A fluorescent probe containing a pyrene group, preparation method and application

Abstract

The present invention discloses a fluorescent probe containing a pyrene group, and its preparation method and application. The fluorescent probe described in the present invention is a ratiometric fluorescent molecule that binds to protamine through electrostatic interaction to induce the emission of an excimer. The addition of trypsin can hydrolyze the protamine, and the fluorescence can be restored to monomer emission, thereby enabling the detection of trypsin. In addition, the probe forms a host-guest complex system with γ-cyclodextrin, exhibiting the fluorescence emission of the excimer. Based on the hydrolysis of cyclodextrin by α-amylase, the fluorescence can be restored to monomer emission, thereby enabling the quantitative detection of α-amylase. The present invention provides a multifunctional fluorescent detection probe with excellent selectivity and sensitivity, which can be used in a diluted serum environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional materials, relates to fluorescent probe materials, and specifically relates to a fluorescent probe containing a pyrene group, a preparation method and an application thereof. Background Art

[0002] With improved living standards and the widespread use of medical knowledge, people are paying more and more attention to health issues. In 2024, my country's average life expectancy reached 78.8 years and continues to increase annually. This is largely due to advances in medical care, which enable earlier disease detection and more precise treatment. Biomacromolecules play a vital role in physiological processes, disease detection, and treatment. Protamine, with its high nutritional and functional properties, can be used as a heparin detoxifier, lower blood pressure, and promote digestion. Trypsin, an important digestive enzyme and a class of enzyme drugs, is also a diagnostic indicator for pancreatic diseases. α-Amylase plays a vital role in carbohydrate digestion and is associated with diseases such as acute pancreatitis. Excessive or insufficient levels of these biomacromolecules can have adverse effects on the human body, necessitating appropriate quantitative methods for their detection.

[0003] Fluorescence sensing methods have the advantages of simplicity, high sensitivity, and rapid response, and have made great progress over the past few decades. Fluorescent probes bind specifically to the analyte, and their interaction leads to significant changes in fluorescence properties, thereby enabling the detection of the analyte. Fluorescence probe methods have been widely used in the detection of various substances. In the field of biomolecular sensing, fluorescence probe methods have been successfully used to detect many small and large biological molecules, such as ATP, amino acids, metal ions, protamine, serum albumin, biogenic amines, heparin, etc. To this day, the design and development of multifunctional fluorescent probe molecules with excellent performance remains a very attractive and challenging topic. Summary of the Invention

[0004] To address the above-mentioned technical problems, the present invention provides a pyrene-containing fluorescent probe, preparation method, and application. The chemical structure of the pyrene-containing fluorescent probe provided by the present invention is shown in Formula I. The probe binds to protamine through electrostatic interaction, enabling quantitative detection of protamine. The host-guest complex formed by the probe and γ-cyclodextrin can detect α-amylase. Using this probe, highly selective and sensitive detection of protamine, trypsin, and α-amylase can be achieved.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a fluorescent probe containing a pyrene group, the chemical structure of which is shown in Formula I:

[0007]

[0008] The present invention also provides a method for preparing the fluorescent probe containing a pyrene group, comprising the following steps:

[0009] Step 1: Dissolve compound 1 and triethylamine in dichloromethane and stir at room temperature. Then add DMAP, EDCI, succinic acid, adipic acid, suberic acid, or sebacic acid and stir to react. Extract with water, remove the organic phase, and remove the solvent by rotary evaporation to obtain compound 2 as a white solid.

[0010] Step 2: Dissolve compound 2, DMAP, and EDCI in dichloromethane and stir at room temperature. Add 1-aminopyrene and triethylamine, and react at low temperature and then at room temperature. Extract with water, remove the organic phase, and remove the solvent by rotary evaporation to obtain compound 3.

[0011] Step 3: Dissolve compound 3 and sodium carbonate in a water-ethanol mixed solvent and reflux to react. After removing the solvent, the fluorescent probe is obtained.

[0012] The chemical structure of compound 1 is shown in Formula II:

[0013]

[0014] The chemical structure of compound 2 is shown in Formula III:

[0015]

[0016] The chemical structural formula of the compound 3 is shown in Formula IV:

[0017]

[0018] In step 1, the molar ratio of compound 1 to succinic acid, adipic acid, suberic acid, or sebacic acid is 1:2 to 1:3; the molar ratio of compound 1 to EDCI is 1:1.05 to 1:1.15; the ratio of compound 1 to DMAP is 1:0.2 to 1:0.4; and the ratio of compound 1 to triethylamine is 1:1.1 to 1:1.3. Step 1 involves the reaction of ethyl glutamate hydrochloride with a diacid to obtain a single-end amidated diacid derivative while retaining a carboxyl group as a functional group for the next amidation reaction.

[0019] The stirring method in step 1 is room temperature stirring, wherein the first stirring time at room temperature is 0.5 to 2 hours, and the second stirring time is 5 to 8 hours; the number of water extractions is 2 to 4 times.

[0020] In step 2, the molar ratio of compound 2 to 1-aminopyrene is 1:1.05 to 1:1.15; the molar ratio of compound 2 to EDCI is 1:1.3 to 1:1.6; the ratio of compound 2 to DMAP is 1:0.2 to 1:0.4; and the ratio of compound 2 to triethylamine is 1:1.1 to 1:1.3. Step 2 involves an amidation reaction between 1-aminopyrene and the retained carboxyl group in compound 2 to obtain a diacid derivative whose ends are respectively amidated with glutamic acid and 1-aminopyrene.

[0021] The stirring method in step 2 is to stir at room temperature first, then stir at low temperature, and finally stir at room temperature again, wherein the first stirring time at room temperature is 0.5 to 2 hours, the low temperature stirring temperature is -8 to -12°C, the stirring time is 0.5 to 2 hours, and the third stirring time is 20 to 30 hours; the number of water extractions is 4 to 6 times.

[0022] The volume ratio of the water-ethanol mixed solvent in step 3 is 1:0.8 to 1:1.2; the molar ratio of compound 3 to sodium carbonate is 1:1 to 1:1.1. Step 3 is a deprotection reaction of the carboxyl group, that is, a hydrolysis reaction of the ester to obtain a fluorescent probe in the form of a sodium salt.

[0023] The stirring method in step 3 is reflux stirring, wherein the first stirring time is 8 to 12 hours, and the second stirring time is 1 to 3 hours.

[0024] The present invention also provides the application of the fluorescent probe containing the pyrene group in the process of detecting protamine and trypsin.

[0025] The present invention also provides the application of the host-guest complex system of the fluorescent probe containing the pyrene group and gamma-cyclodextrin in detecting alpha-amylase.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a pyrene-containing fluorescent probe molecule synthesized from glutamic acid, 1-aminopyrene, succinic acid, adipic acid, suberic acid, or sebacic acid. The probe binds to protamine through electrostatic binding, resulting in an excimer emission peak, enabling quantitative detection of protamine. Furthermore, trypsin hydrolyzes protamine, causing the fluorescence emission to return to monomeric emission, enabling quantitative detection of trypsin. Furthermore, the probe forms a host-guest complex with γ-cyclodextrin, exhibiting excimer emission. α-amylase hydrolyzes γ-cyclodextrin, causing the fluorescence to return to monomeric emission, enabling quantitative detection of α-amylase.

[0028] The probe exhibits multiple detection functions, good selectivity and sensitivity, and has the potential to be applied in complex media. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1a Schematic diagram of the chemical structure of the fluorescent probe molecule containing a pyrene group in Example 1 of the present invention;

[0030] Figure 1b This is the H NMR spectrum (600 MHz, D2O) of the fluorescent probe molecule in Example 1 of the present invention;

[0031] Figure 2 The fluorescence spectrum of the fluorescent probe molecule in Example 1 of the present invention;

[0032] Figure 3 The fluorescence change of the fluorescent probe solution before and after the addition of protamine in Example 2 of the present invention;

[0033] Figure 4 is the ratio of the emission intensity of the fluorescent probe excimer to the peak emission intensity of the monomer in Example 2 of the present invention (I 489 / I 385 ) and the linear relationship between the concentration of protamine;

[0034] Figure 5 The effect of the fluorescent probe in Example 2 of the present invention on detecting protamine at different pH values;

[0035] Figure 6 The selectivity of the fluorescent probe for protamine in Example 3 of the present invention;

[0036] Figure 7 is the linear response range of the fluorescent probe to protamine in diluted fetal bovine serum (FBS) solution in Example 4 of the present invention;

[0037] Figure 8 The fluorescence of the complex system of the fluorescent probe and protamine in Example 5 of the present invention changes with the addition of trypsin;

[0038] Figure 9 is a linear fitting graph of the fluorescence intensity ratio of the complex system of the fluorescent probe and protamine and the trypsin concentration in Example 5 of the present invention;

[0039] Figure 10 The selectivity of the fluorescent probe and protamine complex in detecting trypsin in Example 5 of the present invention;

[0040] Figure 11 The fluorescence spectra and photos of the fluorescent probe in Example 6 of the present invention after adding α-, β-, and γ-cyclodextrin respectively;

[0041] Figure 12 The fluorescence change over time after adding α-amylase to the composite system of the fluorescent probe and γ-cyclodextrin in Example 7 of the present invention;

[0042] Figure 13 The fluorescence changes over time after adding different concentrations of α-amylase to the composite system of the fluorescent probe and γ-cyclodextrin in Example 7 of the present invention;

[0043] Figure 14 The selectivity of the composite system of the fluorescent probe and γ-cyclodextrin in Example 8 of the present invention for α-amylase in the presence of an interfering substance;

[0044] Figure 15 The fluorescence changes over time after adding different concentrations of α-amylase to the composite system of the fluorescent probe and γ-cyclodextrin in Example 9 of the present invention. DETAILED DESCRIPTION

[0045] To make the purpose, technical solutions and advantages of the present invention clearer, the following examples will further illustrate the specific steps and features of the invention. These examples are for illustration only and are not intended to limit the present invention. The methods used in the present invention are conventional methods in the art unless otherwise specified. The reagents and materials involved in the present invention are commercially available unless otherwise specified.

[0046] Example 1 Synthesis of fluorescent probe (n=1)

[0047]

[0048] See Figure 1a , which is a schematic diagram of the chemical structure of a fluorescent probe containing a pyrene group in an embodiment of the present invention. The preparation process includes the following steps:

[0049] Compound 1 (1.2 g, 5 mmol) and triethylamine (0.6 g, 6 mmol) were dissolved in dichloromethane (50 mL). After stirring at room temperature for 30 min, EDCI (5.5 mmol, 1.05 g), DMAP (0.15 mmol, 0.018 g), and succinic acid (10 mmol, 1.18 g) were added and stirred at room temperature for 6 h. After the reaction was completed, the reaction solution was washed with acidic water (pH 1-2) (2×50 mL) and water (2×50 mL) in sequence. The organic phase was collected and the solvent was removed to obtain compound 2. The yield was 78%. The product was characterized by mass spectrometry. ESI-MS: m / z 301.7 [(M-H) － ].

[0050] Compound 2 (5 mmol, 1.5 g), EDCI (7.5 mmol, 1.4 g), and DMAP (0.15 mmol, 0.018 g) were dissolved in dichloromethane (50 mL). After stirring at room temperature for 30 minutes, 1-aminopyrene (5.5 mmol, 1.2 g) and triethylamine (6 mmol, 0.6 g) were added. The mixture was stirred in an ice bath for 30 minutes, then returned to room temperature and stirred for 24 hours. The solution was washed sequentially with acidic water (pH 1-2) (2 × 50 mL), alkaline water (pH 8-9) (2 × 50 mL), and water (2 × 50 mL). The organic phase was collected and the solvent removed to obtain compound 3. The yield was 50%.

[0051] Compound 3 (0.5 g, 1 mmol) was dissolved in ethanol (20 mL), and a solution of sodium carbonate (0.12 g, 1.1 mmol) in water (20 mL) was added. The mixture was refluxed overnight. The solvent was removed to obtain the sodium salt of the fluorescent probe molecule (n = 1). The solution was characterized by mass spectrometry. ESI-MS: m / z 221.9 [(M-2Na) 2－ The sodium salt product was characterized by H NMR spectroscopy, such as Figure 1b shown.

[0052] Fluorescence spectra of aqueous solutions with different concentrations are shown in Figure 2. Figure 2 The maximum emission wavelengths of the fluorescent probe molecule aqueous solution are 385nm and 405nm (the excitation wavelength is 340nm).

[0053] Example 2 Fluorescence response of fluorescent probe (n=1) to protamine

[0054] Prepare 5 mL of HEPES buffer solution (10 mmol / L, pH 7.4) of the probe at a probe concentration of 10 μmol / L. Record the changes in the probe's fluorescence spectrum after adding different concentrations of protamine (excitation wavelength is 340 nm). With the addition of protamine (0-14 μg / mL), an excimer emission peak appears at 489 nm. Under a 360 nm UV lamp, the sample's luminescence color changes from blue to blue-green. Figure 3 The ratio of the emission peak intensity at 489 nm to the emission peak intensity at 385 nm (I 489 / I 385 ) showed a linear relationship with the concentration of protamine in the range of 1-14 μg / mL, such as Figure 4 The detection limit of the fluorescent probe molecule for protamine in HEPES buffer solution was calculated to be 13.8 ng / mL.

[0055] To further determine the applicable pH range of the fluorescent probe, pure water was used to prepare the solution and concentrated hydrochloric acid or sodium hydroxide was used to adjust the pH value. Figure 5As shown, the probe achieves fluorescence response to protamine in the pH range of 4-10.

[0056] Example 3 Selectivity of fluorescent probe (n=1) for protamine

[0057] By comparing the probes under the same conditions for various proteins with different pI values, including hemoglobin, bovine serum albumin, pepsin, ovalbumin, lysozyme (14 μg / mL), two amino acids (lysine and arginine) (14 μg / mL), glucose, and two common ions (Zn 2+ and Ca 2+ The selectivity of the probe for protamine determination was investigated by measuring the fluorescence response of )(140μmol / L). Figure 6 As shown, except for protamine, the other analytes mentioned above have little effect on the fluorescence emission of the probe, indicating that the probe has good selectivity for protamine.

[0058] Example 4 Fluorescence Detection of Protamine by Fluorescent Probe (n=1) in Serum Solution

[0059] The detection ability of the probe for protamine in 0.1% fetal bovine serum (FBS) solution was further investigated. 489 / I 385 ) was linearly correlated with the protamine concentration in the range of 7-16 μg / mL (y=0.00369x-0.01109, R 2 =0.9972), such as Figure 7 The calculated detection limit was 0.64 μg / mL. Spiking and recovery experiments were conducted by adding different amounts of protamine (8, 11, 12, and 13 μg / mL) to the probe solution. The results shown in Table 1 indicate that the probe exhibits good stability and recovery in 0.1% FBS, demonstrating its potential for detecting protamine in complex biological systems.

[0060] Table 1 Detection of protamine by fluorescent probe (n=1) in 0.1% FBS solution

[0061]

[0062] Example 5 Response of the Fluorescent Probe (n=1)-Protamine System (10 μmol / L-14 μg / mL) to Trypsin

[0063] Trypsin can catalyze the hydrolysis of protamine, the complex between the probe and protamine will dissociate, and the fluorescence emission will return to monomer emission. As the concentration of trypsin increases (0-1μg / mL), the initial hydrolysis rate increases, and I 489 / I 385 The rate of descent increases accordingly, as Figure 8 As shown. Recorded 12 minutes later I 489 / I 385 The relationship between the concentration of trypsin and the concentration of trypsin was found to be linear in the range of 0-1 μg / mL, such as Figure 9 This shows that the system can achieve quantitative detection of trypsin.

[0064] In addition, the selectivity of the fluorescent probe molecule-protamine complex in detecting trypsin was investigated. Figure 10 As shown in the figure, except for trypsin, proteins such as lysozyme, bovine serum albumin, ovalbumin, pepsin (1 μg / mL) and biomolecules such as glucose (10 μmol / L) have little effect on the fluorescence of the fluorescent probe molecule-protamine complex, which can be ignored. This shows that this system has a high selectivity for trypsin.

[0065] Example 6 Fluorescence Emission of a Host-Guest Complex of a Fluorescent Probe (n=4) and γ-Cyclodextrin

[0066] Given that pyrene groups can interact with cyclodextrins as a host-guest, and the formation of a host-guest complex affects the fluorescence of the pyrene group, when α-, β-, and γ-cyclodextrins (2 mmol / L) were added to a fluorescent probe (n=4) solution (0.2 mmol / L), it was found that only γ-cyclodextrin could make the excimer peak (excitation wavelength 356 nm) observable in the fluorescence spectrum, as shown in Figure 2. Figure 11 As shown in Figure 2, under 360nm ultraviolet light, the fluorescence color of the solution changes from blue to green, as shown in Figure 2. Figure 11 This indicates that the addition of γ-cyclodextrin induces the formation of excimers, and the change in fluorescence color is observed.

[0067] Example 7 Fluorescence Response of the Host-Guest Complex of Fluorescent Probe (n=4) and γ-Cyclodextrin to α-Amylase

[0068] The addition of γ-cyclodextrin can induce a change in fluorescence color, and removing cyclodextrin can restore the color from green to blue. α-amylase can cut the 1,4-glycosidic bond, destroying the cyclic structure of cyclodextrin, thereby dissociating its host-guest complex. Figure 12 As shown in the figure, α-amylase (10 U / mL) was added to a fluorescent probe (n=4)-γ-cyclodextrin (0.2 mmol / L-0.8 mmol / L) system in HEPES buffer. Over time, γ-cyclodextrin was gradually hydrolyzed, and the excimer emission intensity gradually decreased, eventually returning to its initial state.

[0069] Different concentrations of α-amylase were added to the HEPES solution of fluorescent probe (n=4)-γ-cyclodextrin (0.2mmol / L-0.8mmol / L), and the changes in fluorescence over time were monitored, such as Figure 13 As shown in the figure, the higher the concentration of α-amylase, the higher the fluorescence intensity ratio (I 485 / I 385 ) The faster it decreases. Take 1 485 / I 385 The value of is linearly related to the concentration of α-amylase (y = 0.1726-0.01944x, R 2 =0.9929), with a linear range of 1-5 μg / mL. The detection limit was 0.055 U / mL. This demonstrates that the system is capable of quantitatively detecting α-amylase.

[0070] Example 8 Selectivity of α-amylase detection using a host-guest complex of fluorescent probe (n=4) and γ-cyclodextrin

[0071] The selectivity and anti-interference ability of the host-guest complex of pyrene-containing fluorescent probe (n=4) and γ-cyclodextrin in detecting α-amylase were further investigated. Trypsin, β-amylase, α-glucosidase, alkaline protease, glucose oxidase, laccase, lysozyme, saccharifying enzyme, pepsin, and lipase were selected for anti-interference experiments. α-amylase (5U / mL) was added to the host-guest complex system of fluorescent probe (n=4) and γ-cyclodextrin (0.2mmol / L-0.8mmol / L), and then the above proteins (5U / mL) were added respectively. The fluorescence emission before and after addition was recorded. Figure 14 As shown in the figure, except for α-amylase, the presence of other enzymes has no obvious effect on the detection of α-amylase, indicating that the system has good selectivity and anti-interference ability for α-amylase.

[0072] Example 9 Application of a Fluorescent Probe (n=4) and a Host-Guest Complex of γ-Cyclodextrin to Detect α-Amylase in Serum Solution

[0073] The ability of a host-guest complex system of a fluorescent probe (n=4) and γ-cyclodextrin in a 0.1% FBS solution to detect α-amylase was investigated. A complex system of a fluorescent probe (n=4) and γ-cyclodextrin (0.2mmol / L-0.8mmol / L) was prepared in a 0.1% FBS solution. Different concentrations of α-amylase were added and the changes in fluorescence emission over time were monitored. Figure 15 As shown, the fluorescence intensity ratio (I 485 / I 385 ) showed an accelerating downward trend with the increase of α-amylase concentration. The fluorescence intensity ratio (I 485 / I 385) was plotted against enzyme concentration, and a linear relationship was found between 0.6 and 5 μg / mL (y = 0.1752 - 0.02248x, R 2 =0.9953). The detection limit was 0.062 U / mL. The recovery results are shown in Table 2. All four concentrations demonstrated good recovery and stability. This demonstrates that the fluorescent probe (n=4) and γ-cyclodextrin complex system is capable of quantitatively detecting α-amylase in low-concentration serum solutions.

[0074] Table 2 Detection of α-amylase by the complex of fluorescent probe (n=4) and γ-cyclodextrin in 0.1% FBS solution

[0075]

[0076] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A fluorescent probe containing a pyrene group, characterized in that: The structural formula of the fluorescent probe is shown in formula (I): Here, n is any integer from 1 to 4.

2. The method for preparing a fluorescent probe containing a pyrene group according to claim 1, wherein: The following steps are involved: Step 1: Dissolve compound 1 and triethylamine in dichloromethane, stir at room temperature, then add 4-dimethylaminopyridine (DMAP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), succinic acid, adipic acid, suberic acid, or sebacic acid, and stir to react; extract with water, take the organic phase, and rotary evaporate to obtain compound 2; Step 2: Dissolve the compound 2, DMAP, and EDCI in dichloromethane, stir at room temperature, then add 1-aminopyrene and triethylamine, and react by stirring at low temperature and room temperature, respectively; extract with water, take the organic phase, and remove the solvent by rotary evaporation to obtain compound 3; Step 3, dissolving the compound 3 and sodium carbonate in a water-ethanol mixed solvent, reflux reaction, and removing the solvent to obtain the fluorescent probe; The chemical structure of compound 1 is shown in Formula II: The chemical structure of compound 2 is shown in Formula III: The chemical structural formula of the compound 3 is shown in Formula IV:

3. The preparation method according to claim 2, characterized in that The molar ratio of compound 1 in step 1 to succinic acid, adipic acid, suberic acid or sebacic acid is 1:2 to 1:3; the molar ratio of compound 1 to EDCI is 1:1.05 to 1:1.15; the ratio of compound 1 to DMAP is 1:0.2 to 1:0.4; and the ratio of compound 1 to triethylamine is 1:1.1 to 1:1.

3.

4. The preparation method according to claim 2, characterized in that The stirring method in step 1 is stirring at room temperature, wherein the first stirring time at room temperature is 0.5 to 2 hours, and the second stirring time is 5 to 8 hours; the number of water extractions is 2 to 4 times.

5. The preparation method according to claim 2, characterized in that The molar ratio of compound 2 and 1-aminopyrene in step 2 is 1:1.05 to 1:1.15; the molar ratio of compound 2 to EDCI is 1:1.3 to 1:1.6; the ratio of compound 2 to DMAP is 1:0.2 to 1:0.4; and the ratio of compound 2 to triethylamine is 1:1.1 to 1:1.

3.

6. The preparation method according to claim 2, characterized in that The stirring method in step 2 is first stirring at room temperature, then stirring at low temperature, and finally stirring at room temperature again, wherein the first stirring time at room temperature is 0.5 to 2 hours, the low temperature stirring temperature is -8 to -12°C, the stirring time is 0.5 to 2 hours, and the third stirring time is 20 to 30 hours; the number of water extractions is 4 to 6 times.

7. The preparation method according to claim 2, characterized in that The volume ratio of the water-ethanol mixed solvent in step 3 is 1:0.8 to 1:1.2; the molar ratio of compound 3 to sodium carbonate is 1:1 to 1:1.

1.

8. The preparation method according to claim 2, characterized in that The stirring method in step 3 is reflux stirring, wherein the first stirring time is 8 to 12 hours, and the second stirring time is 1 to 3 hours.

9. Use of the fluorescent probe containing a pyrene group according to any one of claims 1 to 8 in the detection of protamine and trypsin.

10. Use of a host-guest complex system of a fluorescent probe containing a pyrene group according to any one of claims 1 to 8 and γ-cyclodextrin in detecting α-amylase.

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