19F-labeled probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of magnetic resonance spectrum detection, in particular to a < 19 > F labeled probe as well as a preparation method and application thereof. The chemical structural formula of the < 19 > F-labeled probe is shown in the specification, and X is halogen. The probe designed by the invention can be used for simultaneous identification of vicinal diol / phenolic substances. The boric acid group in the probe has relatively strong affinity with vicinal diol / phenolic substances, and can be subjected to specific chemical reaction to generate boric acid ester and cause the change of 19F formant, so that simultaneous detection of vicinal diol / phenolic substances is realized. The ortho-position fluorine atom and the meta-position fluorine atom in the probe structure are sensitive to changes of the space structure and the electron cloud density respectively, identification and distinguishing of a series of vicinal diol / phenol substances are improved, and the problem that due to serious overlapping of formants of endogenous substances of MRS, signal identification becomes very difficult is solved.

Description

Technical Field

[0001] This invention relates to the field of magnetic resonance spectroscopy detection technology, specifically to a... 19 F-labeled probes, their preparation methods, and applications. Background Technology

[0002] The identification and detection of diols / phenols are of great significance in biology and medicine. For example, selectively detecting glucose concentration in urine is highly important for diagnosing diabetes; while detecting catechol neurotransmitters such as dopamine, adrenaline, and noradrenaline can be used to detect Alzheimer's disease, neuroblastoma, and pheochromocytoma. Therefore, researchers have developed many fluorescent probes to detect these important bioanalytes. Although fluorescence-based receptors exhibit high sensitivity to diols / phenols, they cannot simultaneously identify and distinguish multiple similar substances because fluorescence emission can only be modulated in one dimension—that is, the signal intensity can be either enhanced or weakened. Therefore, there is an urgent need to develop a class of probes capable of simultaneously distinguishing a range of similar diols / phenols in complex biological systems.

[0003] Magnetic resonance spectroscopy, or MRS for short, is a non-invasive detection method and an indispensable tool for studying biomolecules and their interactions. It enables the non-invasive measurement of metabolites and physiological changes in vivo. Based on the different magnetic nuclei, MRS mainly includes... 1 H, 19 F, 13 C 31 P-type magnetic resonance spectroscopy. Among them, 1 H and 19 F has an abundance of nearly 100% in nature and a high gyromagnetic ratio, resulting in high NMR sensitivity and wide application. Currently, MRS is mainly limited to the detection of a few endogenous substances, and the resonance peaks of these endogenous substances are usually concentrated in a relatively small region, which leads to severe overlap of resonance peaks, making the identification of their signals extremely difficult. Summary of the Invention

[0004] To address the problem of severe overlap of resonance peaks in MRS (Medium-Ray Suppression Rating Spectroscopy), which makes signal identification extremely difficult, the present invention aims to provide... 19 F-labeled MRS probes, their preparation methods, and applications.

[0005] This invention aims to achieve the detection of analytes by enabling a specific chemical reaction between an exogenous probe and the analyte to generate new substances, accompanied by the formation of new resonance peaks. Guided by this strategy, this invention targets... 19 FMRS designed and synthesized corresponding probes to achieve specific detection of substances in vivo.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] The first aspect of the present invention provides 19 The chemical structure of the F-labeled probe is shown below:

[0008]

[0009] Where X is a halogen.

[0010] In a preferred embodiment, its chemical structural formula is shown below:

[0011]

[0012] The second aspect of the present invention provides a method described in the first aspect. 19 The method for preparing F-labeled probes includes the following steps:

[0013] (2,3-difluoro-4-methylphenyl)boronic acid, N-bromosuccinimide and benzoyl peroxide were refluxed in the first solvent system under a protective atmosphere. After the reaction was completed, intermediate compound F1 was prepared.

[0014] Under a protective atmosphere, intermediate compound F1 and pyridine were reacted with stirring in a second solvent system at a reaction temperature of 65–75 °C. After the reaction was completed, the following compound was prepared: 19 F-labeled probes;

[0015] The chemical structural formula of intermediate compound F1 is:

[0016]

[0017] In a preferred embodiment, the molar ratio of (2,3-difluoro-4-methylphenyl)boronic acid, N-bromosuccinimide, and benzoyl peroxide is 1:1 to 1.2:0.03 to 0.06.

[0018] The molar ratio of intermediate compound F1 to pyridine is 1:1 to 1.1.

[0019] In a preferred embodiment, the first solvent is carbon tetrachloride;

[0020] The second solvent is N,N-dimethylformamide (DMF).

[0021] In a preferred embodiment, the process further includes a first purification step following the reflux reaction, specifically:

[0022] The solution after reflux reaction is transferred into water, extracted with dichloromethane, the organic phase is collected, and distilled to remove the solvent, thus obtaining the final product.

[0023] In a preferred embodiment, a second purification is further included after the stirring reaction, specifically:

[0024] Add acetone and diethyl ether to the solution after stirring to precipitate the crude product. Centrifuge and wash the crude product with acetone and diethyl ether to obtain the final product.

[0025] A third aspect of the present invention provides a method described in the first aspect. 19 The use of F-labeled probes in the preparation of reagents for detecting vicinal diols / phenols.

[0026] In a preferred embodiment, the vicinal diol is a monosaccharide;

[0027] The phenolic substances mentioned are catechol neurotransmitters.

[0028] In a preferred embodiment, the vicinal diol is glucose or galactose; the phenolic substance is dopamine, adrenaline, or noradrenaline.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention designs a 19 An F-labeled probe that can be used for the simultaneous identification of vicinal diols / phenols.

[0031] 2. The boric acid group in the probe of this invention has a strong affinity for vicinal diols / phenolic substances, and can undergo a specific chemical reaction to generate borate esters and cause… 19 The changes in the F resonance peak enable the simultaneous detection of vicinal diols / phenols. The ortho and meta fluorine atoms in the probe structure are sensitive to changes in spatial structure and electron cloud density, respectively, improving the identification and differentiation of a range of vicinal diols / phenols. Attached Figure Description

[0032] Figure 1 This is a schematic diagram illustrating the detection principle of probe FN for vicinal diols / phenols in Example 1.

[0033] Figure 2 This is a synthetic route diagram of the probe FN in Example 1.

[0034] Figure 3 Compound F1 of Example 1 1 H NMR spectrum.

[0035] Figure 4 Compound F1 of Example 1 13 C10 NMR spectrum.

[0036] Figure 5 For the probe FN of Example 11 H NMR spectrum.

[0037] Figure 6 For the probe FN of Example 1 13 C10 NMR spectrum.

[0038] Figure 7 The image shows the FT-IR spectrum of the probe FN in Example 1.

[0039] Figure 8 The image shows the HR-MS spectrum of the probe FN in Example 1.

[0040] Figure 9 For different concentrations of FN 19 F nuclear magnetic resonance signal detection results.

[0041] Figure 10 The results of the FN probe in Example 1 for identifying carbohydrates are shown.

[0042] Figure 11 The result is the reaction between probe FN and sucrose in Example 1.

[0043] Figure 12 The results of the identification of catechol neurotransmitters by probe FN in Example 1 are shown.

[0044] Figure 13 The probe FN of Example 1 in the presence of common cations 19 The variation of the F resonance peak.

[0045] Figure 14 The probe FN of Example 1 in the presence of common anions 19 The variation of the F resonance peak.

[0046] Figure 15 The image shows the changes in the resonance peak signal of probe FN in Example 1 in the presence of a series of amino acids.

[0047] Figure 16 The image shows the change of the resonance peak signal of probe FN in Example 1 in the presence of ROS / RNS.

[0048] Figure 17 The results of the detection of norepinephrine secreted by PC12 cells using probe FN in Example 1 are shown.

[0049] Figure 18 In the diagram, Figure A shows the cytotoxicity experiment; Figure B shows the trend of mouse body weight change; and Figure C shows the H&E staining of major organs.

[0050] Figure 19 This is a comparison chart of blood indicator assessments. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0052] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Currently, MRS is mainly limited to the detection of a few endogenous substances, and the resonance peaks of these endogenous substances are usually concentrated in a relatively small region, leading to severe overlap of resonance peaks and making signal identification extremely difficult. The introduction of exogenous probes promises to solve this problem. By having an exogenous probe undergo a specific chemical reaction with the analyte to generate new substances, accompanied by the generation of new resonance peaks, the detection of the analyte can be achieved. Guided by this strategy, this patent addresses... 19 F MRS designed and synthesized corresponding probes to achieve specific detection of substances in vivo.

[0054] 19 FMRS is playing an increasingly important role in biological and drug research. Compared to 1 HMRS, 19 F MRS has significant advantages: 19 F possesses 100% natural abundance, a high gyromagnetic ratio, a wide chemical shift range (>350), and is free from background signal interference in biological systems. These characteristics make it... 19 F MRS exhibits very high sensitivity for magnetic resonance spectroscopy detection. Furthermore, 19 The F resonance peak is highly sensitive to minute changes in chemical structure, which makes the simultaneous detection of diols / phenols possible. The key challenge that needs to be addressed next is how to construct... 19 The F-labeled probe exhibits a specific chemical reaction with vicinal diols / phenols.

[0055] During our research, we discovered that boric acid groups exhibit strong reactivity and affinity for the ovoid dihydroxyl groups in ovoid diols / phenols. Therefore, this patent introduces boric acid functional groups into the probe, enabling it to react specifically with ovoid diols / phenols. Based on the above analysis, we will... 19 F-labeling of phenylboronic acid derivatives yields a novel probe molecule, FN, for the simultaneous detection and differentiation of vicinal diols and phenols. To further enhance the probe's ability to detect and differentiate vicinal diols and phenols, we introduced F-labeling at the ortho and meta positions of the boronic acid group. 19 F, such as Figure 1 As shown, adjacent 19F is more sensitive to changes in spatial structure and is used to sense changes in spatial structure after a reaction; [the following is a separate sentence fragment:] [The ... 19 F is more sensitive to changes in electron cloud density and is used to sense changes in electron cloud density after a reaction. When the probe FN reacts with vicinal diols / phenols, both the spatial structure and electron cloud density of the borate group change to varying degrees, which leads to… 19 The changes in the F MRS signal peaks can be used to distinguish these substances, achieving the simultaneous identification of a series of similar substances using only one probe molecule.

[0056] First aspect of the invention

[0057] The first aspect of the present invention provides 19 The chemical structure of the F-labeled probe is shown below:

[0058]

[0059] Where X is a halogen. For example, X can be Br, Cl, or I.

[0060] In a preferred embodiment, its chemical structural formula is shown below:

[0061]

[0062] The boric acid group in the probe of this invention has a strong affinity for vicinal diols / phenolic substances, and can undergo a specific chemical reaction to generate borate esters and cause… 19 The changes in the F resonance peak enable the simultaneous detection of vicinal diols / phenols. The ortho and meta fluorine atoms in the probe structure are sensitive to changes in spatial structure and electron cloud density, respectively, improving the identification and differentiation of a range of vicinal diols / phenols.

[0063] Second aspect of the invention

[0064] The second aspect of the present invention provides a method described in the first aspect. 19 The method for preparing F-labeled probes includes the following steps:

[0065] Specific chemical reaction equations are as follows: Figure 2 As shown;

[0066] (1) Preparation of intermediate compound F1

[0067] Under a protective atmosphere, (2,3-difluoro-4-methylphenyl)boronic acid, N-bromosuccinimide, and benzoyl peroxide were refluxed in the first solvent system. After the reaction was completed, intermediate compound F1 was prepared. The chemical structural formula of intermediate compound F1 is as follows:

[0068]

[0069] (2) Preparation 19 F-labeled probes

[0070] Under a protective atmosphere, intermediate compound F1 and pyridine were reacted with stirring in a second solvent system at a reaction temperature of 65–75 °C. After the reaction was completed, the following compound was prepared: 19 F-labeled probes.

[0071] Step (1) Preparation of intermediate compound F1:

[0072] In a preferred embodiment, the molar ratio of (2,3-difluoro-4-methylphenyl)boronic acid, N-bromosuccinimide, and benzoyl peroxide is 1:1 to 1.2:0.03 to 0.06.

[0073] In a preferred embodiment, the first solvent is carbon tetrachloride.

[0074] In a preferred embodiment, the process further includes a first purification step following the reflux reaction, specifically:

[0075] The solution after reflux reaction is transferred into water, extracted with dichloromethane, the organic phase is collected, and distilled to remove the solvent, thus obtaining the final product.

[0076] Step (2) Preparation 19 F-labeled probes

[0077] In a preferred embodiment, the molar ratio of intermediate compound F1 to pyridine is 1:1 to 1.1.

[0078] In a preferred embodiment, the second solvent is N,N-dimethylformamide (DMF).

[0079] In a preferred embodiment, a second purification is further included after the stirring reaction, specifically:

[0080] Add acetone and diethyl ether to the solution after stirring to precipitate the crude product. Centrifuge and wash the crude product with acetone and diethyl ether to obtain the final product.

[0081] In a preferred embodiment, the protective atmosphere is a nitrogen atmosphere.

[0082] Third aspect of the invention

[0083] A third aspect of the present invention provides a method described in the first aspect. 19 The use of F-labeled probes in the preparation of reagents for detecting vicinal diols / phenols.

[0084] In a preferred embodiment, the vicinal diol is a monosaccharide; the phenolic substance is a catechol neurotransmitter.

[0085] In a preferred embodiment, the vicinal diol is glucose or galactose; the phenolic substance is dopamine, adrenaline, or noradrenaline.

[0086] This invention experimentally demonstrates that the probe FN can effectively identify and distinguish vicinal diols, such as glucose and galactose. Furthermore, FN also exhibits good ability to distinguish catechol neurotransmitters, such as dopamine, adrenaline, and noradrenaline. More importantly, this probe can detect noradrenaline secreted in situ by PC12 cells. The good biocompatibility of FN lays the foundation for subsequent in vivo experiments. This work provides a new method for the simultaneous detection of a series of similar substances in complex biological systems. Specific Implementation

[0088] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0089] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in order to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0090] Unless otherwise specified, the methods described in the following embodiments are conventional methods; the reagents and materials mentioned are commercially available unless otherwise specified.

[0091] (1) Main reagents: (2,3-difluoro-4-methylphenyl)boronic acid (98%, Adamas); N-bromosuccinimide (98%, Adamas); benzoyl peroxide (RG, Adamas); norepinephrine (98%, Maclean); dopamine hydrochloride (98%, Maclean); adrenaline (98%, Maclean); pyridine (RG, Acros); carbon tetrachloride (AR, Sinopharm Shanghai); dichloromethane (AR, Sinopharm Shanghai).

[0092] (2) Main experimental instruments: Liquid chromatography-nuclear magnetic resonance mass spectrometer, 400MHz DRX500, Bruker, used for characterization of proton and carbon spectra of compounds; High resolution mass spectrometer, MicroTOFII10257, Bruker, used for characterization of molecular weight of compounds; Multifunctional microplate reader, SpectraMaxiD5, Molecular Devices, used for detection of cell viability; Fourier transform infrared spectrometer, ThermoFisher, NicoletiS10, used for characterization of functional groups of compounds; Small animal in vivo magnetic resonance imaging system, 7T, Bruker, used for spectroscopic testing on small animals.

[0093] The present invention will be further described in detail below with reference to the embodiments.

[0094] Example 1

[0095] A sort of 19 The method for preparing F-labeled probes includes the following steps:

[0096] (1) Synthesis of compound F1: In a dry 100 mL flask, 1.64 mmol of (2,3-difluoro-4-methylphenyl)boric acid, 1.72 mmol of N-bromosuccinimide, and 0.082 mmol of benzoyl peroxide were added, and the mixture was dissolved in 5 mL of CCl4. Under a nitrogen atmosphere, the mixture was heated under reflux overnight. After the reaction was complete, it was poured into water. The mixture was extracted three times with dichloromethane. The organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the desired product F1.

[0097] Compound F1 1 The H NMR spectrum is shown in [reference]. Figure 3 , 13 The C NMR spectrum is shown below. Figure 4 .

[0098] 1 HNMR (400MHz, CDCl3) δ = 8.50 (s, 1H), 7.47 (d, J = 8.0Hz, 1H), 7.30 (s, 1H), 4.72 (s, 2H). 13 CNMR (100MHz, CDCl3) δ = 133.3, 130.0, 129.7, 129.0, 126.2, 123.6, 26.4.

[0099] (2) Synthesis of compound FN: In a 100 mL dry flask, 0.48 mmol of compound F1, 0.51 mmol of pyridine, and 1 mL of anhydrous DMF were added. Nitrogen gas was introduced, and the mixture was heated and stirred at 70 °C for 24 hours until the visible color turned slightly yellow. 20 mL of acetone and 10 mL of diethyl ether were added sequentially to slowly induce precipitation of the crude product. After centrifugation, the crude product was washed twice with 5 mL of acetone under vigorous stirring. Finally, it was washed again with 10 mL of diethyl ether under sonication. The product was dried under vacuum to obtain the final compound FN.

[0100] probe FN 1 The H NMR spectrum is shown in [reference]. Figure 5 , 13 The C NMR spectrum is shown below. Figure 6 FT-IR spectrum can be found Figure 7 HR-MS spectra can be found Figure 8 .

[0101] 1 HNMR (400MHz, CDCl3) δ = 9.13 (d, J = 8.0Hz, 2H), 8.55 (m, 1H), 8.20 (t, J = 8.0Hz, 2H), 7.41 (s, 1H), 7.28 (s, 1H), 5.99 (s, 2H). 13 CNMR (100MHz, CDCl3) δ = 148.2, 139.2, 129.7, 133.4, 129.8, 129.7, 129.0, 125.2, 123.6, 56.9. HRMS(ESI,m / z):calcd for C 12 H 11 BF2NO2[M] + ,250.0845,found 250.0853.

[0102] IR (cm) -1 ):3436,1621,1388,1028.

[0103] Example 2

[0104] A sort of 19 The method for preparing F-labeled probes includes the following steps:

[0105] (1) Synthesis of compound F1: In a dry 100 mL flask, 1.64 mmol of (2,3-difluoro-4-methylphenyl)boric acid, 1.96 mmol of N-bromosuccinimide, and 0.098 mmol of benzoyl peroxide were added, and the mixture was dissolved in 5 mL of CCl4. Under a nitrogen atmosphere, the mixture was heated under reflux overnight. After the reaction was complete, it was poured into water. The mixture was extracted three times with dichloromethane. The organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the desired product F1.

[0106] (2) Synthesis of compound FN: In a 100 mL dry flask, 0.48 mmol of compound F1, 0.5 mmol of pyridine, and 1 mL of anhydrous DMF were added. Nitrogen gas was introduced, and the mixture was heated and stirred at 70 °C for 24 hours until the visible color turned slightly yellow. 20 mL of acetone and 10 mL of diethyl ether were added sequentially to slowly induce precipitation of the crude product. After centrifugation, the crude product was washed twice with 5 mL of acetone by vigorous stirring. Finally, it was washed again with 10 mL of diethyl ether under sonication. The product was dried under vacuum to obtain the final compound FN.

[0107] Example 3

[0108] A sort of 19 The method for preparing F-labeled probes includes the following steps:

[0109] (1) Synthesis of compound F1: In a dry 100 mL flask, 1.64 mmol of (2,3-difluoro-4-methylphenyl)boric acid, 1.82 mmol of N-bromosuccinimide, and 0.052 mmol of benzoyl peroxide were added, and the mixture was dissolved in 5 mL of CCl4. Under a nitrogen atmosphere, the mixture was heated under reflux overnight. After the reaction was complete, it was poured into water. The mixture was extracted three times with dichloromethane. The organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the desired product F1.

[0110] (2) Synthesis of compound FN: In a 100 mL dry flask, add 0.48 mmol of compound F1, 0.53 mmol of pyridine, and 1 mL of anhydrous DMF. Purge with nitrogen, then heat and stir at 70 °C for 24 hours until the visible color turns slightly yellow. Continuously add 20 mL of acetone and 10 mL of diethyl ether to slowly induce precipitation of the crude product. After centrifugation, wash twice with 5 mL of acetone with vigorous stirring. Finally, wash again with 10 mL of diethyl ether under sonication. Dry under vacuum to obtain the final compound FN.

[0111] Compound FN was successfully prepared in Examples 1-3. The following uses the probe FN provided in Example 1 of this invention as an example to specifically verify its spectral performance.

[0112] Test Example 1: Sensitivity Test of Probe FN

[0113] We explored the probe FN at different concentrations 19 Signal changes in the F resonance peak were observed. Different concentrations of test solutions were prepared using PBS buffer to detect the FN probe. The concentrations of the test solutions in the PBS buffer system were 0.5 mM, 0.1 mM, 50 μM, and 20 μM. The test results are shown below. Figure 9 . Figure 9 For different concentrations of FN 19 F nuclear magnetic resonance signal detection results.

[0114] Depend on Figure 9 The experimental results showed that when the probe concentration was 0.5 mM, there were two very obvious... 19 The magnetic resonance spectral peaks of F are -133.7 ppm and -144.5 ppm, corresponding to the ortho and meta positions in the probe, respectively. 19 F. When the concentration of the probe decreases, 19 The signal intensity of F also gradually weakened. It could still be observed when the probe concentration was reduced to 50 μM. 19 The resonance peak of F, when the concentration is further reduced to 20 μM 19 The signal for F is already relatively weak.

[0115] The experimental results above show that even at concentrations as low as 100 μM, a distinct resonance peak can still be observed, indicating that... 19 F-magnetic resonance has very high sensitivity; in actual detection, its sensitivity exceeds [a certain threshold]. 1 H, this is mainly due to 19 The F-magnetic resonance signal is unaffected by water, which makes... 19 F MRS has higher sensitivity in aqueous solutions.

[0116] Test Example 2: Identification of vicinal diols / phenols by probe FN

[0117] (1) Identification of vicinal diols by probe FN

[0118] We explored the detection of sugars in vicinal diols using the FN probe.

[0119] Common monosaccharides, glucose and galactose, were selected. Results are shown below. Figure 10 and Figure 11 . Figure 10 The results of the FN probe in Example 1 for identifying carbohydrates are shown. Figure 11 The result is the reaction between probe FN and sucrose in Example 1.

[0120] Depend on Figure 10The results showed that after adding glucose to the PBS solution containing FN, the original FN content decreased. 19 The intensity of the F resonance peak decreases significantly, accompanied by the formation of a new resonance peak. (Adjacent) 19 The chemical shift of F shifted from -135.2 ppm to -135.4 ppm at higher fields, meta position. 19 The chemical shift of F shifted from -144.5 ppm to -146.4 ppm in the higher field.

[0121] Depend on Figure 10 The results showed that when FN was mixed with galactose and reacted, the original 19 The intensity of the F resonance peak also decreased significantly, and was also accompanied by new [symbols / symbols]. 19 The F resonance peaks were generated at 135.5 ppm and 146.1 ppm, respectively, both shifting towards the higher field.

[0122] We also explored the detection of polysaccharides using the probe FN. A common polysaccharide, sucrose, was selected.

[0123] Depend on Figure 11 The experimental results showed that after adding sucrose, 19 The F resonance peak showed no significant changes, and no new resonance peaks appeared.

[0124] The experimental results above show that the probe FN has a good ability to identify and distinguish reducing monosaccharides, but cannot distinguish non-reducing polysaccharides.

[0125] (2) Identification of phenolic substances by probe FN

[0126] We also explored the identification of catechol neurotransmitters using the probe FN. Catechol neurotransmitters, such as dopamine, norepinephrine, and epinephrine, are structurally very similar, making specific identification and differentiation difficult using a single probe molecule. However, the ortho-dihydroxyl groups in the structure of these catechol neurotransmitters can react with the borate groups in the FN, and different neurotransmitters can elicit different reactions. 19 The different shifts of the F resonance peaks enable the differentiation of different neurotransmitters. Experimental results are shown below. Figure 12 . Figure 12 The results of the identification of catechol neurotransmitters by probe FN in Example 1 are shown.

[0127] Depend on Figure 12 The results showed that when the probe FN reacted with adrenaline (EP), the original... 19 The magnetic resonance peak of F basically disappeared, accompanied by the generation of two new resonance peaks, which shifted to higher fields to -136.0 ppm and -145.8 ppm, respectively.

[0128] When dopamine (DA) and norepinephrine (NE) are added to a solution of FN, the original [results] can be observed. 19 The magnetic resonance peaks of F also shifted to higher fields, resulting in new... 19 The resonance peaks of F. These newly generated... 19 The chemical shifts of the resonance peaks of F are not the same, thus enabling the simultaneous identification and differentiation of dopamine, adrenaline, and noradrenaline.

[0129] Test Example 3: Anti-interference performance of probe FN

[0130] (1) Anti-interference performance of probe FN against anions or cations

[0131] We investigated whether common anions or cations would interfere with the detection of vicinal diols / phenols by the probe FN.

[0132] We selected a series of common anions or cations, including Zn. 2+ Fe 2+ Ca 2+ Cu 2+ Mg 2+ Cl - , AcO - These ions were added to a solution containing FN and their effects were observed. 19 Changes in the F resonance peak. Results are shown in... Figure 13 and Figure 14 . Figure 13 The probe FN of Example 1 in the presence of common cations 19 The variation of the F resonance peak. Figure 14 The probe FN of Example 1 in the presence of common anions 19 The variation of the F resonance peak.

[0133] Depend on Figure 13 and Figure 14 The results showed that when the aforementioned anions or cations were present, the resonance peak of FN remained essentially unchanged, and no new peaks were observed. 19 The appearance of the F resonance peak indicates that the presence of these anions or cations does not interfere with the detection of FN.

[0134] (2) Anti-interference performance of probe FN against amino acids

[0135] We also investigated whether the presence of some common amino acids would interfere with FN's identification of the analytes.

[0136] In this experiment, amino-terminal amino acids selected included phenylalanine (Phe), alanine (Ala), threonine (Thr), serine (Ser), lysine (Lys), glutathione (GSH), and cysteine ​​(Cys). These common amino acids were incubated with the probe FN, and changes in their resonance peaks were observed. Results are shown below. Figure 15 . Figure 15 The image shows the changes in the resonance peak signal of probe FN in Example 1 in the presence of a series of amino acids.

[0137] Depend on Figure 15 The results showed that after the addition of the above amino acids, no significant changes were observed in the resonance peak of the probe FN, nor were any new amino acids observed. 19 The appearance of the F resonance peak indicates that the presence of these amino acids does not affect the detection of the analyte by FN.

[0138] Test Example 4: Stability of Probe FN

[0139] Reactive oxygen species (ROS / RNS) typically possess strong oxidizing properties and may affect probe structure. Therefore, we investigated whether the presence of some common ROS / RNS species affects the probe FN. The results are shown below. Figure 16 . Figure 16 The image shows the change of the resonance peak signal of probe FN in Example 1 in the presence of ROS / RNS.

[0140] Depend on Figure 16 The experimental results show that when H2O2, ·OH, NO and ONOO are added to the FN solution... - When ROS / RNS species are present, the probe FN 19 The resonance peak of F did not change significantly, nor did any new resonance peak appear.

[0141] The experimental results above show that the probe FN has good chemical stability. The presence of ROS / RNS does not damage the structure of the probe FN and does not interfere with the detection of vicinal diols / phenols by FN.

[0142] Test Example 5: Detection at the Cellular Level

[0143] We explored the detection of the probe FN at the cellular level. PC12 cells, which secrete large amounts of norepinephrine, were used in this experiment. We co-incubated 100 μM of the probe FN with PC12 cells for a period of time, followed by nuclear magnetic resonance spectroscopy scanning. The results are shown below. Figure 17 . Figure 17 The results of the detection of norepinephrine secreted by PC12 cells using probe FN in Example 1 are shown.

[0144] Depend on Figure 17The results showed that after reacting with norepinephrine, the original FN probe... 19 The resonance peak of F has basically disappeared, and two new peaks have appeared in the high-field region. 19 The F magnetic resonance peaks were generated with chemical shifts of -136.0 ppm and -145.8 ppm, which are consistent with the chemical shift values ​​in the solution system.

[0145] The above experiments demonstrate that the FN probe can detect norepinephrine at the cellular level.

[0146] Test Example 6: Biocompatibility Assessment

[0147] We investigated the biosafety of the probe FN.

[0148] (1) Cytotoxicity assessment

[0149] Cytotoxicity was evaluated using CCK-8 assay. The toxicity of probe FN on cells was studied by CCK-8 assay, and the effect of different concentrations of FN solutions on the proliferation ability of PC12 cells was tested.

[0150] Mouse cells were incubated at 37°C and 5% CO2 for 48 hours. After cell adhesion and growth, solutions containing different concentrations of FN were added to different wells. The cells were then incubated at 37°C and 5% CO2 for 2 hours. After culture, CCK-8 was added to the wells, and incubation was continued at 37°C and 5% CO2 for 4 hours. After the culture was completed, the supernatant was removed, and the absorbance was measured at 450 nm.

[0151] The concentrations of FN solutions used were: 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM, 800 μM, and 1000 μM. After incubating cells with FN for 24 h, cell viability was calculated by absorbance. Results are shown below. Figure 18 . Figure 18 Figure A in the diagram represents a cytotoxicity experiment.

[0152] Cell viability (%) = (average absorbance of experimental group / average absorbance of blank group) × 100%.

[0153] Experiments have found that, for example Figure 18 As shown in Figure A, when the concentration of probe FN is as high as 1000 μM and the incubation time is as long as 48 h, the cell survival rate is still as high as 90% or more, which indicates that probe FN has low cytotoxicity.

[0154] (2) Biosafety of the probe at the in vivo level

[0155] The biocompatibility of FN was further investigated by intravenous injection in rats.

[0156] ICR mice (male, 4 weeks old) were housed in a barrier environment for 1 week to acclimatize. They were then randomly divided into 3 groups of 5 mice each: (1) Group I (control group): 100 μL PBS was injected via the tail vein; (2) Group II: 3-day short-term toxicity group; (3) Group III: 30-day medium- to long-term toxicity group.

[0157] For groups II and III, 500 μM FN solution was injected into ICR mice via the tail vein. The body weight of mice in group III was recorded every two days. Three and 30 days after injection, respectively, one mouse from groups II and III was sacrificed, and major organs such as the heart, liver, spleen, lungs, and kidneys were harvested for histopathological analysis using hematoxylin and eosin (H&E) staining. Simultaneously, blood samples from other mice in each group were collected for routine blood tests and blood biochemical assays. Results are shown below. Figure 18 and Figure 19 . Figure 18 Figure B shows the trend of mouse body weight change; Figure C shows the H&E staining of major organs. Figure 19 This is a comparison chart of blood indicator assessments.

[0158] The experimental results showed that, over a period of 30 days, the weight change trend of the rats was not significantly different from that of the control group. Figure 18 Figure B. Furthermore, blood biochemical parameters and hematoxylin-eosin (H&E) staining of major organs also showed no significant differences compared to the control group, such as... Figure 18 The C diagram and Figure 19 .

[0159] All of the above results indicate that the probe FN has low toxicity and high biosafety.

[0160] In summary, the embodiments of the present invention have designed and synthesized 19 F-labeled probes (FN) are used for the simultaneous identification of vicinal diols / phenols. The boric acid group in FN has a strong affinity for vicinal diols / phenols, and can undergo a specific chemical reaction to form borate esters, thereby causing... 19 The variation of the F resonance peak enables the simultaneous detection of vicinal diols / phenols. The ortho and meta fluorine atoms in the FN structure are sensitive to changes in spatial structure and electron cloud density, respectively, improving the identification and differentiation of a range of vicinal diols / phenols.

[0161] Experiments have confirmed that the probe FN can effectively identify and distinguish vicinal diols such as glucose and galactose. Furthermore, FN also demonstrates good ability to distinguish catechol neurotransmitters such as dopamine, adrenaline, and noradrenaline. More importantly, this probe can detect noradrenaline secreted in situ by PC12 cells. The good biocompatibility of FN lays the foundation for subsequent in vivo experiments. This work provides a new method for the simultaneous detection of a series of similar substances in complex biological systems.

[0162] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A kind 19 An F-labeled probe, characterized in that, Its chemical structural formula is shown below: Where X is a halogen.

2. As described in claim 1 19 An F-labeled probe, characterized in that, Its chemical structural formula is shown below:

3. A version according to claim 2 19 The method for preparing F-labeled probes is characterized by, Includes the following steps: (2,3-difluoro-4-methylphenyl)boronic acid, N-bromosuccinimide and benzoyl peroxide were refluxed in the first solvent system under a protective atmosphere. After the reaction was completed, intermediate compound F1 was prepared. Under a protective atmosphere, intermediate compound F1 and pyridine were reacted with stirring in a second solvent system at a reaction temperature of 65–75 °C. After the reaction was completed, the following compound was prepared: 19 F-labeled probes; The chemical structural formula of intermediate compound F1 is:

4. The method according to claim 3 19 The method for preparing F-labeled probes is characterized by, The molar ratio of (2,3-difluoro-4-methylphenyl)boronic acid, N-bromosuccinimide, and benzoyl peroxide is 1:1 to 1.2:0.03 to 0.

06. The molar ratio of intermediate compound F1 to pyridine is 1:1 to 1.

1.

5. The method according to claim 3 19 The method for preparing F-labeled probes is characterized by, The first solvent is carbon tetrachloride; the second solvent is N,N-dimethylformamide.

6. The method according to claim 3 19 The method for preparing F-labeled probes is characterized by, The reflux reaction is followed by a first purification, which includes: The solution after reflux reaction is transferred into water, extracted with dichloromethane, the organic phase is collected, and distilled to remove the solvent, thus obtaining the final product.

7. The method according to claim 3 19 The method for preparing F-labeled probes is characterized by, The reaction is followed by a second purification process, specifically: Add acetone and diethyl ether to the solution after stirring to precipitate the crude product. Centrifuge and wash the crude product with acetone and diethyl ether to obtain the final product.

8. A device as claimed in claim 1 19 The use of F-labeled probes in the preparation of reagents for detecting vicinal diols / phenols.

9. The use according to claim 8, characterized in that, The vicinal diols are monosaccharides; the phenolic substances are catechol neurotransmitters.

10. The use according to claim 9, characterized in that, The vicinal diol is glucose or galactose; the phenolic substance is dopamine, adrenaline, or noradrenaline.

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