A tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore, a synthesis method thereof, and applications thereof

Abstract

The present invention discloses a tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore, a synthesis method thereof and applications thereof, belonging to the field of biomedicine. In the present invention, triphenylamine is used as an energy donor, which is connected to a tetraazacyclic ring with three amide arms through an amide bond to obtain a ligand TPA-Cy, and the ligand reacts with Tb<supgt;3+< / supgt; to obtain a tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore, namely TPA-Cy-Tb. This complex can enhance fluorescence in aqueous solution to respond and recognize the biomarker dipicolinic acid of anthrax spores, and the lowest detection limit reaches 72 nM. The prepared TPA-Cy-Tb can be used to prepare a fluorescence probe for the quantitative detection of anthrax spores in the environment. The present invention has the advantages of cheap synthesis raw materials, simple synthesis process, easy separation procedure, high yield, stability and easy preservation. The fluorescence probe of the complex of the present invention can specifically be activated by DPA to have a fluorescence response and is used for the quantitative detection of anthrax spores, and has excellent biological application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore, a synthesis method thereof, and its application in the rapid and sensitive detection of biomarkers for anthrax infectious diseases. Background Art

[0002] Anthrax is a serious infectious disease caused by the Gram-positive rod-shaped bacterium Bacillus anthracis, commonly known as Bacillus anthracis spores, which can cause anthrax fever and lead to a fatal infection of organisms. Inhaling 10 4 Bacillus anthracis spores can be fatal if not effectively treated within 24 - 48 h. As a potential biological warfare agent, Bacillus anthracis spores have attracted wide attention worldwide. 2,6-Pyridinedicarboxylic acid (DPA), which accounts for about 5 - 15% of the dry weight of Bacillus anthracis spores, is usually used as a biomarker for Bacillus anthracis spores. Therefore, the detection of Bacillus anthracis spores can be achieved by inventing an indicator that responds rapidly and sensitively to 2,6-pyridinedicarboxylic acid.

[0003] Currently, the detection of Bacillus anthracis spores can be roughly divided into biological methods and chemical methods. Biological methods include polymerase chain reaction (PCR) and immunoassay. The PCR detection method uses chain polymerization to amplify bacterial DNA. However, they require expensive reagents, complex pretreatment, and long testing time. Chemical methods include high-performance liquid chromatography, surface-enhanced Raman spectroscopy (SERS), electrochemical detection, and fluorescence detection. Compared with the first three detection methods, the fluorescence detection method has the advantages of rapidity, sensitivity, high selectivity, and portability, and has a lower limit of detection (LOD).

[0004] Due to the unique photophysical properties of lanthanide ions, such as large Stokes shifts, narrow f-f transition spectral bands, and long fluorescence lifetimes, background fluorescence interference can be effectively avoided, and the signal-to-noise ratio and detection sensitivity can be improved. These superior photophysical properties have enabled lanthanide ions to be widely studied in the fields of fluorescence detection, fluorescence imaging, and time-resolved imaging.

[0005] The use of lanthanide complexes for the detection of Bacillus anthracis spores has been reported. However, due to their rigid planar structures, these probes will inevitably cause aggregation-caused quenching (ACQ) in water, resulting in fluorescence loss. There are few reports on novel AIE lanthanide complex fluorescence probes, and strong fluorescence enhancement will occur in response to DPA. Summary of the Invention

[0006] Aiming at the deficiencies and problems of the prior art, the purpose of the present invention is to provide a tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore, a synthesis method thereof, and its application.

[0007] On the one hand, the present invention provides a tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore, and its chemical structural formula is as follows:

[0008] 。

[0009] On the other hand, the present invention provides a synthesis method of the above tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore, and the reaction formula of the synthesis method is as follows:

[0010]

[0011] The synthesis method includes the following steps:

[0012] (1) Synthesis of compound 2: Dissolve compound 1 in the first solvent, add sodium carbonate, and dropwise add a chloroform solution containing bromoacetyl bromide at 0 °C. Then heat the reaction mixture to room temperature, stir and react for a period of time, and obtain a white solid through separation and purification.

[0013] (2) Synthesis of precursor TPA-Cy: Dissolve compound 2 and compound 3 in the second solvent, add potassium carbonate and an appropriate amount of potassium iodide, stir and reflux and react for a period of time, and obtain a pale yellow solid through separation and purification.

[0014] (3) Synthesis of TPA-Cy-Tb: Dissolve TPA-Cy in the third solvent, and dropwise add a methanol solution containing TbCl3 . 6H2O while stirring, heat to reflux and react overnight, and obtain TPA-Cy-Tb through separation and purification.

[0015] Preferably, the molar ratio of compound 1 to bromoacetyl bromide in step (1) is 1:1 - 1.5, the molar ratio of compound 2 to compound 3 in step (2) is 1:0.8 - 1, and the molar ratio of TPA-Cy to Tb 3+ in step (3) is 1:1 - 1.2.

[0016] Preferably, the first solvent is chloroform, the second solvent is acetonitrile, and the third solvent is methanol.

[0017] Preferably, the reaction time in step (1) is 8 h, the reaction time in step (2) is 48 h, and the reaction time in step (3) is 12 - 24 h.

[0018] Preferably, the purification techniques in the above steps include washing, extraction, filtration, drying, recrystallization, and column chromatography.

[0019] The present invention discloses the application of the above-mentioned tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore. The Tb(III) complex TPA-Cy-Tb is prepared into a fluorescent probe, which can quantitatively detect anthrax spores in the environment. When quantitatively detecting anthrax spores in the environment, pyridine dicarboxylic acid (DPA) is used as a biomarker for anthrax spores, and TPA-Cy-Tb has a fluorescence enhancement response to pyridine dicarboxylic acid.

[0020] The sensitization mechanism of TPA-Cy-Tb is as follows: Triphenylamine belongs to an aggregation-induced emission dye, which can act as a photosensitizer and transfer energy to Tb 3+ ion luminescence.

[0021] The sensitization mechanism and recognition mechanism of TPA-Cy-Tb are as follows: Due to the presence of coordinated water molecules in TPA-Cy-Tb, based on Tb 3+ the luminescence at 545 nm is very weak; when pyridine dicarboxylic acid is present, TPA-Cy-Tb displaces the coordinated water molecules, resulting in enhanced fluorescence based on Tb 3+ at 545 nm.

[0022] The principle of using the TPA-Cy-Tb fluorescent probe of the present invention for quantitatively detecting anthrax spores in the environment is as follows: One coordination site of Tb 3+ is occupied by water molecules with fluorescence quenching effect. TPA with AIE properties serves as an energy donor, and then DPA replaces the coordination site where water molecules are located, thereby turning on the fluorescence response for quantitative detection of the biological contaminant anthrax spores.

[0023] Furthermore, the method for quantitatively detecting anthrax spores in the environment by using the Tb(III) complex TPA-Cy-Tb prepared into a fluorescent probe is as follows: Dissolve TPA-Cy-Tb in DMSO to prepare a 2×10 -3 M stock solution, and prepare a 20 μM TPA-Cy-Tb test solution with HEPES buffer solution (pH = 7.4). Add 20 μL of water in nature to the test solution to measure its fluorescence intensity, calculate the content of pyridine dicarboxylic acid according to the linear relationship, and obtain the content of anthrax spores according to the proportion of this substance in anthrax spores.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention provides a novel AIE lanthanide complex. The complex with this structure effectively avoids the quenching caused by aggregation in water (ACQ), reduces fluorescence loss, and will be accompanied by strong fluorescence enhancement under the response to DPA.

[0026] (2)The tetrazacyclic Tb(III) complex containing triphenylamine fluorophore prepared by the present invention is an AIE-type lanthanide complex, which has the advantages of cheap synthesis raw materials, simple synthesis process, easy separation procedure, high yield, stability and easy preservation.

[0027] (3)The present invention uses a tetrazacyclic Tb(III) complex containing triphenylamine fluorophore as a fluorescent probe, which can specifically be turned on for fluorescence response by DPA and is used for the quantitative detection of anthrax spores, having excellent biological application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 1H NMR spectrum (CDCl3) of compound 2 in Example 1;

[0029] Figure 2 1H NMR spectrum (DMSO-d6) of TPA-Cy in Example 1;

[0030] Figure 3 13C NMR spectrum (DMSO-d6) of TPA-Cy in Example 1;

[0031] Figure 4 Mass spectrum of TPA-Cy in Example 1;

[0032] Figure 5 Mass spectrum of TPA-Cy-Tb in Example 1;

[0033] Figure 6 Fluorescence spectra of TPA-Cy-Tb, TPA-Cy-Tb + DPA, TPA-Cy, and Tb in Example 2 3+ ;

[0034] Figure 7 Fluorescence lifetime diagrams of TPA-Cy-Tb in water (10 mM HEPES buffer, pH = 7.4) and heavy water in Example 3;

[0035] Figure 8 Fluorescence response diagrams of TPA-Cy-Tb to different anions in Example 4;

[0036] Figure 9 Fluorescence response diagrams of TPA-Cy-Tb to different concentrations of DPA in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0037] Example 1: Synthesis of AIE lanthanide complex TPA-Cy-Tb

[0038] The synthesis method of the AIE lanthanide complex TPA-Cy-Tb of the present invention comprises the following steps:

[0039] (1)Synthesis of Intermediate Compound 2: Compound 3 can be synthesized according to the literature method or purchased from the market. Dissolve Compound 1 (0.74 g, 2.84 mmol) in chloroform (30 mL), add sodium carbonate (0.35 g, 3.3 mmol), stir the mixture vigorously, and dropwise add a solution of bromoacetyl bromide (0.31 mL, 3.57 mmol) in chloroform (10 mL) at 0 0 °C. Then heat the mixture to room temperature and stir for 8 h. Filter, remove the solvent using a rotary evaporator, and purify the crude product by silica gel chromatography (petroleum ether:dichloromethane = 5:1) to obtain 0.63 g of a yellow solid with a yield of 85%.

[0040] The colorless liquid compound obtained above was determined by a nuclear magnetic resonance instrument (Varian instrument 400 MHz), and the results are as Figure 1 shown, and the data are as follows:

[0041] 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.05 (s, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.29 - 7.19 (m, 4H), 7.07 (q, J = 2.5 Hz, 4H), 7.08 - 6.99 (m, 2H), 7.04 - 6.95 (m, 2H), 4.01 (s, 2H).

[0042] (2)Synthesis of Precursor TPA-Cy: Dissolve Compound 2 (0.38 g, 1.00 mmol) and Compound 3 (0.30 g, 0.87 mmol) in acetonitrile, add potassium carbonate (0.27 g, 1.50 mmol) and an appropriate amount of potassium iodide (0.25 g, 1.50 mmol). Stir and reflux for 48 h, filter, and evaporate the solvent. Purify the crude product by silica gel chromatography (dichloromethane:methanol = 20:1) to obtain 0.26 g of a pale yellow solid with a yield of 68%.

[0043] The colorless liquid compound obtained above was determined by a nuclear magnetic resonance instrument (Varian instrument 400 MHz), and the results are as Figure 2 and Figure 3 shown, and the data are as follows:

[0044] 11H NMR (400 MHz, DMSO-d6) δ (ppm): 10.10 (s, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.29 - 7.19 (m, 4H), 7.07 (q, J = 2.5 Hz, 4H), 7.08 - 6.99 (m, 2H), 7.04 - 6.95 (m, 2H), 6.95 (d, J = 8.2 Hz, 6H), 3.14 (s, 6H), 2.75 - 2.63 (m, 7H), 2.68 (s, 9H). 13 13C NMR (101 MHz, DMSO-d6) δ (ppm): 173.46, 173.41, 170.05, 147.76, 142.97, 134.89, 129.86, 125.60, 123.33, 122.84, 121.37, 61.64, 58.36, 57.37.

[0045] The mass spectrometry detection results are as follows Figure 4 shown, MS (m / z): calcd for C 34 H 45 N9O4 644.36, found: 644.8083.

[0046] (3)Synthesis of TPA-Cy-Tb: Dissolve the precursor TPA-Cy (50 mg, 0.078 mmol) in methanol (3 mL), and dropwise add a methanol solution (3 mL) of TbCl3·6H2O (28 mg, 0.078 mmol). Reflux overnight, evaporate the solvent to about 1 mL, dropwise add ether until the solution starts to become turbid, leave it in an ether atmosphere overnight, filter to collect the precipitated solid, and wash the precipitate with ether. 32 mg of white solid was obtained with a yield of 64%. . The mass spectrometry detection results are as follows

[0047] shown, MS (m / z): calcd for C Figure 5 H 34 H 45 N9O4Tb (802.72), found: 801.15.

[0048] The reaction formula of this example is as follows

[0049]

[0050] Example 2: Verification of energy transfer of AIE lanthanide complex TPA-Cy-Tb

[0051] The TPA-Cy-Tb obtained in Example 1 was dissolved in DMSO to prepare a 2×10 -3 M mother liquor, and 2×10 -3 M TPA-Cy and Tb mother liquors were prepared. They were made into 20 μM TPA-Cy-Tb, TPA-Cy, and Tb test solutions with HEPES buffer solution (pH = 7.4), and their fluorescence intensities were measured. 20 μL of 0.01 M DPA was added to TPA-Cy-Tb, and its fluorescence intensity was measured. As 3+ shown, before coordination, the fluorescence intensity of TPA-Cy was 294 a.u., and Tb 3+ had no fluorescence. After coordination, the fluorescence intensity of the ligand decreased to 97.18 a.u., and Tb Figure 6 showed characteristic fluorescence emission peaks. The experimental results showed that the probe TPA-Cy-Tb transferred part of the energy in TPA to Tb 3+ , enabling Tb 3+ to have characteristic fluorescence emission peaks. The calculation formula for the energy transfer efficiency is ϕ 3+ =1 - I 3+ / I Et The energy transfer efficiency of this complex was 66.9%. DA / I D

[0052] Example 3: Fluorescence Lifetimes of TPA-Cy-Tb in Water (10 mM HEPES Buffer, pH = 7.4) and Heavy Water

[0053] The TPA-Cy-Tb obtained in Example 1 was dissolved in DMSO to prepare a 2×10 -3 M mother liquor. A 20 μM TPA-Cy-Tb test solution 1 was prepared with HEPES buffer solution (pH = 7.4), and a 20 μM TPA-Cy-Tb test solution 2 was prepared with heavy water. The fluorescence lifetimes at 545 nm were measured respectively. Figure 7 is the normalized luminescence decay curve, and the decay curve was fitted with a double-exponential curve. According to the modified Horrocks equation q = 5[(1 / τH2O - 1 / τD2O) - 0.06], the coordinated water q = 1.3 was calculated, indicating that the number of coordinated water molecules was approximately 1.

[0054] Example 4: Selectivity Test of the AIE Lanthanide Complex TPA-Cy-Tb for Pyridine Dicarboxylic Acid (DPA)

[0055] The TPA-Cy-Tb obtained in Example 1 was dissolved in DMSO to prepare a 2×10 -3 ​M mother liquor, and the TPA-Cy-Tb test solution with a concentration of 20 μM was prepared with HEPES buffer solution (pH = 7.4). Then, 20 μM of 0.01 M of F - , Cl - , Br - , I - , SO4 2- , CO3 2- , HCO3 2- , SO3 2 , ClO - , DPA, CH3COO - , Pi and other anions were used for fluorescence titration experiments. As shown in a) of Figure 8 , it was found that the characteristic fluorescence emission spectrum of Tb in TPA-Cy-Tb was significantly enhanced only after the addition of DPA. 3+ , and the fluorescence emission spectrum of Tb in TPA-Cy-Tb was significantly enhanced only after the addition of DPA. Figure 8 b) in Figure 8 is the fluorescence emission peak at 545 nm after the addition of different anions. After the addition of DPA, the fluorescence intensity increased by 6.6 times compared with that without DPA. The experiment shows that the probe TPA-Cy-Tb has high selectivity for DPA, indicating that the probe can be used for the detection of anthrax spores in the environment.

[0056] Example 5: Response of AIE lanthanide complex TPA-Cy-Tb to DPA

[0057] The TPA-Cy-Tb obtained in Example 1 was dissolved in DMSO to prepare a mother liquor with a concentration of 2×10 -3 M, and the TPA-Cy-Tb test solution with a concentration of 20 μM was prepared with HEPES buffer solution (pH = 7.4). Fluorescence titration experiments were carried out by adding DPA. As shown in a) of Figure 9 , it was found that the fluorescence emission spectrum of TPA-Cy-Tb changed with the addition of DPA. Figure 9 , and it was found that the fluorescence emission spectrum of TPA-Cy-Tb changed with the addition of DPA. Figure 9 b) in Figure 9 is the fluorescence emission peak at 545 nm, which increased with the increase of the concentration of DPA and reached saturation at 75 μM, and the fluorescence intensity increased by 6.6 times. In the fluorescence emission spectrum test, the linear relationship curve between the fluorescence intensity of 20 μM probe and 20 μM - 45 μM DPA is shown in c) of Figure 9 . There is a good linear relationship between the fluorescence intensity and the content of DPA, and its linear equation is Y = 18.736X - 312.19. According to the 3σ / K rule, the detection limit of the probe TPA-Cy-Tb for DPA was calculated to be 72 nM (σ = 0.45, K = 18.736). The experiment shows that there is a good linear relationship between the probe TPA-Cy-Tb and the content of DPA, indicating that the probe can be used for the quantification of anthrax spores in the environment.

Claims

1. A tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore, characterized in that, The Tb(III) complex is TPA-Cy-Tb, and its chemical structure is shown in Formula (I): Formula (I).

2. The synthesis method of a tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore as claimed in claim 1, characterized in that, The synthesis method uses 4-aminotriphenylamine as a raw material to obtain a tetraaza-cyclic ligand containing triphenylamine through amidation and nucleophilic substitution reactions; then, the tetraaza-cyclic ligand with triphenylamine as the antenna group is coordinated with TbCl3 . Tb in 6H2O 3+ to obtain the Tb(Ⅲ) complex, namely TPA-Cy-Tb; the reaction formula of the synthesis method is as follows: 。 3. The synthesis method of a tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore according to claim 2, characterized in that, The synthesis method comprises the following steps: (1) Synthesis of Compound 2: Dissolve Compound 1 in the first solvent CHCl3, add sodium carbonate, and stir vigorously. Dropwise add a chloroform solution containing bromoacetyl bromide at 0 °C. Then heat the reaction mixture to room temperature and stir for the reaction. After separation and purification, a white solid Compound 2 is obtained; (2) Synthesis of the precursor TPA-Cy: Dissolve Compound 2 and Compound 3 in the second solvent CH3CN, add potassium carbonate and an appropriate amount of potassium iodide, stir and reflux for the reaction, and separate and purify to obtain a pale yellow solid TPA-Cy; (3) Synthesis of TPA-Cy-Tb: Dissolve the TPA-Cy in the third solvent methanol, and dropwise add a methanol solution containing TbCl3 . ·6H2O thereto under stirring, heat the mixture under reflux overnight for reaction, and separate and purify to obtain TPA-Cy-Tb.

4. The synthesis method according to claim 3, characterized in that, The molar ratio of compound 1 to bromoacetyl bromide in step (1) is 1:1 - 1.5; the molar ratio of compound 2 to compound 3 in step (2) is 1:0.8 - 1, and the molar ratio of TPA-Cy to TbCl3 . ·6H2O in step (3) is 1:1 - 1.

2.

5. The synthesis method according to claim 3, characterized in that, The reaction time in step (1) is 8 h, the reaction time in step (2) is 48 h, and the reaction time in step (3) is 12 - 24 h.

6. Use of the tetraazacyclic Tb(III) complex containing a triphenylamine fluorophore according to claim 1 in the preparation of a fluorescent probe.

7. The application according to claim 6, wherein: The fluorescent probe is used for the quantitative detection of anthrax spores in the environment.

8. The application according to claim 7, characterized in that Using pyridinedicarboxylic acid as a biomarker for anthrax spores, the specific method for the quantitative detection of anthrax spores in the environment is as follows: Dissolve the TPA-Cy-Tb in DMSO to prepare a stock solution, and prepare a TPA-Cy-Tb test solution with a HEPES buffer solution. Add natural water to the test solution to measure its fluorescence intensity, calculate the content of pyridinedicarboxylic acid according to the linear relationship, and obtain the content of anthrax spores according to the proportion of this substance in anthrax spores.

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