A diphenyl ether derivative specifically recognizing tryptophan and its preparation method and application

Abstract

The present invention belongs to the technical field of aggregation-induced enhanced luminescent materials and aggregation-induced luminescent materials, and discloses a diphenyl ether derivative that specifically recognizes tryptophan, and a preparation method and application thereof. Three compounds are successfully prepared by the Suzuki coupling method, and are separated and purified by column chromatography. Among the diphenyl ether derivatives obtained by the present invention, NDPO and TDPO have excellent AIE effects, and SDPO has excellent AEE effects. The luminescence intensity can be increased by 29 times, 4 times and 34 times, respectively. The appropriate measuring solvent for the compound is determined by the solvation effect. The compounds SDPO and TDPO can be used as fluorescent probes to have efficient and specific recognition of tryptophan.

Description

Technical Field

[0001] The present invention belongs to the technical field of aggregation-induced enhanced emission (AEE) materials and aggregation-induced emission (AIE) materials, and the present invention relates to a diphenyl ether derivative specifically recognizing tryptophan, and a preparation method and application thereof. Background Art

[0002] In 2001, Tang Benzhong's team first proposed the concept of aggregation-induced emission (AIE). The characteristics of molecules with AIE properties are that they emit weak fluorescence or even no fluorescence in dilute solutions, but emit strong fluorescence in the aggregated state. In 2002, Park's team discovered the phenomenon of aggregation-induced enhanced emission (AEE). Compared with ACQ, AIE and AEE molecules have a wider range of applications as fluorescent probes, and their solid quantum yield is high and their photostability is good. In recent years, scientists are no longer limited to synthesizing AIE or AEE molecules with pure carbon-hydrogen structures and metal complex structures, but have synthesized novel fluorescent groups containing heteroatoms. New materials based on AIE or AEE have been successfully applied in many fields such as organic optoelectronic devices, organic light-emitting diodes (OLEDs), chemical and biosensors. Therefore, it is reasonable to synthesize diphenyl ether organic fluorescent materials with heteroatoms.

[0003] Tryptophan (Trp), also known as α-amino-β-indolepropionic acid, is one of the essential amino acids for the human body and is involved in a variety of physiological processes. Tryptophan plays an important role in regulating growth, emotions, behavior and immune response. It can effectively prevent pellagra, depression, improve sleep and regulate emotions. Tryptophan exists in various natural matrices and can only be ingested exogenously. Currently, the main methods for detecting tryptophan are enzymatic hydrolysis, reversed-phase liquid chromatography-tandem mass spectrometry, etc. Compared with traditional methods, fluorescent probes are one of the most convenient detection methods, but they have not been widely used because they cannot specifically identify a single natural amino acid. Therefore, it is necessary to synthesize a fluorescent probe that can efficiently, sensitively and specifically identify a single amino acid. Summary of the invention

[0004] The present invention is to solve the above-mentioned problems existing in the prior art. The present invention provides a diphenyl ether derivative that specifically recognizes tryptophan, and a preparation method and application thereof. Three compounds are successfully prepared by the Suzuki coupling method, and are separated and purified by column chromatography. Among the diphenyl ether derivatives obtained by the present invention, NDPO (Formula I) and TDPO (Formula III) have excellent AIE effect, and SDPO (Formula II) has excellent AEE effect. The luminescence intensity can be increased by 29 times, 4 times and 34 times respectively. The suitable measurement solvent of the compound is determined by the solvation effect. The compounds SDPO and TDPO can be used as fluorescent probes to have efficient and specific recognition of tryptophan (the detection limit of SDPO is 0.14um, and the detection limit of TDPO is 0.091um).

[0005] The above object of the present invention is achieved through the following technical solutions:

[0006] A diphenyl ether derivative with AIE characteristics, whose general structural formula is shown in Formula I and Formula III; and a diphenyl ether derivative with AEE characteristics, whose general structural formula is shown in Formula II;

[0007]

[0008] The preparation method of the above-mentioned diphenyl ether derivatives, the synthetic route is as follows:

[0009]

[0010] Follow the steps below:

[0011] Place tribromobenzene compounds (0.5g, 1mmol), 4-phenoxyphenylboronic acid (0.96g, 4.5mmol), anhydrous potassium carbonate (0.83g, 6mmol) and Pd(PPh3)4 (0.2g, 0.174mmol) in a flask, add 120ml 1,4-dioxane and 20ml distilled water, stir and heat to react for 24 hours under nitrogen protection. After the reaction is completed, cool to room temperature, extract, dry, filter, and spin out the solvent. Use petroleum ether / dichloromethane as eluent for column chromatography purification, and recrystallize from ethanol to obtain crystals or powder.

[0012] The diphenyl ether derivatives prepared by the above preparation method are used as fluorescent probes for identifying small molecules, specifically, as fluorescent probes for identifying tryptophan.

[0013] The beneficial effects of the present invention compared with the prior art are:

[0014] Among the diphenyl ether derivatives prepared by the present invention, SDPO has aggregation-induced enhanced emission (AEE) characteristics, and NDPO and TDPO have aggregation-induced emission (AIE) characteristics. Compounds SDPO and TDPO can be used as fluorescent probes to identify small biological molecules, and can efficiently and specifically identify tryptophan, with the detection limit of the former reaching 0.14 μM and the latter reaching 0.091 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the compound prepared in Example 1 of the present invention 1 H NMR spectrum.

[0016] Figure 2 is the compound prepared in Example 1 of the present invention 13 C NMR spectrum.

[0017] Figure 3 Graphs showing the fluorescence spectrum (a) and fluorescence variation trend (b) of the compound prepared in Example 1 of the present invention in a THF / C2H5OH mixture.

[0018] Figure 4 It is the fluorescence spectrum diagram of the compound prepared in Example 1 of the present invention in different solvents.

[0019] Figure 5 It is a fluorescence spectrum diagram of the compound prepared in Example 1 of the present invention as a fluorescent probe interacting with different amino acids.

[0020] Figure 6 It is a PL diagram of the compound prepared in Example 1 of the present invention as a fluorescent probe interacting with tryptophan at different concentration gradients.

[0021] Figure 7 It is a fluorescence intensity trend diagram of the compound prepared in Example 1 of the present invention as a fluorescent probe.

[0022] Figure 8 This is a fluorescence spectrum diagram of a competitive experiment of the compound prepared in Example 1 of the present invention as a fluorescent probe for amino acids.

[0023] Fig. 9 This is a graph showing the detection limit of the compound prepared in Example 1 of the present invention as a fluorescent probe for detecting tryptophan.

[0024] Fig.10 This is a graph exploring the sensitivity of the compound prepared in Example 1 of the present invention as a fluorescent probe for detecting tryptophan.

[0025] Fig.11 is the compound prepared in Example 2 of the present invention 1 H NMR spectrum.

[0026] Fig.12 is the compound prepared in Example 2 of the present invention 13 C NMR spectrum.

[0027] Fig.13 Graphs showing the fluorescence spectrum (a) and fluorescence variation trend (b) of the compound prepared in Example 2 of the present invention in a THF / C2H5OH mixture.

[0028] Fig.14 It is the fluorescence spectrum diagram of the compound prepared in Example 2 of the present invention in different solvents.

[0029] Fig.15 It is a fluorescence spectrum diagram of the compound prepared in Example 2 of the present invention as a fluorescent probe interacting with different amino acids.

[0030] Fig.16It is a PL diagram of the compound prepared in Example 2 of the present invention as a fluorescent probe interacting with tryptophan at different concentration gradients.

[0031] Fig.17 It is a fluorescence intensity trend diagram of the compound prepared in Example 2 of the present invention as a fluorescent probe.

[0032] Fig.18 This is a fluorescence spectrum diagram of a competitive experiment of the compound prepared in Example 2 of the present invention as a fluorescent probe for amino acids.

[0033] Fig.19 This is a graph showing the detection limit of the compound prepared in Example 2 of the present invention as a fluorescent probe for detecting tryptophan.

[0034] Fig. 20 This is a graph exploring the sensitivity of the compound prepared in Example 2 of the present invention as a fluorescent probe for detecting tryptophan.

[0035] Fig.21 is the compound prepared in Example 3 of the present invention 1 H NMR spectrum.

[0036] Fig. 22 is the compound prepared in Example 3 of the present invention 13 C NMR spectrum.

[0037] Fig.23 Graphs showing the fluorescence spectrum (a) and fluorescence variation trend (b) of the compound prepared in Example 3 of the present invention in a THF / C2H5OH mixture. DETAILED DESCRIPTION

[0038] The present invention is described in detail below by specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0039] Example 1

[0040] The preparation method of the diphenyl ether derivative with AIE properties, TDPO, is carried out in the following steps:

[0041] 4-phenyl ether phenylboronic acid (0.96g, 4.5mmol), 1,3,5-triiodo-2,4,6-trimethylbenzene (0.5g, 1mmol), anhydrous potassium carbonate (0.83g, 6mmol), Pd(PPh3)4 (0.2g, 0.174mmol) were weighed and placed in a reaction flask, and then 1,4-dioxane (120ml) and distilled water (20ml) were added to the reaction flask, stirred until the reactants were completely dissolved, vacuumed, nitrogen protected, constant temperature 90℃, magnetic stirring reaction for 24h. After the reaction was completed, it was naturally cooled to room temperature, extracted three times with 30ml of dichloromethane, and then poured into saturated brine for washing, the organic phase was collected, an appropriate amount of anhydrous sodium sulfate was added, dried for 3h, filtered and dried, and the crude product was purified by column chromatography, and the eluent was petroleum ether / dichloromethane (5 / 1). Finally, dichloromethane and anhydrous ethanol were used for recrystallization to obtain 0.468 g of white needle-shaped crystals with a yield of 75.1%.

[0042] The prepared compound 1 H NMR spectrum Figure 1 As shown, the structural formula is as follows:

[0043]

[0044] Experimental study 1: AIE performance experiment of the compound prepared in Example 1.

[0045] The compound is highly soluble in most organic solvents but insoluble in water. In order to detect the AIE activity of the compound prepared in Example 1, deionized water was added to its THF solution to prepare THF-H2O solutions with different concentrations ranging from 0% to 90% by volume, and the PL spectra of the mixtures were recorded as follows: Figure 3 As shown. Figure 3 As shown in (b), the compound TDPO emits weak fluorescence in pure THF solution. As the water content increases, the fluorescence intensity gradually increases. When the water content is as high as 90%, the emission intensity reaches the highest, which is 34 times higher than the original value. The compound emits almost no fluorescence in pure solution, while the fluorescence intensity is enhanced in mixed solution. This fluorescence feature is a typical aggregation-induced emission (AIE) effect.

[0046] Experimental study 2: Solvent effect of TDPO

[0047] The emission behavior of the test compound TDPO in different solvents was used to select a suitable solvent. Figure 4As shown, the emission behavior of 100 μM compound in different solvents is different, and 8 solvents (1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, dichloromethane, ethylene glycol, xylene) were selected. The probe has a good dispersion state in DMF, and the probe shows a high fluorescence intensity in DMF. DMF solvent was selected in the following tests.

[0048] Experimental study 3: TDPO as a fluorescent probe interacting with different amino acids

[0049] The compound with a concentration of 10 μM was prepared with DMF as solvent. The excitation wavelength was determined to be 300 nm by UV test, and the slit width was 5:5. 100 μM of different amino acids were added to the sensor, including tryptophan (Trp), leucine (Leu), serine (Ser), methionine (Met), alanine (Ala), citrulline (Ccp), threonine (Thr), proline (Pro), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), valine (Val), and cysteine ​​(Cys). The fluorescence emission spectrum was measured. The fluorescence spectrum is shown in the figure below. Figure 5 The results showed that the fluorescence intensity was significantly enhanced only when tryptophan was added, while no significant fluorescence response was observed when other amino acids were added under the same conditions.

[0050] Experimental study 4: TDPO as a fluorescent probe interacting with tryptophan at different concentration gradients

[0051] The probe response range was determined by adding tryptophan of different concentration gradients (0, 10, 20, ... 120 μM) to the probe solution with a concentration of 10 μM. Figure 6 As shown in FIG. 1 , as the concentration of amino acids increases, the fluorescence intensity gradually increases. The linear relationship diagram of the probe detecting tryptophan is obtained by linear fitting (see FIG. Figure 7 According to the formula LOD = 3σ / k (where σ is the standard deviation when tryptophan is not added, and K is the slope of the linear relationship between the fluorescence intensity of the sensor and the concentration of tryptophan), the detection limit can be calculated to be 0.091 μM (as shown in FIG. Fig. 9 The detection limit is low, indicating that the probe can well detect trace amounts of tryptophan. We use the Stern-Volmer equation I0 / I=K SV [Q]+1, (where I0 is the initial fluorescence intensity of the fluorescent probe, I is the fluorescence intensity after adding tryptophan, and [Q] is the molar concentration of tryptophan.) Fig.10 As shown, the sensitivity (K SV ) is 31060M -1

[0052] Experimental study 5: Competition experiment of TDPO as a fluorescent probe for amino acids

[0053] In order to determine whether the compounds have specific and selective recognition for tryptophan, the fluorescence spectra of leucine (Leu), serine (Ser), methionine (Met), alanine (Ala), citrulline (Ccp), threonine (Thr), proline (Pro), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), valine (Val), cysteine ​​(Cys), and tryptophan (Trp) were studied. Figure 8 shown.

[0054] The results show that: Figure 8 As shown, 20 μM of other amino acids and Trp (5 times each) were alternately added to the probe solution, and the fluorescence intensity was measured. It was clearly observed that the addition of other amino acids had little effect on the fluorescence intensity, but the addition of tryptophan significantly increased the fluorescence intensity. It can be inferred that the probe can achieve efficient and rapid detection while specifically recognizing Trp.

[0055] Embodiment 2:

[0056] The preparation method of the diphenyl ether derivative with AEE characteristics, SDPO, of the present invention is carried out in the following steps:

[0057] 4-phenyl ether phenylboronic acid (1.44g, 6.75mmol), 1,3,5-tribromotoluene (0.5g, 1.5mmol), anhydrous potassium carbonate (1.24g, 9mmol), Pd(PPh3)4 (0.2g, 0.174mmol) were weighed and placed in a reaction flask, and then 1,4-dioxane (120ml) and distilled water (20ml) were added to the reaction flask, stirred until the reactants were completely dissolved, vacuumed, nitrogen protected, constant temperature 90℃, and magnetic stirring reacted for 24h. After the reaction was completed, it was naturally cooled to room temperature, extracted three times with 30ml of dichloromethane, and then poured into saturated brine to wash clean, the organic phase was collected, an appropriate amount of anhydrous sodium sulfate was added, dried for 3h, filtered and dried, and the crude product was purified by column chromatography, and the eluent was petroleum ether / dichloromethane (5 / 1). Finally, dichloromethane and anhydrous ethanol were used for recrystallization to obtain 0.7g of white needle-shaped crystals. Yield: 78.8%.

[0058] The prepared compound 1 H NMR spectrum Figure 1 As shown, the structural formula is as follows:

[0059]

[0060] Experimental Study 6: AEE performance experiment of the compound prepared in Example 2.

[0061] In order to detect the optical properties of the compound prepared in Example 2, deionized water was added to its THF solution to prepare THF-H2O solutions with different concentrations ranging from 0% to 90% by volume, and the PL spectra of the mixtures were recorded as follows: Fig.13 As shown. Fig.13 As shown in (b), the compound SDPO emits weak fluorescence in pure THF solution. As the water content increases, the fluorescence intensity gradually increases. When the water content is as high as 80%, the emission intensity reaches the highest, which is 4 times higher than the original value. The compound can emit fluorescence in pure solution, and the fluorescence intensity is enhanced in mixed solution. This fluorescence feature is a typical aggregation-induced enhanced emission (AEE) effect.

[0062] Experimental study 7: Solvent effect of SDPO

[0063] The emission behavior of the test compound SDPO in different solvents was used to select a suitable solvent. Fig.14 As shown, the emission behaviors of 100 μM compounds in different solvents are different, and 8 solvents (1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, dichloromethane, ethylene glycol, and xylene) were selected. The probe has a good dispersion state in DMF, and the probe exhibits a higher fluorescence intensity in DMF. DMF solvent was selected in the subsequent tests.

[0064] Experimental study 8: SDPO as a fluorescent probe interacting with different amino acids

[0065] The compound with a concentration of 10 μM was prepared with THF as the solvent. The excitation wavelength was determined to be 300 nm by UV test, and the slit width was 5:5. 100 μM of different amino acids were added to the sensor, including tryptophan (Trp), leucine (Leu), serine (Ser), methionine (Met), alanine (Ala), citrulline (Ccp), threonine (Thr), proline (Pro), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), valine (Val), and cysteine ​​(Cys). Fluorescence emission spectrum was measured. The fluorescence spectrum is shown in the figure below. Fig.15 The results showed that the fluorescence intensity was significantly enhanced only when tryptophan was added, while no significant fluorescence response was observed when other amino acids were added under the same conditions.

[0066] Experimental study 9: TDPO as a fluorescent probe interacting with tryptophan at different concentration gradients

[0067] The probe response range was determined by adding tryptophan of different concentration gradients (0, 10, 20, ... 120 μM) to the probe solution with a concentration of 10 μM. Fig.16 As shown in FIG. 1 , as the concentration of amino acids increases, the fluorescence intensity gradually increases. The linear relationship diagram of the probe detecting tryptophan is obtained by linear fitting (see FIG. Fig.17 As shown in Figure 2, according to the formula LOD = 3σ / k, the detection limit is 0.14μM. The detection limit at the nanoscale is low, indicating that the probe can well detect trace amounts of tryptophan. We use the Stern-Volmer equation I0 / I = K SV [Q]+1, such as Fig. 20 As shown, the sensitivity (K SV ) is 13380M -1

[0068] Experimental study 10: Competition experiment of TDPO as a fluorescent probe for amino acids

[0069] In order to determine whether the compounds have specific and selective recognition for tryptophan, the fluorescence spectra of leucine (Leu), serine (Ser), methionine (Met), alanine (Ala), citrulline (Ccp), threonine (Thr), proline (Pro), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), valine (Val), cysteine ​​(Cys), and tryptophan (Trp) were studied. Figure 8 shown.

[0070] The results show that: Figure 8 As shown, 20 μM of other amino acids and Trp (5 times each) were alternately added to the probe solution, and the fluorescence intensity was measured. It was clearly observed that the addition of other amino acids had little effect on the fluorescence intensity, but the addition of tryptophan significantly increased the fluorescence intensity. It can be inferred that the probe can achieve efficient and rapid detection while specifically recognizing Trp.

[0071] Embodiment 3:

[0072] The preparation method of the diphenyl ether derivative with AEE characteristics, SDPO, of the present invention is carried out in the following steps:

[0073] 4-phenyl ether phenylboronic acid (1.54g, 7.2mmol), 1,3,5-tribromobenzene (0.5g, 1.6mmol), anhydrous potassium carbonate (1.32g, 9.6mmol), Pd(PPh3)4 (0.2g, 0.174mmol) were weighed and placed in a reaction flask, and then 1,4-dioxane (120ml) and distilled water (20ml) were added to the reaction flask, stirred until the reactants were completely dissolved, vacuumed, nitrogen protected, constant temperature 90℃, and magnetic stirring reacted for 24h. After the reaction was completed, it was naturally cooled to room temperature, extracted three times with 30ml of dichloromethane, and then poured into saturated brine to wash clean, the organic phase was collected, an appropriate amount of anhydrous sodium sulfate was added, dried for 3h, filtered and dried, and the crude product was purified by column chromatography, and the eluent was petroleum ether / dichloromethane (5 / 1). Finally, dichloromethane and anhydrous ethanol were used for recrystallization to obtain 0.755g of white needle-shaped crystals. Yield: 81.7%.

[0074] The prepared compound 1 H NMR spectrum Figure 1 As shown, the structural formula is as follows:

[0075]

[0076] Experimental study 11: AIE performance experiment of the compound prepared in Example 3.

[0077] In order to detect whether the compound prepared in Example 3 has AIE properties, deionized water was added to its THF solution to prepare THF-H2O solutions with different concentrations ranging from 0% to 90% by volume, and the PL spectra of the mixtures were recorded as follows: Fig.23 As shown. Fig.23 As shown in (b), the compound NDPO emits weak fluorescence in pure THF solution. As the water content increases, the fluorescence intensity gradually increases. When the water content is as high as 90%, the emission intensity reaches the highest, which is 29 times higher than the original value. The compound emits almost no fluorescence in pure solution, while the fluorescence intensity is enhanced in mixed solution. This fluorescence feature is a typical aggregation-induced emission (AIE) effect.

[0078] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A diphenyl ether derivative having AIE properties, characterized in that: Its structure is shown in formula III: Formula III.

2. A method for preparing a diphenyl ether derivative having AIE properties as claimed in claim 1, characterized in that: The synthetic route is as follows: 。 3. A diphenyl ether derivative having AEE characteristics, characterized in that: Its general structural formula is shown in Formula II; Formula II.

4. A method for preparing a diphenyl ether derivative having AEE characteristics as claimed in claim 3, characterized in that: The synthetic route is as follows: 。

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