A biphenylpyridine salt compound, its preparation method and its application as a fluorescent probe in detecting microcystin

Abstract

The present invention belongs to the technical field of ecotoxicology research, and in particular to a biphenyl pyridine salt compound and its preparation method and its application as a fluorescent probe in detecting microcystin (MC-LR). The biphenyl pyridine salt compound provided by the present invention has a structure shown in Formula I. The biphenyl pyridine salt compound of the structure shown in Formula I provided by the present invention can, under the condition of the presence of microcystin, rely on the linear structure in the molecular structure to be interlocked with the cyclic structure of microcystin through hydrogen bond interaction, thereby changing the spectral properties of the biphenyl pyridine salt compound of the structure shown in Formula I itself, showing a strong fluorescence response, thereby realizing the detection of MC-LR in the aqueous phase by changes in fluorescence intensity; At the same time, the biphenyl pyridine salt compound 18BDQ of the structure shown in Formula I provided by the present invention can realize specific detection of MC-LR, and the operating method is simple, with broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of ecotoxicology research, and particularly relates to a biphenyl pyridine salt compound, a preparation method thereof, and an application of the biphenyl pyridine salt compound as a fluorescent probe in detecting microcystin (MC-LR). Background Art

[0002] Cyanobacterial blooms caused by eutrophication have severely polluted water bodies and aquatic life. Among cyanobacterial toxins, microcystins (MC-LR) are the most toxic. Microcystins are widely distributed in lakes and threaten the environment on which humans depend. For example, ingestion of MC-LR can cause liver disease, reproductive disorders, and neurotoxicity.

[0003] The existing technology for detecting microcystin (MC-LR) is mainly through high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA), but the above two methods have the defects of complex procedures and expensive instruments, which limits their practical application. Summary of the Invention

[0004] The present invention aims to provide a biphenylpyridinium salt compound, a preparation method thereof, and use thereof as a fluorescent probe in detecting microcystin (MC-LR). The biphenylpyridinium salt compound provided by the present invention can be used as a fluorescent probe to achieve specific detection of microcystin (MC-LR) in water bodies, and the method is simple.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a biphenylpyridine salt compound having a structure shown in Formula I:

[0007]

[0008] The present invention provides a method for preparing a biphenylpyridine salt compound having a structure represented by Formula I described in the above technical solution, comprising the following steps:

[0009] 4-methylpyridine and 1-iodooctadecane are subjected to a salt-forming reaction in an organic solvent to obtain an intermediate compound having a structure shown in Formula II;

[0010]

[0011] The intermediate compound with the structure shown in formula II and p-phenylbenzaldehyde are subjected to a condensation reaction in an organic solvent to obtain the biphenylpyridinium salt compound with the structure shown in formula I.

[0012] Preferably, the molar ratio of the 4-methylpyridine to the iodooctadecane is 1:(1.0-1.25).

[0013] Preferably, the salt-forming reaction is carried out under heating reflux conditions, the heating reflux temperature is 55-60° C., and the holding time of the salt-forming reaction is 4-6 hours; the salt-forming reaction is carried out in a protective gas atmosphere.

[0014] Preferably, the molar ratio of the intermediate compound of the structure represented by Formula II to the p-phenylbenzaldehyde is 1:(1.0-1.25).

[0015] Preferably, the condensation reaction is carried out under heating reflux conditions, the heating reflux temperature is 80-90° C., and the insulation time of the condensation reaction is 12-15 hours; the condensation reaction is carried out in a protective gas atmosphere.

[0016] Preferably, after the condensation reaction, a condensation reaction liquid is obtained, and the method further comprises: removing the organic solvent from the condensation reaction liquid and then purifying it by column chromatography, and drying the obtained purified eluent to obtain a pure product of the biphenylpyridinium salt compound with the structure shown in Formula I; the eluent used in the column chromatography purification is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 40 to 50:1.

[0017] The present invention provides the use of the biphenyl pyridinium salt compound described in the above technical solution or the biphenyl pyridinium salt compound prepared by the preparation method described in the above technical solution as a fluorescent probe in the detection of microcystin (MC-LR).

[0018] Preferably, the application is to detect the concentration of microcystin (MC-LR) in the aqueous phase.

[0019] The present invention provides a biphenylpyridine salt compound having a structure shown in Formula I. The biphenylpyridinium salt compound of the structure represented by Formula I (abbreviated as 18BDQ) provided by the present invention has a weak fluorescence signal in an aqueous environment. However, in the presence of microcystin (MC-LR), the biphenylpyridinium salt compound of the structure represented by Formula I can, by virtue of the linear structure of the molecular structure, intercalate with the cyclic peptide structure of microcystin (MC-LR) through hydrogen bonding interactions, thereby changing the spectral properties of the biphenylpyridinium salt compound of the structure represented by Formula I itself, exhibiting a strong fluorescence response with a response multiple exceeding 20 times, thereby enabling detection of MC-LR in the aqueous phase through changes in fluorescence intensity. At the same time, the recognition mechanism of the biphenylpyridinium salt compound of the structure represented by Formula I provided by the present invention for MC-LR lies in the "intersection" of the cyclic structure of MC-LR with the linear structure of the biphenylpyridinium salt compound of the structure represented by Formula I. Since other organic molecules in water cannot achieve this characteristic "intersection", the biphenylpyridinium salt compound of the structure represented by Formula I provided by the present invention, 18BDQ, can achieve specific detection of MC-LR, and the operation method is simple, with broad application prospects.

[0020] The present invention provides a method for preparing a biphenylpyridine salt compound having a structure represented by Formula I as described in the above technical solution, comprising the following steps: mixing 4-methylpyridine, iodooctadecane, and an organic solvent to undergo a salt-forming reaction to obtain an intermediate compound having a structure represented by Formula II; and mixing the intermediate compound having a structure represented by Formula II, p-phenylbenzaldehyde, and an organic solvent to undergo a condensation reaction to obtain a biphenylpyridine salt compound having a structure represented by Formula I. The preparation method provided by the present invention obtains a biphenylpyridine salt compound having a linear structure in a monosomal structure through salt-forming and condensation reactions. The preparation method provided by the present invention is simple and suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the nuclear magnetic resonance (NMR) spectrum of compound 18BDQ prepared in an example of the present invention;

[0022] Figure 2 This is a high-resolution mass spectrum of compound 18BDQ prepared in an example of the present invention;

[0023] Figure 3 This is the fluorescence spectrum of compound 18BDQ prepared in an example of the present invention under different MC-LR concentration conditions;

[0024] Figure 4 for Figure 3 Fitting curve diagram of the change trend of the corresponding fluorescence emission peak under different MC-LR concentration conditions;

[0025] Figure 5This is a fluorescence spectrum of the compound 18BDQ prepared in an example of the present invention in a selectivity experiment in an aqueous environment;

[0026] Figure 6 for Figure 5 The histogram of the highest emission peak under different ion conditions;

[0027] Figure 7 This is a reaction flow chart for preparing compound 18BDQ in an example of the present invention. DETAILED DESCRIPTION

[0028] The present invention provides a biphenylpyridine salt compound having a structure shown in Formula I:

[0029]

[0030]

[0031] The present invention provides a biphenylpyridinium salt compound having a structure represented by Formula I. The compound structure comprises a linear structure and a biphenylpyridinium structure, wherein the biphenylpyridinium structure serves as a luminescent group. The linear structure can intercalate with the cyclic peptide structure of microcystin (MC-LR) through hydrogen bonding interactions to alter the spectral properties of the biphenylpyridinium salt compound having the structure represented by Formula I, exhibiting a strong fluorescence response with a response multiple exceeding 20 times, thereby enabling detection of MC-LR in an aqueous phase through changes in fluorescence intensity.

[0032] The present invention provides a method for preparing a biphenylpyridine salt compound having a structure represented by Formula I described in the above technical solution, comprising the following steps:

[0033] 4-methylpyridine and 1-iodooctadecane are subjected to a salt-forming reaction in an organic solvent to obtain an intermediate compound having a structure shown in Formula II;

[0034]

[0035] The intermediate compound with the structure shown in formula II and p-phenylbenzaldehyde are subjected to a condensation reaction in an organic solvent to obtain the biphenylpyridinium salt compound with the structure shown in formula I.

[0036] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0037] In the present invention, 4-methylpyridine and iodooctadecane are subjected to a salt-forming reaction in an organic solvent (hereinafter referred to as the first organic solvent) to obtain an intermediate compound with a structure shown in formula II.

[0038] In the present invention, the first organic solvent is preferably ethanol. In the present invention, ethanol is used as the first organic solvent. Ethanol has good solubility for iodooctadecane, which is conducive to the smooth progress of the salt-forming reaction.

[0039] In the present invention, the molar ratio of the 4-methylpyridine to the iodooctadecane is preferably 1:(1.0-1.25), more preferably 1:(1.1-1.15).

[0040] In the present invention, the ratio of the volume of the first organic solvent to the amount of iodooctadecane is preferably (40-60) mL:10 mmol, more preferably (45-55) mL:10 mmol.

[0041] In the present invention, the salt-forming reaction is preferably carried out under heating reflux conditions, and the heating reflux temperature is preferably 55-60°C, more preferably 60°C.

[0042] In the present invention, the holding time of the salt-forming reaction is preferably 4 to 6 hours, more preferably 4.5 to 5 hours.

[0043] In the present invention, the salt-forming reaction is preferably carried out in a protective gas atmosphere, and the protective gas is preferably nitrogen.

[0044] In the present invention, after the salt-forming reaction, a salt-forming reaction liquid is obtained. In the present invention, the salt-forming reaction liquid is preferably washed after removing the solvent to obtain a crude intermediate compound having the structure shown in Formula II, which is directly used in the condensation reaction without purifying the crude intermediate compound having the structure shown in Formula II. In the present invention, the specific embodiment of removing the solvent is preferably evaporation under reduced pressure. In the present invention, the solvent used for the washing is preferably ethanol. In the present invention, the washing is preferably performed multiple times with a small amount of ethanol.

[0045] After obtaining the intermediate compound of the structure shown in Formula II, the present invention conducts a condensation reaction between the intermediate compound of the structure shown in Formula II and p-phenylbenzaldehyde in an organic solvent (hereinafter referred to as the second organic solvent) to obtain the biphenylpyridinium salt compound of the structure shown in Formula I.

[0046] In the present invention, the second organic solvent is preferably ethanol. In the present invention, ethanol is used as the second organic solvent. Ethanol has good solubility for the intermediate compound, which is conducive to the smooth progress of the condensation reaction.

[0047] In the present invention, the molar ratio of the intermediate compound of the structure represented by Formula II to the p-phenylbenzaldehyde is preferably 1:(1.0-1.25), and more preferably 1:(1.1-1.15).

[0048] In the present invention, the ratio of the volume of the second organic solvent to the amount of substance of the intermediate compound is preferably (40-60) mL:10 mmol, more preferably (45-55) mL:10 mmol.

[0049] In the present invention, the condensation reaction is preferably carried out under heating reflux conditions, and the heating reflux temperature is preferably 80-90°C, more preferably 90°C.

[0050] In the present invention, the holding time of the condensation reaction is preferably 12 to 15 hours, more preferably 12 hours.

[0051] In the present invention, the condensation reaction is preferably carried out in a protective gas atmosphere, and the protective gas is preferably nitrogen.

[0052] In the present invention, a condensation reaction liquid is obtained after the condensation reaction. The present invention preferably further comprises: removing the organic solvent from the condensation reaction liquid and then performing column chromatography purification, and drying the obtained purified eluent to obtain a pure product of the biphenylpyridine salt compound of the structure described in Formula I. In the present invention, the specific implementation method of removing the solvent is preferably evaporation under reduced pressure. In the present invention, the eluent used for the column chromatography purification is preferably a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane and methanol is preferably 40 to 50:1, more preferably 50:1. In the present invention, the drying temperature is preferably 40°C.

[0053] The present invention provides the use of a biphenylpyridinium salt compound of the structure shown in Formula I described in the above technical solution or a biphenylpyridinium salt compound of the structure shown in Formula I prepared by the preparation method described in the above technical solution as a fluorescent probe in the detection of microcystin (MC-LR).

[0054] In the present invention, the application is preferably: detecting the concentration of the microcystin (MC-LR) in the aqueous phase.

[0055] In the present invention, the specific application method of the biphenyl pyridinium salt compound of the structure shown in Formula I for detecting the concentration of the microcystin (MC-LR) in the aqueous phase preferably includes the following steps:

[0056] Mixing the biphenyl pyridinium salt compound of the structure shown in Formula I and the aqueous solution of microcystin (MC-LR) to obtain a test solution;

[0057] Measuring the fluorescence spectrum of the solution to be tested, obtaining the fluorescence emission peak intensity of microcystin (MC-LR), and obtaining a measured fluorescence emission peak intensity value;

[0058] The measured fluorescence absorption peak intensity value is substituted into the microcystin (MC-LR) concentration-fluorescence emission peak intensity value standard curve or standard equation to obtain the concentration of the microcystin aqueous solution.

[0059] In the present invention, a biphenylpyridinium salt compound having a structure shown in Formula I and an aqueous solution of microcystin (MC-LR) are mixed to obtain a test solution. In the present invention, the aqueous solution of microcystin (MC-LR) is preferably 0 to 110 μM.

[0060] After obtaining the test solution, the present invention measures the fluorescence spectrum of the test solution to obtain the fluorescence emission peak intensity of microcystin (MC-LR) and obtain the measured fluorescence emission peak intensity value. The present invention preferably measures the fluorescence spectrum of the test solution under conditions where the excitation wavelength is preferably 380 nm.

[0061] After obtaining the measured fluorescence emission peak intensity value, the present invention inserts the measured fluorescence emission peak intensity value into the microcystin (MC-LR) concentration-fluorescence emission peak intensity value standard curve or standard equation to obtain the concentration of the microcystin (MC-LR) aqueous solution.

[0062] In the present invention, the method for obtaining the microcystin (MC-LR) concentration-fluorescence emission peak intensity value standard curve preferably comprises the following steps:

[0063] A series of aqueous solutions of microcystin (MC-LR) with known concentrations are used to obtain a series of test solutions with known microcystin (MC-LR) concentrations according to the above method for obtaining the test solution;

[0064] measuring the fluorescence spectra of the series of test solutions with known microcystin (MC-LR) concentrations to obtain a series of fluorescence emission peak intensity values;

[0065] A standard curve of microcystin (MC-LR) concentration-fluorescence emission peak intensity value was drawn using the microcystin (MC-LR) concentration of the aqueous phase solution with a series of known concentrations of microcystin (MC-LR) as the independent variable and the corresponding series of fluorescence emission peak intensity values as the dependent variable.

[0066] In one or more specific embodiments of the present invention, the molar concentration of the aqueous solution of microcystin (MC-LR) with a series of known concentrations is specifically 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, and 110 μM.

[0067] In the present invention, the method for obtaining the standard equation of microcystin (MC-LR) concentration-fluorescence emission peak intensity value preferably includes the following steps: linearly simulating the standard curve of microcystin (MC-LR) concentration-fluorescence emission peak intensity value to obtain the standard equation of microcystin (MC-LR) concentration-fluorescence emission peak intensity value.

[0068] In the present invention, the standard equation is shown in Formula 1:

[0069] y=25.5520x+65.9486Equation 1, R 2 =0.9656;

[0070] In formula 1, y is the fluorescence peak and x is the concentration of microcystin (MC-LR).

[0071] The linear structure of the biphenyl pyridinium salts provided by the present invention integrates with the macrocyclic structure of MC-LR to achieve specific detection of microcystin (MC-LR). This detection is achieved by altering the spectral properties of the biphenyl pyridinium salts themselves in the presence of microcystin (MC-LR).

[0072] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0073] Example 1

[0074] according to Figure 7 Preparation as shown in the reaction flow chart: iodooctadecane (3.84 g, 10.0 mmol) was dissolved in ethanol (EtOH, 60 mL) and stirred to dissolve. 4-methylpyridine (1.0 g, 10.0 mmol) was added to the mixture, and the mixture was refluxed at 60°C for 6 h under nitrogen as a protective gas to produce a light yellow system. After the reaction, the solvent was evaporated under reduced pressure to obtain a light yellow solid, which was washed several times with a small amount of ethanol to obtain an intermediate compound with the structure shown in Formula II. Due to its high purity, the intermediate can be directly used in the next reaction.

[0075] The intermediate (1.05 g, 3.0 mmol) and p-phenylbenzaldehyde (0.6 g, 3.0 mmol) were dissolved in ethanol (EtOH, 60 mL) and refluxed at 90 degrees Celsius for 12 hours under nitrogen protection, causing the reaction system to turn reddish-brown. After completion of the reaction, ethanol, the preferred solvent of the present invention, was evaporated to dryness under negative pressure. The product was purified by column chromatography using dichloromethane and methanol in a 50:1 volume ratio as eluents. The resulting reddish-brown solid was a fluorescent probe having the structure of Formula I (abbreviated as 18BDQ). The yield was 35%.

[0076] The nuclear magnetic resonance data of the biphenylpyridinium salt compound of the structure shown in Formula 1 prepared in this embodiment are:

[0077] H NMR(500MHz,DMSO-d6)δ8.95(dd,J=42.4,6.5Hz,2H),8.29(d,J

[0078] =6.4Hz,2H),8.11(d,J=16.3Hz,1H),7.89–7.74(m,4H),7.71–7.56(m,2H),7.51(t,J=7.6Hz,2H),7.42( t,J=7.4Hz,2H),4.51(q,J=6.7,6.2Hz,2H),1.91(p,J=7.7Hz,2H),1.22(s,30H),0.84(t,J=6.8Hz,3H).

[0079] Figure 1 is the nuclear magnetic resonance (NMR) spectrum of compound 18BDQ, Figure 2 This is the high-resolution mass spectrum of compound 18BDQ. Figure 1 and Figure 2 It can be seen that the target product prepared in this example is indeed compound 18BDQ.

[0080] Test Example 1

[0081] The solvation effect of compound 18BDQ prepared in Example 1 was measured as follows:

[0082] Compound 18BDQ was dissolved in DMSO to obtain 5 mL of a 2 mM 18BDQ stock solution. 25 μL of the 18BDQ stock solution was then added to five identical 5 mL volumetric flasks. The solution was diluted to volume with DMF, acetonitrile, PBS buffer (pH 7.2-7.4), methanol, DMSO, and purified water, respectively. Fluorescence detection was then performed under an excitation wavelength of 380 nm.

[0083] The maximum emission peaks of compound 18BDQ in the organic phase and in the aqueous phase both appear around 5 nm.

[0084] Test Example 2

[0085] Compound 18BDQ prepared in Example 1 was dissolved in DMSO to obtain 5 mL of a 2 mM 18BDQ stock solution. MC-LR was dissolved in pure water to obtain a 0.5 mM MC-LR stock solution. 10 μL of the 2 mM 18BDQ stock solution was added to 21 identical 5 mL centrifuge tubes, and 0 μL, 20 μL, 40 μL, and 80-440 μL of the MC-LR stock solution were added, respectively. The volume was then adjusted to 2 mL with pure water to obtain test solutions. The concentrations of compound 18BDQ and MC-LR in the test solutions were 10 μM and 0-110 μM, respectively. Fluorescence detection was performed on each test solution at an excitation wavelength of 380 nm.

[0086] Figure 3 The fluorescence spectra of compound 18BDQ in water environment at different MC-LR concentrations are shown in Figure 2. Figure 3 It can be seen that an emission peak appears at 525 nm, and as the concentration of MC-LR in the test solution increases, the fluorescence intensity gradually increases, which shows that the compound 18BDQ provided by the invention can realize the detection of MC-LR in water environment.

[0087] Figure 4 for Figure 3 The changing trend diagram and fitting curve diagram of the corresponding highest emission peak under different microcystin (MC-LR) concentration conditions.

[0088] Test Example 3

[0089] The selectivity of compound 18BDQ prepared in Example 1 was analyzed as follows:

[0090] Depend on Figure 3 When the concentration of 18BDQ in the test solution was 10 μM, the fluorescence intensity increased with the increase of MC-LR concentration. The equilibrium was reached when the MC-LR concentration was 90 μM. Therefore, a variety of metal ions and amino acids were selected to test the selectivity of the compound 18BDQ to MC-LR. The specific operation was as follows: 2+ Cr 3+ , K + Mg 2+ 、Na + 、Zn 2+ ), amino acids (Ala, Arg, Cys, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Try, Val) concentrations were all 90 μM; the test solution with a concentration of compound 18BDQ was 10 μM, and fluorescence detection was performed under an excitation wavelength of 380 nm. Figure 5 The fluorescence spectra of 18BDQ in different solutions are shown in Figure 2. Figure 5It can be seen that an emission peak appears at 525 nm, and the response peak of 18BDQ to MC-LR is much higher than that to metal ions and amino acids, indicating that compound 18BDQ has good selectivity for MC-LR. Figure 6 for Figure 5 The histograms of the maximum peak values corresponding to different solutions show more clearly the selectivity of the reaction compound 18BDQ for MC-LR.

[0091] The above test examples show that the fluorescent probe provided by the present invention can detect MC-LR in aqueous phase with excellent selectivity, is simple to operate, and has broad application prospects.

[0092] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A biphenylpyridine salt compound, characterized in that: Having the structure shown in formula I:

2. The method for preparing the biphenylpyridinium salt compound of the structure shown in Formula I according to claim 1, characterized in that: The following steps are involved: 4-methylpyridine and 1-iodooctadecane are subjected to a salt-forming reaction in an organic solvent to obtain an intermediate compound having a structure shown in Formula II; The intermediate compound with the structure shown in formula II and p-phenylbenzaldehyde are subjected to a condensation reaction in an organic solvent to obtain the biphenylpyridinium salt compound with the structure shown in formula I.

3. The preparation method according to claim 2, characterized in that The molar ratio of the 4-methylpyridine to the 1-iodooctadecane is 1:(1.0-1.25).

4. The preparation method according to claim 2 or 3, characterized in that The salt-forming reaction is carried out under heating reflux conditions, the heating reflux temperature is 55-60° C., and the holding time of the salt-forming reaction is 4-6 hours; the salt-forming reaction is carried out in a protective gas atmosphere.

5. The preparation method according to claim 2, characterized in that The molar ratio of the intermediate compound having the structure shown in formula II to the p-phenylbenzaldehyde is 1:(1.0-1.25).

6. The preparation method according to claim 2 or 5, characterized in that The condensation reaction is carried out under heating reflux conditions, the heating reflux temperature is 80-90° C., and the insulation time of the condensation reaction is 12-15 hours; the condensation reaction is carried out in a protective gas atmosphere.

7. The preparation method according to claim 2, characterized in that After the condensation reaction, a condensation reaction liquid is obtained, and the method further comprises: removing the organic solvent from the condensation reaction liquid and then performing column chromatography purification, and drying the obtained eluate to obtain a pure product of the biphenylpyridinium salt compound with the structure shown in Formula I; the eluent used in the column chromatography purification is a mixed solvent of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 40 to 50:

1.

8. Use of the biphenylpyridinium salt compound according to claim 1 or the biphenylpyridinium salt compound prepared by the preparation method according to any one of claims 2 to 7 as a fluorescent probe for detecting microcystins for purposes other than disease diagnosis and treatment.

9. The use according to claim 8, characterized in that The application is to detect the concentration of microcystin in the water phase.

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