Colorimetric molecular probe and preparation method and application thereof

Abstract

The present invention relates to the technical field of copper ion detection, and discloses a colorimetric molecular probe, a preparation method thereof, and an application thereof. The structural formula of the colorimetric molecular probe is as follows. The colorimetric molecular probe provided by the present invention is a Schiff base structure, which can be synthesized by condensing dicyanoisophorone with p-hydroxybenzaldehyde, then using the Duff reaction to add an aldehyde group, and then condensing with p-toluenesulfonyl hydrazide. The colorimetric molecular probe provided by the present invention only requires four steps to synthesize the target product, and the post-processing process is relatively simple; the probe can realize the sensing detection of copper ions through ultraviolet spectroscopy and color changes, and has good selectivity, strong anti-interference ability, low detection limit, and can be used for reversible detection of copper ions.

Description

Technical Field

[0001] The invention relates to the technical field of copper ion detection, and in particular to a colorimetric molecular probe and a preparation method and application thereof. Background Art

[0002] Copper is an essential trace element in nature, present in various environments and living organisms, and is an essential element for basic physiological processes in humans and animals. It plays a key role in many enzyme catalysis and redox reactions and is necessary for health and normal function. Appropriate amounts of copper ions can effectively participate in the biosynthesis and cofactors of a variety of enzymes, such as tyrosinase, cytochrome oxidase, etc., which helps to effectively inhibit the proliferation and growth of human bacteria and enhance the body's immune function. However, insufficient intake can lead to bone deformities, pigmentation, abnormal cholesterol metabolism and serious neurological problems. Of course, excessive intake of copper in the environment can cause its long-term insoluble precipitation in the body, causing harm to organisms, leading to cirrhosis, movement disorders, sensory nerve disorders and many other diseases. Therefore, a balanced intake of copper in the body is essential to maintain good health. According to the World Health Organization (WHO) report, the average daily copper intake of adults should not exceed 10-12 mg / day. Therefore, the development of new methods for detecting copper compounds is of great research significance to protect ecosystems and humans from copper ions.

[0003] At present, the widely used methods for metal ion detection include inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectroscopy (AAS), electrochemical methods and voltammetry. However, these methods require expensive and complex instruments and skilled professionals in the process of detecting metal ions. The analysis process is cumbersome, the detection and analysis time is long, and the analysis cost is high, which are the disadvantages of the above analysis methods. This obviously does not meet the requirements of rapid on-site evaluation required by modern environmental monitoring. In recent years, molecular probe detection and analysis methods based on chemical sensors have attracted special attention. They are simple to operate, have high selectivity and sensitivity, low detection limit, fast analysis time, are non-destructive to samples, and can also perform convenient visual qualitative identification.

[0004] In recent years, many molecular probes that recognize copper ions have been reported, but the synthesis steps of these probes are cumbersome, and their selectivity and sensitivity are low, resulting in reduced applicability of the probes.

[0005] Therefore, there is an urgent need to develop a colorimetric molecular probe with good selectivity, high sensitivity and simple synthesis. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art and to provide a colorimetric molecular probe for specific recognition of copper ions and a preparation method and application thereof.

[0007] In order to achieve the above object, the first aspect of the present invention provides a colorimetric molecular probe, wherein the structural formula of the colorimetric molecular probe is as follows:

[0008]

[0009] The second aspect of the present invention provides a method for preparing the colorimetric molecular probe according to the first aspect, wherein the method comprises the following steps:

[0010] (1) subjecting dicyanoisophorone to a first reaction with p-hydroxybenzaldehyde to obtain compound II;

[0011] (2) subjecting compound II to a second reaction with hexamethylenetetramine to obtain compound III;

[0012] (3) allowing compound III to undergo a third reaction with p-toluenesulfonyl hydrazide to obtain the colorimetric molecular probe.

[0013] The third aspect of the present invention provides the use of the colorimetric molecular probe described in the first aspect or the colorimetric molecular probe obtained by the method described in the second aspect in copper ion sensing detection.

[0014] Through the above technical solution, the beneficial technical effects achieved by the present invention are as follows:

[0015] 1) The synthesis of the colorimetric molecular probe provided by the present invention can be completed in only four steps, the raw materials are economical and easily available, and the post-processing process is relatively simple.

[0016] 2) The present invention realizes that the probe can sense and detect copper ions through both ultraviolet spectrum change and color change, and has good selectivity, strong anti-interference ability to other metal ions, and low detection limit (detection limit is 7.19×10 -8 M), and Na 2 EDTA enables reversible detection of copper ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the infrared spectrum of the colorimetric molecular probe H-6 synthesized in Example 1 of the present invention.

[0018] Figure 2 This is a high-resolution mass spectrum of the colorimetric molecular probe H-6 synthesized in Example 1 of the present invention.

[0019] Figure 3 This is the 1HNMR spectrum of the colorimetric molecular probe H-6 synthesized in Example 1 of the present invention.

[0020] Figure 4 This is the nuclear magnetic resonance hydrogen spectrum 13CNMR graph of the colorimetric molecular probe H-6 synthesized in Example 1 of the present invention.

[0021] Figure 5 :(a) is the probe H-6 (concentration of 1×10 -5 mol / L) in DMF / H 2 O (6:4, v / v) for different metal ions. (b) is the selectivity of probe H-6 and probe H-6+Cu 2+ UV spectrum.

[0022] Figure 6 :(a) is the normalized UV spectra of probe H-6 and different ions, (b) is the ratio of UV absorption intensity (A) of probe H-6 and different ions 486 / A 436 ).

[0023] Figure 7 It is the influence of coexisting ions on the determination of copper ions.

[0024] Figure 8 :(a) is the probe H-6 (concentration of 1×10 -5 mol / L) in DMF / H 2 O (6:4, v / v) with different concentrations of Cu 2+ UV spectrum of A 486 / A 436 The UV absorption intensity and Cu 2+ The linear relationship between the concentration of 1% and 2% was (0-15 μM).

[0025] Fig. 9 This is the reversible change diagram of probe H-6's recognition of copper ions.

[0026] Fig.10 Probe H-6+Cu 2+ High-resolution mass spectrum.

[0027] Fig.11 Probe H-6+Cu 2+ NMR titration results.

[0028] Fig.12 For probe H-6 and Cu 2+ The combination mode. DETAILED DESCRIPTION

[0029] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] The first aspect of the present invention provides a colorimetric molecular probe H-6, wherein the structural formula of the colorimetric molecular probe is as follows:

[0031]

[0032] The colorimetric molecular probe H-6 provided by the present invention has a Schiff base structure, which can be obtained by condensing dicyanoisophorone with p-hydroxybenzaldehyde, adding an aldehyde group by using the Duff reaction, and then condensing with p-toluenesulfonyl hydrazide to generate the target probe H-6. 2+ Complexation will cause changes in the UV spectrum and solution color, which can be used to specifically identify copper ions.

[0033] The color change of the colorimetric molecular probe solution can be observed under sunlight, and it is a colorimetric molecular probe with a colorimetric sensing function. Compared with other probes that only detect metal ions quantitatively through ultraviolet spectroscopy, the colorimetric molecular probe H-6 can also perform ultraviolet ratio quantitative detection through ultraviolet spectroscopy. Based on its specificity, the probe can be used as a specific indicator for the presence of copper ions in colorimetric detection aqueous solutions, and can perform real-time qualitative and quantitative visual colorimetric detection. And more importantly, the sensing detection of copper ions can achieve rapid, reversible and repeated detection. Therefore, the probe provided by the present invention is a simple, rapid and sensitive copper ion colorimetric detection reagent, which has broad application prospects in the field of water sample environmental detection.

[0034] The second aspect of the present invention provides a method for preparing the colorimetric molecular probe according to the first aspect, wherein the method comprises the following steps:

[0035] (1) subjecting dicyanoisophorone to a first reaction with p-hydroxybenzaldehyde to obtain compound II;

[0036] (2) subjecting compound II to a second reaction with hexamethylenetetramine to obtain compound III;

[0037] (3) allowing compound III to undergo a third reaction with p-toluenesulfonyl hydrazide to obtain the colorimetric molecular probe.

[0038] During the preparation of the colorimetric molecular probe of the present invention, the reaction that occurs is:

[0039]

[0040] In the present invention, compound II can be obtained by separating and purifying the product obtained by spinning the reaction liquid of the first reaction to dryness through column chromatography; compound III can be obtained by cooling the reaction liquid of the second reaction to room temperature and then slowly pouring it into ice water, filtering the obtained crude product and separating and purifying it through column chromatography; the target compound, i.e., the colorimetric molecular probe H-6, can be obtained by cooling the reaction liquid of the third reaction to room temperature to produce a large amount of precipitation, filtering, and purifying the filter cake by beating with ethanol.

[0041] In some embodiments of the present invention, the molar ratio of dicyanoisophorone to p-hydroxybenzaldehyde is 1:1.03.

[0042] In some embodiments of the present invention, the catalyst for the first reaction is piperidine.

[0043] In some embodiments of the present invention, the solvent of the first reaction is anhydrous ethanol.

[0044] In some embodiments of the present invention, the conditions of the first reaction include: reflux; reaction time is 5-6h.

[0045] In some embodiments of the present invention, in step (2), the molar ratio of compound II to hexamethylenetetramine is 1:1.

[0046] In some embodiments of the present invention, the solvent for the second reaction is trifluoroacetic acid.

[0047] In some embodiments of the present invention, the conditions of the second reaction include: reflux; reaction time is 6-8h.

[0048] In some embodiments of the present invention, in step (3), the molar ratio of compound III to p-toluenesulfonyl hydrazide is 1:1.1.

[0049] In some embodiments of the present invention, the solvent of the third reaction is super dry ethanol.

[0050] In some embodiments of the present invention, the conditions of the third reaction include: reflux; reaction time is 4-8h.

[0051] In some embodiments of the present invention, in steps (1)-(3), the reaction is monitored by TLC.

[0052] In some embodiments of the present invention, the dicyanoisophorone is obtained by the reaction of isophorone and malononitrile, and can also be commercially available.

[0053] The third aspect of the present invention provides the use of the colorimetric molecular probe described in the first aspect or the colorimetric molecular probe obtained by the method described in the second aspect in copper ion sensing detection. The colorimetric molecular probe of the present invention can quickly and intuitively detect harmful metal ions through color changes.

[0054] In some embodiments of the present invention, the colorimetric molecular probe is used for sensing and detecting copper ions in a water environment system.

[0055] In some embodiments of the present invention, the sensing detection is ultraviolet ratio detection, visual qualitative detection and copper ion reversible detection.

[0056] In some embodiments of the present invention, the sensing assay is a selective sensing assay and a competitive sensing assay.

[0057] The present invention will be described in detail below through examples.

[0058] In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if no manufacturer is specified, are conventional products that can be obtained through commercial channels.

[0059] Example 1

[0060] (1) Synthesis of dicyanoisophorone (Compound I):

[0061]

[0062] 6.91g isophorone (50.1mmol) was dissolved in 100mL anhydrous ethanol, and then 3.84g malononitrile (60.1mmol) and 0.3mL piperidine (3.0mmol) were added in sequence. The mixture was heated and stirred at reflux under a nitrogen atmosphere, and the isophorone was completely reacted by TLC monitoring for about 6h. The reaction was stopped, and the reaction was allowed to stand and cool to room temperature, and the reaction solution was then poured into 200mL ice water to generate a large amount of off-white precipitates. The solid obtained by suction filtration was separated and purified by column chromatography to obtain 7.23g of white crystalline solid, and product compound I was obtained.

[0063] (2) Synthesis of Compound II:

[0064]

[0065] p-Hydroxybenzaldehyde (10.2 mmol) was added to a round-bottom flask containing 40 mL of anhydrous ethanol, followed by the addition of 10.5 mmol of compound I and 3.0 mmol of piperidine, and the mixture was heated to reflux until the TLC spot plate detected the disappearance of the raw material spot, and the reaction was stopped for about 5 h. The product obtained after the reaction solution was spin-dried was separated and purified by column chromatography to obtain the product compound II.

[0066] (3) Synthesis of Compound III:

[0067]

[0068] Compound II (1.5 mmol) was dissolved in 10 mL trifluoroacetic acid, and then 1 equivalent of hexamethylenetetramine was added and heated to reflux. TLC monitored the reaction to be complete and the reaction was stopped for about 6 h. The reaction solution was cooled to room temperature and then slowly poured into 50 mL ice water, and the crude product obtained by filtration was separated and purified by column chromatography to obtain compound III.

[0069] (4) Synthesis of target compound H-6:

[0070]

[0071] 100 mg of compound III (0.2 mmol) was added to a round-bottom flask containing 20 mL of ultra-dry ethanol, followed by 1.1 equivalents of p-toluenesulfonyl hydrazide, and heated under reflux for 4 h until the reaction was complete as monitored by TLC. The reaction was stopped, and the reaction solution was cooled to room temperature. A large amount of precipitate was generated, which was filtered, and the filter cake was purified by ethanol pulping to obtain the target compound H-6.

[0072] The target compound H-6 obtained in this example is a new compound that has not been reported before. After purification, it was characterized by infrared spectroscopy (IR), high resolution mass spectrometry (HRMS), and nuclear magnetic resonance spectroscopy (1H NMR, 13C NMR). The infrared spectrum of probe H-6 is shown in Figure 1 As shown, wave number 3209.60cm -1 The peak is attributed to the stretching vibration of NH. The cyano stretching vibration peak of dicyanoisophorone is 2223.96 cm -1 Peak at 1602.87cm -1 and 1581.14cm -1 The peaks at are respectively attributed to the stretching vibration peaks of C=O and C=N. The high-resolution mass spectrum of probe H-6 is shown in Figure 2 As shown in the figure, the molecular ion peak with a mass-to-charge ratio of 487.1807 was measured, and the theoretical value of the H-6 mass-to-nuclear ratio was 487.1804. The relative error between the two is 6.15e -7 , within the error tolerance range. Probe H-6 H NMR spectrum 1 H NMR Figure 3 As shown, 1H NMR (500MHz, DMSO) δ11.58 (s, 1H), 10.71 (s, 1H), 8.16 (s, 1H), 7.78 (d, J = 5.1 Hz, 1H), 7.76 (s, 2H), 7.68 (dd, J = 8.7, 1.9 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.24 (dd, J = 37.9, 16.1 Hz, 2H), 6.90 (d, J = 8.6 Hz, 1H), 6.83 (s, 1H), 2.61 (s, 2H), 2.54 (s, 2H), 2.36 (s, 3H), 1.01 (s, 6H). Probe H-6 hydrogen nuclear magnetic resonance spectrum 13 C NMR Figure 4 As shown, 13C NMR (101 MHz, DMSO) δ 170.78, 158.37, 156.82, 145.65, 144.13, 137.92, 136.39, 130.89, 130.27, 128.29, 128.14, 127.87, 127.64, 122.39, 120.01, 117.44, 114.48, 113.69, 75.93, 42.79, 38.65, 32.14, 27.91, 21.48.

[0073] Application Example 1

[0074] The colorimetric molecular probe H-6 synthesized in Example 1 that can recognize copper ions was dissolved in DMF to a concentration of 1×10 -5 mol / L DMF / H 2 O (6:4, v / v) solution was used to detect its UV selectivity for different metal ions.

[0075] Figure 5 The probe H-6 solution has a UV absorption peak at 436nm. 3+ , K + , Fe 3+ , Fe 2+ 、Na + Cr 3+ , Ca 2+ 、Zn 2+ Mg 2+ , Mn 2+ , Ba 2+ , Hg 2+ , Pb 2+ 、Cd 2+ 、Co 2+ 、Ni 2+ 、Ag + and Cu 2+After adding various ions, the ultraviolet peak at 436nm undergoes complex changes, some blue shifts, some red shifts, and the absorbance increases and decreases, indicating that the probe H-6 interacts with various metal ions. 2+ After that, the UV spectrum of the probe H-6 solution changed most significantly. The original absorption peak at 436 nm disappeared, and a new absorption peak appeared at 486 nm.

[0076] In order to better analyze the changes in the UV peak movement of other ions and probe H-6 solution, we performed a normalized analysis.

[0077] like Figure 6 As shown in (a), except Cu 2+ 17 ions other than Al 3+ , K + , Fe 3+ , Fe 2+ 、Na + Cr 3+ , Ca 2+ 、Zn 2+ Mg 2 + , Mn 2+ , Ba 2+ , Hg 2+ , Pb 2+ 、Cd 2+ 、Co 2+ 、Ni 2+ 、Ag + ), so that the maximum UV absorption peak of the probe H-6 solution moves within the range of 422-455nm, and the corresponding solution color is yellow-green, while Cu 2+ The probe solution was red-shifted by 50 nm and the solution appeared pink, which showed that the probe H-6 could identify Cu by observing the solution color with the naked eye. 2+ In addition, the absorbance ratio at 436 nm and 486 nm after the addition of ions to probe H-6 was also analyzed. Figure 6 (b) shows that when Cu was added to the probe H-6 solution 2+ The ratio of UV absorption intensity (A 486 / A 436 ) increased, which was obviously different from the ratio UV absorption intensity of probe H-6 itself (A 486 / A 436 ), while other ions did not cause significant changes in the ratiometric UV absorption intensity.

[0078] Application Example 2

[0079] To further determine whether the coexistence of other metal ions affects the selectivity for copper ions, competitive UV spectroscopy experiments were performed.

[0080] Add Cu to the probe H-6 solution 2+ ions other than Cu 2+ , compared with adding Cu 2+ The ratio of the UV absorption intensity of the solution before and after (A 486 / A 436 ) value. Figure 7 As shown, except Al 3+ Cr 3+ , Fe 3+ The ratio of the ultraviolet absorption intensity (A 486 / A 436 ) values ​​increased to around 1.6 and would not interfere with the recognition of Cu by probe H-6. 2+ Although probe H-6 is sensitive to Al 3+ Cr 3+ , Fe 3+ Cu cannot be identified in the presence 2+ However, probe H-6 can still effectively detect Cu in the presence of most other ions. 2+ .

[0081] Ultraviolet spectrum analysis showed that the coexistence with other metal ions did not affect the probe's recognition of copper ions.

[0082] Application Example 3

[0083] In order to study whether the probe H-6 can 2+ For quantitative detection, we performed UV concentration titration experiments on the probe.

[0084] Probe H-6 (concentration 1×10 -5 mol / L) in DMF / H 2 CuO (6 / 4, v / v) solution was gradually added 2+ , and record the ratio of UV absorption intensity (A 486 / A 436 ) value. Figure 8 As shown in (a), the 436nm absorption peak of probe H-6 itself increases with the Cu 2+ As the concentration increases, it slowly red-shifts to 486nm. 2+ After reaching 20 μM, the absorbance at 486 nm reached a plateau. An isosbestic point appeared at 455 nm, indicating that the probe H-6 and Cu 2+ The absorbance ratio at 486nm and 436nm is similar to that of Cu 2+ (0-15μM) has a good linear relationship (R 2 =0.9922). According to the formula, the probe H-6 has a strong affinity for Cu 2+The detection limit was 7.19×10 -8 The above results show that the probe H-6 can detect Cu in a certain range through UV spectroscopy. 2+ Perform quantitative testing.

[0085] Application Example 4

[0086] Fig. 9 The concentration is 1×10 -5 mol / L probe H-6 solution. The same concentration (1×10 -5 mol / L) copper ions, the UV spectrum red-shifted, and the absorption peak at 436nm red-shifted to 500nm. Then, 1×10 -5 mol / L Na 2 The violet spectrum of EDTA solution was restored, and 1×10 -5 mol / L Cu 2 + Then its UV spectrum red-shifted again, proving that the colorimetric probe has good reversibility.

[0087] Test Case

[0088] In order to verify the interaction of probe H-6 with Cu 2+ The coordination of H-6+Cu 2+ High-resolution mass spectrometry of solutions. Fig.10 Probe H-6+Cu 2+ The high-resolution mass spectrum shows a molecular ion peak with a mass-to-charge ratio of 548.0949, which can be attributed to [H-6+Cu 2+ -H + ] + The theoretical value of the mass-to-nuclear ratio is 548.0943, and the relative error between the two is 1.09×e -6 , within the allowable error range, further illustrates that the probe H-6 and Cu 2+ The complexation ratio is 1:1.

[0089] In order to further understand the relationship between probe H-6 and Cu 2+ Complexation mode, NMR titration experiments were carried out. Fig.11 As shown, the proton signal H on the amide bond at 11.58 ppm a and the hydroxyl proton signal H at 10.71 ppm b , as Cu 2+ With the addition of Cu 2+ This causes the hydroxyl group to deprotonate and coordinate with the oxygen atom.

[0090] Combining the above complexation ratio determination, high-resolution mass spectrometry characterization and nuclear magnetic resonance titration results, we speculated that the probe H-6 and Cu 2+ Modes of interaction, such as Fig.12 As shown. 2+ The probe was complexed with the oxygen atom on the hydroxyl group of H-6 and the nitrogen atom on the carbon-nitrogen double bond, so that the probe was added to Cu 2+ After that, the UV spectrum and its color change significantly.

[0091] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A colorimetric molecular probe, characterized in that The structural formula of the colorimetric molecular probe is as follows:

2. A method for preparing a colorimetric molecular probe according to claim 1, characterized in that: The method comprises the following steps: (1) subjecting dicyanoisophorone to a first reaction with p-hydroxybenzaldehyde to obtain compound II; (2) subjecting compound II to a second reaction with hexamethylenetetramine to obtain compound III; (3) subjecting compound III to a third reaction with p-toluenesulfonyl hydrazide to obtain the colorimetric molecular probe; 3. The method according to claim 2, wherein: In step (1), the molar ratio of dicyanoisophorone to p-hydroxybenzaldehyde is 1:1.03; and / or, the catalyst for the first reaction is piperidine; And / or, the solvent of the first reaction is anhydrous ethanol; And / or, the conditions of the first reaction include: reflux; reaction time is 5-6h.

4. The method according to claim 2 or 3, wherein: In step (2), the molar ratio of compound II to hexamethylenetetramine is 1:1; And / or, the solvent of the second reaction is trifluoroacetic acid; And / or, the conditions of the second reaction include: reflux; The reaction time is 6-8h.

5. The method according to any one of claims 2 to 3, wherein: In step (3), the molar ratio of compound III to p-toluenesulfonyl hydrazide is 1:1.1; And / or, the solvent of the third reaction is super dry ethanol; And / or, the conditions of the third reaction include: reflux; reaction time is 4-8h.

6. The method according to any one of claims 2 to 3, wherein: In steps (1)-(3), the reaction was monitored by TLC; And / or, the dicyanoisophorone is obtained by reacting isophorone with malononitrile.

7. Use of the colorimetric molecular probe according to claim 1 or the colorimetric molecular probe obtained according to any one of claims 2 to 6 in copper ion sensing detection.

8. The use according to claim 7, wherein: The colorimetric molecular probe is used for sensing and detecting copper ions in a water environment system.

9. The use according to claim 7 or 8, wherein: The sensing detection includes ultraviolet ratio detection, visual qualitative detection and copper ion reversible detection.

10. The use according to claim 7 or 8, wherein: The sensing assay is a selective sensing assay and a competitive sensing assay.

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