Reactive fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of noradrenaline detection, and particularly relates to a reaction type fluorescent probe and a preparation method and application thereof.The reaction type fluorescent probe is obtained by taking 6-(benzothiazole-2-yl) naphthalene-2-alcohol as a fluorophore, introducing thiophenol carbonate as a recognition group through a specific organic synthesis reaction and finally conducting synthesis; the capability of specifically recognizing noradrenaline is realized. The reactive fluorescent probe disclosed by the invention can effectively avoid interference of amino acids such as tryptophan and glutamic acid and metal ions such as potassium ions and calcium ions, so that high-specificity and high-sensitivity visual detection of noradrenaline in a complex matrix is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of norepinephrine detection, and in particular relates to a reaction-type fluorescent probe and a preparation method and application thereof. Background Art

[0002] Rapid screening for the stimulant (norepinephrine, or NE) in horse urine is a crucial tool for ensuring fair competitions and promoting the healthy development of equestrian sports. Fluorescence probes are a commonly used method for detecting NE in horse urine. Detection is achieved through a change in the fluorescent signal caused by the binding of a fluorescent probe to the target analyte.

[0003] The document "Visualization and Drug Intervention of Specific Norepinephrine Signaling Pathway" was published in the "Second National Youth Symposium on Photofunctional Materials of the Chinese Chemical Society" in 2020. The document used the "protection-deprotection" strategy to design a fluorescent probe that can specifically detect norepinephrine in brain tissue. A cyanine dye containing a well-water-soluble group (sulfonic acid group) was used as a fluorophore, and the p-toluenethiophenol recognition group was connected with a carbonate as the center. The unique β-hydroxyethylamine structure in norepinephrine can react with the probe through nucleophilic substitution reaction and intramolecular nucleophilic cyclization to form a five-membered ring compound and release the fluorophore, thereby realizing the specific fluorescence detection of norepinephrine.

[0004] However, due to the lack of specificity of their recognition groups, these fluorescent probes are susceptible to interference from other biomolecules (such as amino acids and metal ions) when applied to the complex matrix of athletic horse urine. This results in low sensitivity and selectivity, making it incapable of accurately detecting norepinephrine in complex biological samples. Therefore, existing technologies for detecting norepinephrine in horse urine suffer from insufficient specificity and sensitivity. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and to provide a reactive fluorescent probe with high specificity and high sensitivity for detecting norepinephrine, as well as a preparation method and application thereof.

[0006] To achieve the above objectives, the technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a reactive fluorescent probe for detecting norepinephrine, wherein the reactive fluorescent probe has the structural formula:

[0008] .

[0009] In a second aspect, the present invention provides a method for preparing a reactive fluorescent probe for norepinephrine detection, the preparation method comprising:

[0010] Step S1, dissolving 6-hydroxy-2-naphthaldehyde, 2-aminobenzenethiol, and p-aminobenzenesulfonic acid monohydrate in anhydrous ethanol to react to obtain 6-(benzothiazol-2-yl)naphthalene-2-ol, wherein the structural formula of the 6-(benzothiazol-2-yl)naphthalene-2-ol is:

[0011] ;

[0012] Step S2: dissolving 6-(benzothiazol-2-yl)naphthalene-2-ol in dichloromethane, first adding triphosgene and triethylamine, and then adding p-methylthiophenol, to react to obtain the aforementioned reactive fluorescent probe.

[0013] The molar ratio of the 6-hydroxy-2-naphthaldehyde, 2-aminobenzenethiol and p-toluenesulfonic acid monohydrate is 1.0:1.1-1.25:0.2-0.25.

[0014] The molar ratio of 6-(benzothiazol-2-yl)naphthalen-2-ol, triphosgene, triethylamine, and p-methylthiophenol is 1.0:0.2-0.25:0.2-0.25:0.8-0.9.

[0015] In a third aspect, the present invention provides a method for detecting norepinephrine in horse urine, the detection method comprising:

[0016] Step A1, centrifuging the horse urine sample, extracting the supernatant by oscillation, and concentrating the supernatant by nitrogen blowing to obtain a concentrate;

[0017] Step A2: Mix the concentrate, DMSO / PBS mixed solution, and the aforementioned reactive fluorescent probe, incubate the mixed system at 37° C. for 30-120 min, and detect the fluorescence intensity to achieve detection of norepinephrine.

[0018] The final concentration of the reactive fluorescent probe in the mixed system is 10 μM.

[0019] The volume ratio of DMSO to PBS in the DMSO / PBS mixed solution is 1:1.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The reactive fluorescent probe of the present invention is synthesized by introducing thiophenol carbonate as a recognition group using 6-(benzothiazol-2-yl)naphthalene-2-ol as a fluorophore through a specific organic synthesis reaction. It has the ability to specifically recognize norepinephrine. The detection mechanism is that the β-hydroxyethylamine structure in norepinephrine undergoes a nucleophilic substitution reaction and an intramolecular nucleophilic reaction with the thiophenol carbonate in the reactive fluorescent probe, causing the thiophenol carbonate to leave and release the fluorophore. The presence of norepinephrine is determined based on the change in the fluorescence signal. This detection mechanism can effectively avoid interference from amino acids such as tryptophan and glutamate and metal ions such as potassium ions and calcium ions, thereby achieving highly specific and sensitive visual detection of norepinephrine in complex matrices.

[0022] 2. When the reactive fluorescent probe of the present invention is used to detect norepinephrine in horse urine, the minimum detection concentration can reach 10 μM, which can meet the detection needs of trace norepinephrine in horse urine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the reaction principle of BN-PN and NE in the present invention.

[0024] Figure 2 Schematic diagram of the reaction principle of step S1 in the present invention.

[0025] Figure 3 Schematic diagram of the reaction principle of step S2 in the present invention.

[0026] Figure 4 1H NMR chart of BN.

[0027] Figure 5 This is the ESI-HRMS spectrum of BN.

[0028] Figure 6 1H NMR spectrum of BN-PN.

[0029] Figure 7 UV and fluorescence images of BN and BN-PN.

[0030] Figure 8 Fluorescence images of BN-PN reacting with NE solutions of different concentrations.

[0031] Figure 9 Fluorescence images of BN-PN reacting with different metal ion and anion solutions.

[0032] Figure 10 Fluorescence images of BN-PN reacting with different amino acid solutions.

[0033] Figure 11 The fluorescence images of BN-PN incubated with horse urine solutions containing different concentrations of NE for 30 minutes.

[0034] Figure 12 The fluorescence images of BN-PN incubated with horse urine solutions containing different concentrations of NE for 60 min.

[0035] Figure 13 The fluorescence images of BN-PN incubated with horse urine solutions containing different concentrations of NE for 90 min.

[0036] Figure 14 The fluorescence images of BN-PN incubated with horse urine solutions containing different concentrations of NE for 120 min. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0038] The reaction principle of the reactive fluorescent probe (BN-PN for short) of the present invention and norepinephrine (NE for short) is as follows: Figure 1 shown.

[0039] Example 1: Preparation of reactive fluorescent probe

[0040] Step S1: 1.0 g (5.8 mM, 1 eq) of 6-hydroxy-2-naphthaldehyde, 1.17 g (6.68 mM, 1.1 eq) of 2-aminobenzenethiol, and 0.22 g (1.16 mM, 0.2 eq) of p-toluenesulfonic acid monohydrate were dissolved in 20 mL of anhydrous ethanol and reacted at 90° C. overnight. After the reaction, the mixture was cooled to room temperature to precipitate a yellow precipitate, which was filtered and dried to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:2 (V / V)) to obtain a yellow solid, namely 6-(benzothiazol-2-yl)naphthalene-2-ol (abbreviated as BN), with a yield of 79%. The reaction principle of step S1 is as follows: Figure 2 As shown;

[0041] The structure of the yellow solid was characterized by 1H NMR and ESI-HRMS, and the 1H NMR and ESI-HRMS patterns were as follows: Figure 4 、 Figure 5 The yellow solid was confirmed to be BN. 1H NMR (400 MHz, DMSO-d6): δ10.13 (s, 1H), 8.56 (s, 1H), 8.09 (ddd, J = 26.4, 19.2, 8.3 Hz, 4H), 7.86 (d, J = 8.7 Hz, 1H), 7.55 (s, 1H), 7.46 (s, 1H), 7.21 (s, 2H); ESI-HRMS (m / z) calculated value: [M+H]+278.0039, found value: 278.0636.

[0042] Step S2: 227 g of yellow solid BN was dissolved in 20 mL of ultra-dry dichloromethane (CH2Cl2) in a round-bottom flask. 0.9 g of triphosgene (C3Cl6O3) and 0.2 g of triethylamine solvent were added under anhydrous and oxygen-free conditions. 0.25 g of p-methylthiophenol was added to the reaction system in the round-bottom flask under ice-water bath conditions and stirred for 1 h. After the reaction, the reaction was quenched with ultrapure water, extracted with dichloromethane, and after removing the solvent, purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:3 (V / V)) to obtain a white solid BN-PN with a yield of 35%. The reaction principle of step S2 is as follows: Figure 3 As shown;

[0043] The structure of the obtained white solid was characterized by 1H NMR. Figure 6 The obtained white solid was confirmed to be BN-PN. 1H NMR (600 MHz, D2O): 8.77 (1H, s), 8.28 (2H, t, J = 9.1), 8.21 (1H, d, J = 8.4), 8.15-8.10 (2H, m), 7.96 (1H, s), 7.58 (4H, d, J = 8.2), 7.53-7.49 (1H, m), 7.34 (2H, d, J = 8.0), 2.37 (3H, s).

[0044] Example 2: Optical property detection of BN-PN

[0045] (1) Preparation of standard solution and NE mother solution

[0046] Prepare a 1:1 dimethyl sulfoxide (DMSO) / phosphate buffered saline (PBS) mixed solution; weigh 2.08 mg of BN and add 3 mL of the DMSO / PBS mixed solution to obtain a 2.5 mM BN stock solution; weigh 3.21 mg of BN-PN and add 3 mL of the DMSO / PBS mixed solution to obtain a 2.5 mM BN-PN stock solution; take 8.46 mg of NE and add 10 mL of the DMSO / PBS mixed solution to obtain a 5 mM NE stock solution.

[0047] (2) Detection of BN-PN response to NE

[0048] 12 μL of each 2.5 mM BN-PN stock solution and 2.5 mM BN stock solution were added to 5 mL EP tubes, followed by 2988 μL of a DMSO / PBS mixture (final assay concentration: 10 μM). UV absorbance and fluorescence intensity were measured using a UV-2600 UV-visible spectrophotometer (Shimadzu Corporation, Japan) and a F4700 fluorescence spectrophotometer (Hitachi, Japan), respectively. The scan speed was 1200 nm / min, the excitation slit was 5.0 nm, the emission slit was 5.0 nm, and the photomultiplier tube (PMT) voltage was 700 V.

[0049] Test results such as Figure 7 As shown, Figure 7 Figure a is the comparison of the ultraviolet absorbance of BN-PN mother solution and BN mother solution, and Figure b is the comparison of the fluorescence intensity of BN-PN mother solution and BN mother solution. Figure 7 It can be seen that the probe BN-PN has weak fluorescence, with a maximum absorption wavelength at 340 nm and a maximum emission wavelength at 420 nm; the maximum absorption wavelength of the fluorophore BN is 355 nm and the maximum emission wavelength is 521 nm.

[0050] Example 3: Fluorescence detection of the reaction between BN-PN and different concentrations of NE

[0051] The NE stock solution was serially diluted to obtain NE solutions of different concentrations (50 μM, 100 μM, 200 μM, 500 μM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM). 12 μL of 2.5 mM BN and BN-PN stock solutions were added to 2988 μL of NE solutions of different concentrations, respectively. After incubation at 37°C for 5 minutes, the fluorescence spectra were measured at an emission wavelength of 350 nm. The test results are shown in Figure 2. Figure 8 As shown, from Figure 8 As the NE concentration increases from 0.5 mM to 5 mM, the fluorescence peaks of the BN-PN probe exhibit correlated changes. Specifically, the fluorescence peak near 400 nm primarily originates from BN-PN molecules that are unbound or weakly bound to NE. As the NE concentration increases, the fluorescence intensity of the peak near 400 nm decreases, while the fluorescence intensity of the peak near 530 nm increases, indicating that the BN-PN probe is bound to NE.

[0052] Example 4: BN-PN specific recognition verification

[0053] (1) Preparation of metal ion and anion solutions

[0054] HSO3 -The solution was prepared by weighing 1.56 mg of NaHSO3, adding 2 mL of ultrapure water, stirring to dissolve, transferring to a 5 mL centrifuge tube, and adding ultrapure water to make up to 3 mL; Ca 2+ The solution is made by weighing 2.21 mg of CaCl2·2H2O and mixing it with HSO3 - The solution was prepared by the same dissolution and volume adjustment steps; Cl - The solution was prepared by weighing 0.87 mg of NaCl and mixing it with HSO3 - The solution was prepared by the same dissolution and volume adjustment steps; CO3 2- Solution Weigh 1.59 mg of Na2CO3 and mix with HSO3 - The solution was prepared by the same dissolution and volume adjustment steps; HS - The solution was prepared by quickly weighing 0.84 mg of NaHS in a nitrogen atmosphere, transferring it to a sealed container containing 2 mL of deoxygenated water, continuously passing nitrogen to expel oxygen, stirring until completely dissolved, and then diluting the volume to 3 mL. + The solution is made by weighing 1.12 mg of KCl and mixing it with HSO3 - The solution was prepared by the same dissolution and volume adjustment steps; Mg 2+ The solution is made by weighing 3.05 mg of MgCl2·6H2O and mixing it with HSO3 - The solution was prepared by the same dissolution and volume adjustment steps; Na + The solution is made by weighing 0.877 mg of NaCl and mixing it with HSO3 - The solution was prepared by the same dissolution and volume adjustment steps; NO2 - The solution was prepared by weighing 1.035 mg of NaNO2 and transferring it to a brown centrifuge tube, adding 2 mL of ultrapure water, shaking and dissolving it, and then making up to volume; SO3 2- The solution was prepared by weighing 1.89 mg of Na2SO3 in a nitrogen atmosphere, transferring it to a brown centrifuge tube containing 2 mL of deoxygenated water, passing nitrogen to remove oxygen, stirring to dissolve, and then adjusting the volume to 3 mL. The solution was sealed and stored in the dark at 4°C. 2- The solution is made by weighing 2.13 mg of Na2SO4 and mixing it with HSO3 - The solution was prepared by the same dissolution and volume adjustment steps.

[0055] (2) Preparation of amino acid solution

[0056] The Phe solution was prepared by weighing 2.48 mg of Phe, adding 2 mL of ultrapure water, sonicating at room temperature until dissolved, and then adding ultrapure water to 3 mL. The Pro solution was prepared by weighing 1.73 mg of Pro, adding 2 mL of ultrapure water, vortexing to dissolve, and then adding ultrapure water to 3 mL. The Glu solution was prepared by weighing 2.21 mg of Glu, dissolving, and then adding ultrapure water to 3 mL. The Gly solution was prepared by weighing 1.68 mg of Gly, sonicating, and then adding ultrapure water to 3 mL. The Met solution was prepared by weighing 2.47 mg of Met, sonicating, and then adding ultrapure water to 3 mL. ; Leu solution was prepared by weighing 2.0 mg of Leu, adding it to a brown centrifuge tube, and adding 3 mL of ultrapure water for ultrasonic dissolution; Trp solution was prepared by weighing 3.06 mg of Trp, adding it to a 3 mL brown centrifuge tube, and adding 3 mL of ultrapure water for ultrasonic dissolution; Ser solution was prepared by weighing 1.58 mg of Ser, vortexing to dissolve it, and then adjusting the volume to 3 mL; Thr solution was prepared by weighing 1.79 mg of Thr, vortexing to dissolve it, and then adjusting the volume to 3 mL; His solution was prepared by weighing 3.14 mg of His, adding 3 mL of ultrapure water, and vortexing until completely dissolved.

[0057] When preparing the above interfering solutions, ultrapure water was used for dissolution and the final volume was adjusted to 3 mL to ensure a concentration of 5 mM. The dissolved amino acids could be assisted by ultrasound or vortexing to accelerate the dissolution.

[0058] (3) BN-PN specific detection

[0059] Take 12 μL of 2.5 mM BN-PN stock solution, 10 μL of each interfering solution and 2978 μL of DMSO / PBS mixed solution; take 12 μL of 2.5 mM BN-PN stock solution, 1.5 mL of 5 mM NE stock solution and 1488 μL of DMSO / PBS mixed solution to serve as NE control group; after incubation at 37°C for 30 minutes, the fluorescence value was measured at an emission wavelength of 350 nm to verify the specific response of the probe BN-PN to NE. The fluorescence detection results of the metal ion and anion solutions are as follows: Figure 9 As shown in Figure 2, the fluorescence detection results of amino acid solution are as follows: Figure 10 As shown. Figure 9 、 Figure 10 As can be seen, probe BN-PN only produces a significant fluorescence signal change in response to NE, while spectral changes caused by other substances are negligible. These results confirm the high selectivity and specificity of probe BN-PN for NE, effectively avoiding interference from amino acids such as tryptophan and glutamate, as well as metal ions such as potassium and calcium.

[0060] Example 5: Detection of NE in Horse Urine by BN-PN

[0061] Horse urine samples without NE were centrifuged at 3000-5000 rpm for 10 min. The supernatant was extracted three times with 2 mL of ethyl acetate by oscillation. The organic phases were combined and concentrated by nitrogen blowdown to obtain a concentrate. The concentrate was dissolved in a DMSO / PBS mixed solution (as a blank sample) and a 5 mM NE stock solution (as a spiked sample). The spiked sample was diluted in a gradient to obtain horse urine solutions containing 200 μM, 100 μM, 50 μM, and 10 μM NE. To 2988 μL of horse urine solutions containing different NE concentrations, 12 μL of 2.5 mM BN-PN solution was added (the final detection concentration of BN-PN was 10 μM). The fluorescence intensity was detected after incubation for 30, 60, 90, and 120 min, respectively.

[0062] The test results after incubation for 30, 60, 90, and 120 min were as follows: Figures 11 to 14 As shown. Figures 11 to 14 It can be seen that the minimum detection concentration of NE by probe BN-PN can reach 10 μM, and as the NE concentration increases, the fluorescence intensity is significantly enhanced, which can meet the detection needs of trace norepinephrine.

[0063] The BN-PN synthesis process described in the present invention is simple, the required raw materials are readily available, and the synthesis yield is high. The detection process uses a limited number of reagents and dosages, reducing detection costs and offering good economic benefits. Sample pretreatment is simple, requiring only centrifugation, extraction, and nitrogen concentration, eliminating the complex derivatization steps required by traditional chromatography-mass spectrometry. Detection is possible after a 30-minute incubation at 37°C, significantly shortening detection time and improving efficiency, making it suitable for rapid on-site screening.

Claims

1. A reactive fluorescent probe, characterized in that: The structural formula of the reactive fluorescent probe is: 。 2. A method for preparing a reactive fluorescent probe, characterized in that: The preparation method comprises: Step S1, dissolving 6-hydroxy-2-naphthaldehyde, 2-aminobenzenethiol, and p-aminobenzenesulfonic acid monohydrate in anhydrous ethanol to react to obtain 6-(benzothiazol-2-yl)naphthalene-2-ol, wherein the structural formula of the 6-(benzothiazol-2-yl)naphthalene-2-ol is: ； Step S2: dissolving 6-(benzothiazol-2-yl)naphthalene-2-ol in dichloromethane solution, first adding triphosgene and triethylamine, then adding p-methylthiophenol, and reacting to obtain the reactive fluorescent probe according to claim 1.

3. The method for preparing a reactive fluorescent probe according to claim 2, wherein: The molar ratio of the 6-hydroxy-2-naphthaldehyde, 2-aminobenzenethiol and p-toluenesulfonic acid monohydrate is 1.0:1.1-1.25:0.2-0.

25.

4. The method for preparing a reactive fluorescent probe according to claim 2, wherein: The molar ratio of 6-(benzothiazol-2-yl)naphthalen-2-ol, triphosgene, triethylamine, and p-methylthiophenol is 1.0:0.2-0.25:0.2-0.25:0.8-0.

9.

5. An application of a reactive fluorescent probe, characterized in that: The reactive fluorescent probe is used for detecting norepinephrine in horse urine, and the detection method includes: Step A1, centrifuging the horse urine sample, extracting the supernatant by oscillation, and concentrating the supernatant by nitrogen blowing to obtain a concentrate; Step A2: Mix the concentrate, the DMSO / PBS mixed solution, and the reactive fluorescent probe according to claim 1, incubate the mixed system at 37° C. for 30-120 minutes, and then detect the fluorescence intensity to detect norepinephrine.

6. The use of a reactive fluorescent probe according to claim 5, characterized in that: The final concentration of the reactive fluorescent probe in the mixed system is 10 μM.

7. The use of a reactive fluorescent probe according to claim 5, characterized in that: The volume ratio of DMSO to PBS in the DMSO / PBS mixed solution is 1:1.

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