Fluorescent probe containing triphenylamine-thiophene-aryl Schiff base structure, preparation method of fluorescent probe and application of fluorescent probe in copper ion detection

Abstract

The invention discloses a fluorescent probe containing a triphenylamine-thiophene-aryl Schiff base structure, a preparation method of the fluorescent probe and application of the fluorescent probe in copper ion detection. The structural formula of the fluorescent probe is as shown in a formula I in the specification. The fluorescent probe (formula I) with copper ion detection capability is prepared by taking triphenylamine thiophene aldehyde as a fluorophore and carrying out Schiff base reaction on the triphenylamine thiophene aldehyde and an aniline derivative. The probe induces fluorescence group release through copper ion mediated carbon-nitrogen double bond cracking, and realizes copper ion detection based on a significantly enhanced fluorescence signal. The probe is short in response time to copper ions in a solution, good in selectivity, high in sensitivity and easy to prepare and store. Formula I

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of metal ion detection, and particularly relates to a fluorescent probe containing a triphenylamine-thiophene-aryl Schiff base structure, a preparation method thereof and application thereof in copper ion detection. BACKGROUND

[0002] Copper is the third most abundant transition metal element in the human body next to iron and zinc, and plays an important role in various physiological processes such as redox reactions, signal transduction, hematopoiesis, and affects the functions of the central nervous system, the immune system and the endocrine system. The normal concentration range of blood copper is 100-150 µg / L (15.7-23.6 µM). When excessive exogenous copper is absorbed or endogenous copper metabolism is disturbed, copper ion homeostasis is imbalanced, which will cause various diseases such as anemia, coronary heart disease, Menkes syndrome, Wilson syndrome and Alzheimer's disease.

[0003] Among the many copper ion detection methods, fluorescent probe technology is attracting more and more attention due to its high sensitivity, good selectivity, wide dynamic response range, and economic and simple advantages. The reported copper ion fluorescent probes can be divided into two categories: molecular recognition type and reaction type. The molecular recognition type mainly recognizes the measured substance through weak interactions such as hydrogen bonds, coordination bonds, and electrostatic interactions; the reaction type is mainly based on the specific chemical reaction between the probe molecule and the measured substance, which induces the change of the probe fluorescence signal (such as activation, quenching or emission wavelength shift), thereby realizing the recognition of the measured substance. The former needs to introduce N, O, S and other heteroatoms into the structure of the probe for Cu 2+ coordination, and the latter needs to introduce Cu 2+ catalytic reaction sites for the breaking or formation of chemical bonds. The reaction type probe has better selectivity than the molecular recognition type probe and performs more outstandingly in complex sample environments. However, the current reported reaction type copper ion probes are mainly designed based on the reaction principles of copper ion-mediated 2-pyridine carboxylate bond hydrolysis, benzyl ether bond cleavage, and spiro lactam ring opening, and the reaction types are relatively single, and the detection performance is uneven. Therefore, it is urgent to explore new reaction sites of Cu 2+ probe recognition, and enrich the Cu 2+ probe molecule library. SUMMARY

[0004] The application provides a copper ion fluorescent probe containing a triphenylamine-thiophene-aryl Schiff base structure, and the structural formula is shown as formula I: Formula I, wherein R1 is a hydroxyl group, and R2, R3, R4 and R5 are independently selected from hydrogen, a hydrocarbon group, an amino group, a hydroxyl group, an alkoxy group, , ; or R5 is hydroxyl, R1, R2, R3, R4 are independently selected from hydrogen, hydrocarbyl, amino, hydroxyl, alkoxy, , ; or R1, R5 is hydroxyl, R2, R3, R4 are independently selected from hydrogen, hydrocarbyl, amino, hydroxyl, alkoxy, , .

[0005] The application also provides a preparation method of the above-mentioned copper ion fluorescent probe, a synthesis route and reaction steps of which are as follows: , Step a1: 4-boronic acid triphenylamine, 5-bromo thiophene-2-formaldehyde, a catalyst, a base are placed in a reaction container, a reaction solvent is added, and a compound DPTC is prepared by refluxing reaction; Step b1: the compound DPTC, R1~R5 substituted aniline, boron trifluoride diethyl ether are placed in a reaction container, a reaction solvent is added, and formula I is prepared by refluxing reaction; or, , Step a2: 4-boronic acid triphenylamine, 2, 5-dibromo thiophene, 5-aldehyde-2-thiophene boronic acid, a catalyst, a base are placed in a reaction container, a reaction solvent is added, and a compound DPBC is prepared by refluxing reaction; Step b2: the compound DPBC, R1~R5 substituted aniline, boron trifluoride diethyl ether are placed in a reaction container, a reaction solvent is added, and formula I is prepared by refluxing reaction.

[0006] In the preparation method of the above-mentioned copper ion fluorescent probe, the catalyst in steps a1 and a2 is palladium tetraphenylphosphine, the base is sodium carbonate aqueous solution, and the solvent is ethylene glycol dimethyl ether.

[0007] In the preparation method of the above-mentioned copper ion fluorescent probe, the amount of substance of 5-bromo thiophene-2-formaldehyde in step a1 is 1~3 times that of 4-boronic acid triphenylamine, the amount of substance of the catalyst is 0.05~0.5 times that of 4-boronic acid triphenylamine, and the amount of substance of the base is 1.5~4 times that of 4-boronic acid triphenylamine.

[0008] In the preparation method of the above-mentioned copper ion fluorescent probe, the amount of substance of R1~R5 substituted aniline in step b1 is 1~3 times that of the compound DPTC, and the amount of substance of boron trifluoride diethyl ether is 0.1~0.7 times that of the compound DPTC.

[0009] In the preparation method of the copper ion fluorescent probe, the amount of substance of the 2,5-dibromothiophene in step a2 is 1-3 times that of the 4-aniline boronic acid, the amount of substance of the 5-aldehyde-2-thiophene boronic acid is 1-3 times that of the 4-aniline boronic acid, the amount of substance of the catalyst is 0.05-0.5 times that of the 4-aniline boronic acid, and the amount of substance of the base is 1.5-4 times that of the 4-aniline boronic acid.

[0010] In the preparation method of the copper ion fluorescent probe, the amount of substance of the R1-R5 substituted aniline in step b2 is 1-3 times that of the compound DPBC, and the amount of substance of the boron trifluoride diethyl ether is 0.1-0.7 times that of the compound DPBC.

[0011] The application further provides application of the copper ion fluorescent probe in detection of copper ions.

[0012] The application further provides application of the copper ion fluorescent probe in preparation of a copper ion detection kit.

[0013] In the application, triphenylamine-thiophene aldehyde is used as a fluorophore, and an ortho-hydroxy aniline derivative is introduced as a copper ion recognition site to prepare a fluorescent probe with excellent Cu 2+ recognition ability as shown in Formula I. For example, the probe with hydroxyl as R1, hydrogen as R2, R3 and R5, and methoxy as R4 can be selectively 'turned on' to emit yellow fluorescence with a wavelength of 545 nm, and the process is hardly disturbed by other metal ions; the response time is short, and the response signal reaches equilibrium within 0.5 min; the sensitivity is high, and 10 eq. Cu 2+ can produce about 98 times of fluorescence enhancement, the linear range is 2 µM-15 µM, and the detection limit is as low as 1.76 nM. 2+ It is worth mentioning that most of the reported Schiff base probes are based on coordination mechanism to recognize Cu 2+ . 1 H NMR and HPLC have confirmed that the fluorescent probe disclosed in the application is based on the breakage reaction of Cu 2+ participation to realize copper ion detection, specifically, Cu 2+ catalyzes the cleavage of the carbon-nitrogen double bond in the probe structure, so that the fluorophore triphenylamine-thiophene aldehyde can be released, thereby obtaining a significantly enhanced fluorescence signal. In addition, the probe for detecting copper ions has the advantages of simple operation, few steps in the preparation process of the probe, and low economic cost. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the compound DPTC prepared in Example 1; Figure 2 is the nuclear magnetic resonance carbon spectrum of the compound DPTC prepared in Example 1. Figure 3 is the high resolution mass spectrum of compound DPTC prepared in Example 1; Figure 4 is the nuclear magnetic resonance hydrogen spectrum of compound DPBC prepared in Example 2; Figure 5 is the high resolution mass spectrum of compound DPBC prepared in Example 2; Figure 6 is the nuclear magnetic resonance hydrogen spectrum of fluorescent probe DPTC-6 prepared in Example 3; Figure 7 is the nuclear magnetic resonance carbon spectrum of fluorescent probe DPTC-6 prepared in Example 3; Figure 8 is the high resolution mass spectrum of fluorescent probe DPTC-6 prepared in Example 3; Figure 9 is the response rate of fluorescent probe DPTC-6 to copper ions in Example 4; Figure 10 is the selectivity of fluorescent probe DPTC-6 to copper ions in Example 5; Figure 11 is the fluorescence titration graph and detection limit of fluorescent probe DPTC-6 to copper ions in Example 6; Figure 12 is the H NMR change of fluorescent probe DPTC-6 before and after the action of copper ions in Example 7; 1 Figure 13 is the HPLC change of fluorescent probe DPTC-6 before and after the action of copper ions in Example 7; Figure 14 is the molecular structure of triphenylamine-thiophene-aryl Schiff base fluorescent probe with different substitutions in Comparative Example 1; Figure 15 is the fluorescence response of triphenylamine-thiophene-aryl Schiff base fluorescent probe with different substitutions to copper ions in Comparative Example 1, the abscissa "1~7" respectively refers to fluorescent probes DPTC1~7, and the abscissa "OAP" refers to fluorescent probe DPTC-OAP. DETAILED DESCRIPTION

[0015] The application will be further described in conjunction with examples, but not limit the scope of the application. The experimental methods used in the application are conventional methods unless otherwise specified. The materials, reagents and the like used in the experiment can be obtained from commercial channels unless otherwise specified. All reagents are commercially available analytical pure, chemical pure or chromatographic pure.

[0016] Example 1 - Synthesis of compound DPTC ​A round bottom flask was charged with 4-boronic acid triphenylamine (1.734 g, 6 mmol), 5-bromothiophene-2-carboxaldehyde (1.149 g, 6 mmol), tetrakis(triphenylphosphine)palladium (0.208 g, 0.18 mmol), 6 mL aqueous sodium carbonate (2 M) and 50 mL ethylene glycol dimethyl ether as the reaction solvent. The reaction was stirred at reflux for 6-7 h. The reaction progress was monitored by thin layer chromatography (TLC). After the reaction was complete, the reaction was cooled to room temperature, filtered to remove the tetrakis(triphenylphosphine)palladium and the filtrate was distilled under reduced pressure to remove the solvent. The crude product was dissolved in ethyl acetate and washed with water three times. The crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether to give the pure product as an orange yellow solid in 85% yield. The product was identified as compound DPTC by proton nuclear magnetic resonance spectroscopy (1H NMR), carbon nuclear magnetic resonance spectroscopy (13C NMR) and high resolution mass spectrometry (HRMS). 1 H NMR), carbon nuclear magnetic resonance spectroscopy (13C NMR) and high resolution mass spectrometry (HRMS). 13 C NMR), carbon nuclear magnetic resonance spectroscopy (13C NMR) and high resolution mass spectrometry (HRMS). Figures 1-3 1 H NMR (400 MHz, DMSO- d 6) δ 9.86 (s, 1H), 7.99 (s, 1H), 7.68(s, 2H), 7.59 (s, 1H), 7.34 (s, 4H), 7.09 (s, 6H), 6.95 (d, J = 5.9 Hz, 2H). 13 CNMR (101 MHz, DMSO- d 6) δ 184.1, 153.4, 149.0, 141.9, 141.9, 141.3, 139.9,130.2, 127.9, 126.00, 124.6, 124.4, 122.2. HRMS (ESI) Calcd for [C 23 H 17 NOS + H] + 356.1109; Found: 356.1105.

[0017] Example 2 - Synthesis of compound DPBC ​A round bottom flask was charged with 4-boronic acid triphenylamine (1.734 g, 6 mmol), 2,5-dibromothiophene (1.451 g, 6 mmol), 5-formyl-2-thiophene boronic acid (0.936 g, 6 mmol), tetrakis(triphenylphosphine)palladium (0.208 g, 0.18 mmol), 6 mL of aqueous sodium carbonate solution (2 M), and 50 mL of ethylene glycol dimethyl ether as the reaction solvent. The reaction was stirred at reflux for 6-7 h. The reaction progress was monitored by thin layer chromatography (TLC). After the reaction was complete, the reaction was cooled to room temperature, filtered to remove the tetrakis(triphenylphosphine)palladium, and the filtrate was distilled under reduced pressure to remove the solvent. The crude product was dissolved in ethyl acetate and washed with water three times. The crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether to give the pure product as an orange solid in 70% yield. The product was identified as compound DPBC by nuclear magnetic resonance hydrogen spectrum (1H NMR), carbon spectrum (13C NMR), and high resolution mass spectrum (HRMS). 1 13 Figure 4 Figure 5 1 H NMR (400 MHz, DMSO- d 6) δ9.88 (s, 1H), 7.99 (d, J = 4.0 Hz, 1H), 7.60 (dd, J = 9.1, 2.8 Hz, 2H), 7.52 (d, J = 4.0 Hz, 1H), 7.46 (d, J = 3.9 Hz, 1H), 7.34 (dd, J = 8.3, 7.5 Hz, 2H), 7.18 –7.05 (m, 3H), 6.98 (d, J = 8.7 Hz, 1H). HRMS (ESI) Calcd for [C 27 H 19 NOS2+ H] + 438.0986; Found: 438.0979.

[0018] Example 3 - Synthesis of fluorescent probe DPTC-6 ​​​​The compound DPTC prepared in Example 1 (1 mmol, 0.355 g), 2-aminophenol (1.5 mmol, 0.164 g), boron trifluoride diethyl ether (40 uL) were placed in a round bottom flask and 10 mL of ethanol was added as the reaction solvent and stirred at reflux for 1-2 h. The reaction progress was monitored by thin layer chromatography (TLC) and after complete reaction, it was cooled to room temperature. The solid was filtered and dissolved in a small amount of dichloromethane in ethanol and then filtered to remove the molecular sieves. The filtrate was directly evaporated under vacuum to get the crude product which was purified by silica gel chromatography using ethyl acetate / pet ether to get an orange yellow solid in 72% yield. The product was identified as compound DPTC-6 by proton nuclear magnetic resonance spectroscopy (1H NMR), carbon nuclear magnetic resonance spectroscopy (13C NMR), high resolution mass spectrometry (HRMS). 1 13 Figures 6-8 1 1H NMR (400 MHz, DMSO- d 6) δ 8.91 (d, J = 88.2 Hz, 1H), 7.65 (dd, J = 12.5,6.3 Hz, 2H), 7.49 (d, J = 3.8 Hz, 1H), 7.36 (t, J = 7.9 Hz, 2H), 7.17 – 7.06 (m,4H), 7.06 – 6.96 (m, 1H), 6.90 (dd, J = 8.0, 1.2 Hz, 1H), 6.83 (td, J = 7.6, 1.3Hz, 1H). 13 13C NMR (101 MHz, DMSO- d 6) δ 153.5, 151.2, 148.1, 148.1, 147.1, 141.5,138.3, 134.9, 130.2, 130.2, 127.4, 127.3, 125.5, 125.1, 124.2, 123.8, 122.9,120.9, 120.0, 116.6. HRMS (ESI) Calcd for [C 29 H 22 N2OS + H] + 447.1526; Found:447.1524.

[0019] Example 4 - Response rate of fluorescent probe DPTC-6 to copper ions ​​​The fluorescent probe DPTC-6 was dissolved in chromatographically pure DMSO to form a mother liquor with a concentration of 10 mM. The mother liquor was diluted into an aqueous solution containing 50% acetonitrile, and then transferred into a quartz cuvette with an optical path of 1 cm. 10 eq. Cu 2+ was added, and the fluorescence spectrum was scanned at time nodes of 0 min, 0.5 min, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc. The results show that the fluorescence signal of the system is significantly enhanced within 10 s, and the fluorescence intensity basically reaches equilibrium after half a minute Figure 9 . The naked eye also observed that the system changed from no fluorescence to strong orange-yellow fluorescence emission. This confirms that DPTC-6 can respond rapidly to copper ions in solution, and is expected to achieve rapid analysis of Cu 2+ .

[0020] Example 5 - Selectivity of the fluorescent probe DPTC-6 to copper ions Various salts were accurately weighed and dissolved in deionized water to form corresponding ion mother liquor with a concentration of 0.1 M, including Cu 2+ , Al 3+ , Mn 2+ , Pb 2+ , Na + , K + , Mg 2+ , Ca 2+ , Ba 2+ , Zn 2+ , Ni 2+ , Li + , Ag + , Hg 2+ , Sr 2+ , Fe 2+ , Cd 2+ , Co 2+ , Cr 2+ , F − , Cl − , Br − , OH − , HS − , SO3 2− , SO4 2− , CO3 2− , HCO3 − , NO2 − , NO3 − , CH3COO− , SCN − , PO4 2− . The probe stock solution prepared in Example 4 was diluted into water solution containing 50% acetonitrile, and different kinds of 10 eq. anions or cations were added, then the fluorescence spectrum was scanned. As shown in Figure 10 , only Cu 2+ can cause significant change of fluorescence spectrum, and the fluorescence intensity at 545 nm is greatly enhanced, while the test solution after adding common anions or cations has similar fluorescence spectrum to the probe itself, which indicates that the probe can respond selectively to Cu 2+ .

[0021] Example 6 - Fluorescence titration experiment of the fluorescent probe DPTC-6 to copper ions The probe stock solution prepared in Example 4 was diluted into water solution containing 50% acetonitrile, and gradient concentrations of Cu 2+ were added into the test solution, then the fluorescence spectrum was scanned. The results show that Figure 11 , with the increasing concentration of Cu 2+ , the fluorescence intensity of the system is continuously enhanced. The fluorescence intensity and the concentration of Cu 2+ show a good linear relationship in the range of 2 µM-15 µM, and accordingly the detection limit of the probe DPTC-6 is calculated to be 1.76 nM (DL=3 σ / k ), indicating that the probe DPTC-6 has high sensitivity.

[0022] Example 7 - Propose and verification of the detection mechanism We speculate that the selective response of the probe to Cu 2+ is due to the Cu 2+ mediated cleavage of C=N double bond, and at the same time the triphenylamine-thiophene aldehyde capable of emitting strong fluorescence is generated. First, the mechanism is verified by nuclear magnetic resonance hydrogen spectrum. The probe DPTC-6 is dissolved in DMSO- d 6, and the nuclear magnetic resonance hydrogen spectrum is collected, and then the nuclear magnetic resonance hydrogen spectrum is collected again after adding Cu 2+ . As shown in Figure 12 , a new peak appears near δ=9.8 ppm in the mixed system, which is close to the position of the aldehyde proton of the fluorophore triphenylamine-thiophene aldehyde DPTC; at the same time, compared with the 1 H NMR of the probe itself DPTC-6, it is found that the characteristic peaks (–HC=N-, -OH) at δ=8.5~9.0 ppm have disappeared. The above experimental phenomena can be explained as that the probe Cu 2+ induces the cleavage of C=N double bond in DPTC-6, and generates the aldehyde compound DPTC and 2-amino-4-methoxy phenol.

[0023] Next, HPLC was used to further verify the hypothesized reaction mechanism. DPTC, DPTC-6, and DPTC-6+Cu were then used. 2+ Inject samples separately, using CH3CN / H2O = 4 / 1 as the mobile phase, at a flow rate of 1 mL / min, and collect chromatograms. For example... Figure 13 As shown, the retention time of DPTC was 9.64 min, and the retention time of DPTC-6 was 15.97 min. Meanwhile, the retention time of DPTC-6 and Cu... 2+ The mixed system eluted at t = 9.65 min and showed no peak at t = 15.97 min. This experimental result also confirms the relationship between DPTC-6 and Cu. 2+ The reaction produced the aldehyde compound DPTC, and the conversion was nearly complete.

[0024] In summary, both 1H NMR spectroscopy and HPLC confirmed the Cu content of our proposed method. 2+ The response mechanism mediating C=N splitting. To our knowledge, this response mechanism has not been reported before.

[0025] Comparative Example 1 – Comparison of the detection capabilities of triphenylamine-thiophene-aryl Schiff base fluorescent probes with different substituents for copper ions The copper ion fluorescent probe shown in Formula 1 requires a hydroxyl group at the ortho position of the C=N double bond, meaning that at least one of the substituents R1 and R5 must be a hydroxyl group. Therefore, the contrast probes DPTC-OAP and DPTC1~7 were synthesized. Figure 14 The probe was first dissolved in chromatographically pure DMSO to prepare a 10 mM stock solution, which was then diluted to an aqueous solution containing 50% acetonitrile. 10 eq. of copper ions were added, and the fluorescence intensity was tested. Figure 15 As shown, when the hydroxyl group is located at the para or meta position of the C=N double bond, or when the ortho position of the C=N double bond does not contain a hydroxyl group (substituted with a methoxy, mercapto, or hydrogen group), the addition of copper ions does not cause a significant increase in fluorescence intensity. Besides requiring a hydroxyl group at the ortho position of the C=N double bond, the remaining substituents on the benzene ring must also be electron-donating groups. When electron-withdrawing groups are present, such as in the probe DPTC-7, copper ions also do not cause a significant increase in fluorescence intensity.

[0026] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A copper ion fluorescent probe containing triphenylamine-thiophene-aryl Schiff base structure, and a structural formula of the copper ion fluorescent probe is shown as formula I: Formula I, wherein, R1is hydroxyl, R2, R3, R4, R5are independently selected from the group consisting of hydrogen, hydrocarbyl, amino, hydroxyl, alkoxy, , ; or R5is hydroxyl, R1, R2, R3, R4are independently selected from the group consisting of hydrogen, hydrocarbyl, amino, hydroxyl, alkoxy, , ; or R1, R5are hydroxyl, R2, R3, R4are independently selected from the group consisting of hydrogen, hydrocarbyl, amino, hydroxyl, alkoxy, , .

2. The copper ion fluorescent probe according to claim 1, characterized in that: R1is hydroxyl, R2, R3, R4, R5are independently selected from alkoxy, , .

3. The copper ion fluorescent probe according to claim 1, characterized in that: R5 is hydroxy, R1, R2, R3, R4 are independently selected from alkoxy, , .

4. The copper ion fluorescent probe according to claim 1, characterized in that: R1, R5 are hydroxyl, R2, R3, R4 are independently selected from alkoxy, , . 5.A preparation method of the copper ion fluorescent probe according to any one of claims 1-4, comprising the following synthesis route and reaction steps: ， Step a1: placing 4-boronic acid triphenylamine, 5-bromothiophene-2-formaldehyde, a catalyst, and a base in a reaction container, adding a reaction solvent, and refluxing to prepare compound DPTC; Step b1: placing compound DPTC, R1-R5 substituted aniline, and boron trifluoride diethyl ether in a reaction container, adding a reaction solvent, and refluxing to prepare formula I; Or, ， Step a2: placing 4-boronic acid triphenylamine, 2, 5-dibromothiophene, 5-aldehyde-2-thiophene boronic acid, a catalyst, and a base in a reaction container, adding a reaction solvent, and refluxing to prepare compound DPBC; Step b2: placing compound DPBC, R1-R5 substituted aniline, and boron trifluoride diethyl ether in a reaction container, adding a reaction solvent, and refluxing to prepare formula I.

6. The method for preparing the copper ion fluorescent probe according to claim 5, characterized in that: In steps a1 and a2, the catalyst is tetrakis triphenylphosphine palladium, the base is sodium carbonate aqueous solution, and the solvent is ethylene glycol dimethyl ether. 7.The copper ion fluorescent probe according to claims 1-4 is applied to detecting copper ions. 8.The copper ion fluorescent probe according to claims 1-4 is applied to preparing a copper ion detection kit.

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