile in an organic solvent, and then adding sodium ethoxide to react to obtain compound 2;

[0013] (3) Compound 2, 4-Boc aminobenzaldehyde and piperidine are mixed and dissolved in an organic solvent, and reacted at 85-90° C. to obtain compound 3;

[0014] (4) dissolving compound 3 in an organic solvent, adding trifluoroacetic acid to remove the amino protection of Boc to obtain the final product;

[0015] The synthetic route is as follows:

[0016]

[0017] The R group is a C1-C3 alkyl group.

[0018] Preferably, in step (3), the organic solvent is ethanol, the reaction process is monitored by TLC, and the mobile phase solvent is petroleum ether and ethyl acetate. After the reaction is completed, the excess solvent is removed by vacuum filtration, the filter cake is washed with cold acetonitrile, vacuum dried, and purified by column chromatography (mobile phase is dichloromethane and methanol) to obtain an orange solid, i.e., the desired product compound 3.

[0019] Preferably, in step (4), the organic solvent is dichloromethane, the reaction is first reacted at 0-5°C, and then continued to react at room temperature, the reaction process is monitored by TLC, and the mobile phase solvent is dichloromethane and methanol. After the reaction is completed, saturated sodium bicarbonate is added to adjust the system to alkalinity, and after extraction with dichloromethane three times, anhydrous sodium sulfate is added to dry, and the filtrate is collected after vacuum filtration, and the solvent is evaporated under reduced pressure, and then purified by column chromatography (mobile phase is dichloromethane and methanol) to obtain a dark yellow solid, that is, the product quinoline-malononitrile derivative fluorescent probe (hereinafter referred to as probe BQM-NH2).

[0020] Preferably, in step (1), the reaction temperature is 85-95°C, the organic solvent is acetonitrile, the reaction process is monitored by TLC, and the mobile phase solvents are dichloromethane and methanol. After the reaction is completed, the reaction mixture is cooled to room temperature, the precipitate is filtered under reduced pressure, washed, and vacuum dried to obtain compound 1.

[0021] Preferably, in step (2), the organic solvent is ethanol, the reaction is first carried out at -3 to 0°C, and then continued at room temperature, the reaction process is monitored by TLC, and the mobile phase solvents are dichloromethane and methanol. After the reaction is completed, the excess solvent is removed by vacuum filtration, the filter cake is washed with cold ethanol, and vacuum dried to obtain a yellow solid, namely compound 2.

[0022] The invention discloses an application of the quinoline-malononitrile derivative fluorescent probe in detecting sulfatase.

[0023] The applications include detecting sulfatases in solution and detecting sulfatases in cells.

[0024] The solution detection method is as follows: adding different concentrations of sulfatase to a reaction system containing a quinoline-malononitrile derivative fluorescent probe, mixing the reaction solution evenly, incubating at 36-38° C., and then measuring the ultraviolet absorption and fluorescence emission spectra.

[0025] The cell detection method is as follows: A549 cells are cultured in a laser confocal dish, fluorescent probes are added into the dish for co-incubation, and imaging is performed using a laser confocal microscope.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The probe selectively detects sulfatase by hydrogen bonding, responds very quickly, has high sensitivity, and achieves the purpose of rapid detection; (2) The detection limit of the quinoline-malononitrile derivative probe with an ethyl group for sulfatase is 1.73U / L; (3) The probe can still achieve rapid detection of sulfatase in strong acid and strong alkaline environments; (4) The preparation method is simple and easy to industrialize; (5) The probe BQM-NH2 has low cytotoxicity and can selectively detect sulfatase at the cellular level, and successfully realizes the application of fluorescence imaging of intracellular sulfatase in A549 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the fluorescent probe of the quinoline-malononitrile derivative prepared in Example 1;

[0028] Figure 2 The mass spectrum of the fluorescent probe of quinoline-malononitrile derivative prepared in Example 1;

[0029] Figure 3This is a fluorescence spectrum diagram of the quinoline-malononitrile derivative fluorescent probe prepared in Example 1 for sulfatase detection;

[0030] Figure 4 The graph showing the variation trend and linear relationship of the fluorescence intensity of the quinoline-malononitrile derivative fluorescent probe prepared in Example 1 in PBS buffer with the concentration of sulfatase;

[0031] Figure 5 This is an imaging diagram of the quinoline-malononitrile derivative fluorescent probe prepared in Example 1 in A549 living cells;

[0032] Figure 6 This is a graph showing the change in fluorescence intensity of sulfatase detected by the quinoline-malononitrile derivative fluorescent probe prepared in Example 1 under different pH conditions. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with embodiments.

[0034] Example 1

[0035] The quinoline-malononitrile derivative of the present invention has the structural formula:

[0036]

[0037] The preparation method comprises the following steps:

[0038] (1) 1.33 mL (10.0 mmol) of 2-methylquinoline and 1.20 mL (15.0 mmol) of iodoethane were dissolved in anhydrous acetonitrile (15 mL), and the mixture was refluxed at 85°C for 12 h under the protection of inert gas N2. The reaction progress was monitored by TLC, and the mobile phase solvent was dichloromethane and methanol in a volume ratio of 60:1. After the reaction was completed, the reaction mixture was cooled to room temperature, and a large amount of solid was precipitated. The excess solvent was filtered off under reduced pressure, and the filter cake was washed with cold acetonitrile (3×15 mL). After drying, a light yellow solid, i.e., compound 1, was obtained.

[0039] (2) 0.3 g (1 mmol) of compound 1 and 0.247 g (3.7 mmol) of malononitrile were dissolved in 25 mL of anhydrous ethanol and stirred continuously at -2°C. Then 0.307 g (3.7 mmol) of sodium ethoxide was added and the reaction was continued at -2°C for 0.5 h. The mixture was then heated to room temperature and stirred for 4 h. The reaction progress was monitored by TLC. The mobile phase solvent was dichloromethane and methanol in a volume ratio of 60:1. After the reaction was completed, the precipitate was filtered under reduced pressure to remove the solvent. The filter cake was washed with cold ethanol (3×15 mL) and dried in vacuo to obtain a yellow solid, namely compound 2.

[0040] (3) Compound 2 0.47 g (2.0 mmol) and 4-Boc aminobenzaldehyde 0.46 g (2.1 mmol) and piperidine 0.6 mL were dissolved in 30 mL ethanol at 85°C under N2 protection and stirred under reflux for 8 h. The reaction process was monitored by TLC, and the mobile phase solvent was petroleum ether and ethyl acetate in a volume ratio of 5:1. After the reaction was completed, the reaction solution was cooled to room temperature, and a large amount of solid precipitated. The excess solvent was removed by vacuum filtration, and the filter cake was washed with cold acetonitrile (3×15 mL), dried in vacuo, and purified by column chromatography (dichloromethane: methanol = 60:1) to obtain an orange solid, i.e., the desired product compound 3.

[0041] 1 HNMR(600MHz,DMSO-d6)δ9.61(s,1H),8.91(dd,J=8.5,1.1Hz,1H),8.07(d,J=8.9Hz,1H),7.94-7.90(m,1H),7.72(d,J=8.7Hz,2H),7. 61-7.58(m,1H),7.56(d,J=8.6Hz,2H),7.37(q,J=15.7Hz,2H),7.00(s,1H),4.56(q,J=7.0Hz,2H),1.50(s,9H),1.41(t,J=7.1Hz,3H).

[0042] (4) 0.32 g (0.7 mmol) of compound 3 was dissolved in 9 mL of anhydrous dichloromethane, and stirred continuously at 0°C under N2 protection, and then 1.8 mL of trifluoroacetic acid was slowly added dropwise. After reacting at 0°C for 30 min, the mixture was transferred to 25°C and continued to react for 2 h. The reaction process was monitored by TLC, and the mobile phase solvent was dichloromethane and methanol in a volume ratio of 60:1. After the reaction was completed, saturated sodium bicarbonate was added to adjust the system to alkalinity, and the mixture was extracted three times with dichloromethane, and then dried with anhydrous sodium sulfate. The filtrate was filtered under reduced pressure and the solvent was removed from the filtrate, and then purified by column chromatography (volume ratio of dichloromethane: methanol = 60:1) to obtain a dark yellow solid, i.e., quinoline-malononitrile derivative (hereinafter referred to as BQM-NH2).

[0043] Figure 1 is the H NMR spectrum of quinoline-malononitrile derivatives, Figure 2 The mass spectrum of quinoline-malononitrile derivative shows that the fluorescent probe BQM-NH2 was successfully synthesized. 1H NMR (600MHz, DMSO-d6) δ8.88(d,J=8.2Hz,1H),8.05(d,J=8.7Hz,1H),7.89(t,J=7.5Hz,1H),7.58(dd,J=21.9,7.7Hz,3H),7.32(d,J=15.5 Hz,1H),7.21(d,J=15.5Hz,1H),7.00(s,1H),6.78(d,J=7.7Hz,2H),4.54(d,J=6.7Hz,2H),1.41(t,J=6.6Hz,3H)..HR-MS(ESI,m / z):calcd for C 22 H 18 N4[M+H] + 339.15315,found:339.16156.

[0044] Example 2

[0045] (1) Weigh 3.4 mg of the probe BQM-NH2 prepared in Example 1 and dissolve it in 10 ml of DMSO to prepare a 1 mM / L stock solution. Dissolve the sulfatase in PBS buffer to prepare a 100 U / mL stock solution. Add 15 μL of the prepared fluorescent probe BQM-NH2 stock solution, 15 μL of DMSO and 1.17 mL of PBS buffer to a 5 mL centrifuge tube, and add 1.8 mL of the prepared sulfatase stock solution to the mixed solution to form a DMSO-PBS mixed solution (BQM-NH2 concentration is 5 μM / L, Fw=99%).

[0046] (2) Place the centrifuge tube in a constant temperature water bath and incubate at 37°C for 30 min. PBS-DMSO solution (pH = 7.4, pH w =99%) to prepare a 5 μM probe BQM-NH2 stock solution. First, the UV absorption spectrum of the probe BQM-NH2 was tested, and then the fluorescence spectrum of the probe BQM-NH2 was tested using the maximum absorption wavelength as the excitation wavelength. Subsequently, the response fluorescence spectrum of the probe BQM-NH2 was measured, and the test results are as follows: Figure 3 shown.

[0047] like Figure 3 As shown in (a), after adding sulfatase, the UV absorption spectrum of probe BQM-NH2 becomes relatively flat, which may be caused by the interference of a large amount of sulfatase in the mixed solution. Figure 3As shown in (b), the fluorescence spectrum of the probe BQM-NH2 mixed with sulfatase was measured with 420nm as the excitation wavelength. After incubation with sulfatase, the fluorescence signal peak of the probe BQM-NH2 slightly blue-shifted and the fluorescence intensity increased sharply, with a fluorescence signal peak appearing at 580nm. The results show that the probe BQM-NH2 can be used as a fluorescent probe to detect the presence of sulfatase.

[0048] Example 3

[0049] Weigh 3.4 mg of the probe BQM-NH2 prepared in Example 1 and dissolve it in 10 mL of DMSO solution to prepare a standard solution with a concentration of 1 mM; prepare the sulfatase into a 100 U / mL standard solution using PBS buffer solution (10 Mm, pH 7.4). Take a 4 mL EP tube and add different final concentrations of sulfatase solution (0-50 U / mL) to the reaction system PBS buffer (10 mM, pH=7.4, 1% DMSO) containing the probe BQM-NH2 (5 μM). Incubate at 37°C in a constant temperature shaker for 30 minutes. Under excitation at a wavelength of 420 nm, collect the fluorescence emission spectrum of the solution in the 450-800 nm band. The test results are as follows: Figure 4 shown.

[0050] Depend on Figure 4 It can be seen that the fluorescence intensity curve of BQM-NH2 prepared in probe Example 1 increases with the increase of sulfatase concentration. In addition, it is calculated that its detection limit is 1.73U / L and the detection range is 0-50U / mL, indicating that the probe has high sensitivity and can be used for the detection of endogenous sulfatase in biological systems.

[0051] Example 4

[0052] A549 cells were placed in a culture flask and cultured in an incubator (37°C, 5% CO2). After 24 hours, the cells were taken out and 2 mL of trypsin digestion solution was added to the culture flask to digest the cells. After digestion was completed, 2 ml of culture medium (DEME medium containing 10% FBS) was added to terminate the digestion. The cell suspension was then centrifuged, and the supernatant was discarded after centrifugation. 1 mL of culture medium was added and the cells were resuspended by blowing with a pipette. The cell suspension was added to a laser confocal dish and placed in a 5% CO2, 37°C incubator overnight to adhere to the wall. The culture medium was discarded, and 2 mL of culture medium was added after washing with PBS buffer. The probe BQM-NH2 (5 μM) prepared in Example 1 was then used for confocal fluorescence imaging of living A549 cells. First, after incubating A549 cells with the probe BQM-NH2 for 30 minutes, the cells showed strong red fluorescence (such as Figure 5a, b, and c), indicating that the probe BQM-NH2 can detect the presence of sulfatase in A549 living cells. In addition, after A549 living cells were treated with dopamine hydrochloride for 30 minutes, the probe BQM-NH2 was added and incubated for another 30 minutes. At this time, the red fluorescence of the cells was weak (as shown in Fig. Figure 5 d, e, and f), which is basically negligible, indicating that the sulfatase in A549 cells was successfully inhibited by dopamine hydrochloride, indicating that the probe BQM-NH2 has a high selectivity for the detection of sulfatase. The above results show that the probe BQM-NH2 can selectively detect sulfatase at the cellular level and is successfully applied to the fluorescence imaging of A549 living cells.

[0053] Example 5

[0054] The probe BQM-NH2 prepared in Example 1 was dissolved in DMSO to prepare a 1 mM stock solution. w =99%) to prepare a 5 μM probe BQM-NH2 stock solution, and test the fluorescence intensity of the probe BQM-NH2 itself and after incubation with sulfatase at 580 nm under pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively. The test results are as follows Figure 6 shown.

[0055] Depend on Figure 6 It can be seen that in the range of pH = 2-10, the probe BQM-NH2 always maintains a relatively weak fluorescence. After incubation with sulfatase, the probe BQM-NH2 can show strong fluorescence at 580nm. The stronger the alkalinity of the solution, the stronger the fluorescence intensity of the probe BQM-NH2 after incubation with sulfatase, that is, the stronger the detection ability of the probe BQM-NH2 for sulfatase. The results show that even under strong acid and strong alkaline conditions, the probe BQM-NH2 can well identify sulfatase, and the detection ability is better under strong alkaline conditions.

Claims

1. A quinoline-malononitrile derivative fluorescent probe, characterized in that: The structural formula is as follows: ; Among them, the R group is ethyl.

2. A method for preparing the quinoline-malononitrile derivative fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) dissolving 2-methylquinoline and iodinated alkane in an organic solvent and reacting them to obtain compound 1; (2) Compound 1 and malononitrile are dissolved in an organic solvent, and then sodium ethoxide is added to react to obtain compound 2; (3) Compound 2, 4-Boc aminobenzaldehyde and piperidine are mixed and dissolved in an organic solvent and reacted at 85-90°C to obtain compound 3; (4) Dissolving compound 3 in an organic solvent, adding trifluoroacetic acid to remove the amino protection of Boc to obtain the final product; The synthetic route is as follows: ; Among them, the R group is ethyl.

3. The method for preparing the quinoline-malononitrile derivative fluorescent probe according to claim 2, characterized in that: In step (3), the organic solvent is ethanol.

4. The method for preparing the quinoline-malononitrile derivative fluorescent probe according to claim 2, characterized in that: In step (4), the organic solvent is dichloromethane, and the removal of the amino protection of Boc is: first reacting at 0-5°C, and then continuing to react at room temperature.

5. The method for preparing the quinoline-malononitrile derivative fluorescent probe according to claim 2, characterized in that: In step (1), the reaction temperature is 85-95° C., and the organic solvent is acetonitrile.

6. The method for preparing the quinoline-malononitrile derivative fluorescent probe according to claim 2, characterized in that: In step (2), the organic solvent is ethanol, and the reaction is first carried out at -3 to 0°C and then continued at room temperature.

7. Use of the quinoline-malononitrile derivative fluorescent probe according to claim 1 in the preparation of a reagent for detecting sulfatase.

8. The use according to claim 7, characterized in that: The application method comprises the following steps: adding sulfatase of different concentrations to a reaction system containing a quinoline-malononitrile derivative fluorescent probe, mixing the reaction solution evenly, incubating the mixture at 36-38° C., and then measuring ultraviolet absorption and fluorescence emission spectra.

9. The use according to claim 7, characterized in that: The application method comprises the following steps: culturing cells in a laser confocal dish, adding fluorescent probes into the dish for co-incubation, and performing imaging with a laser confocal microscope.

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