CN113527193B

CN113527193B - Probe for nuclear magnetic fluorine spectrum detection and application of probe in detection of sulfhydryl compounds

Info

Publication number: CN113527193B
Application number: CN202010308265.3A
Authority: CN
Inventors: 李从刚; 柴兆斐; 吴琼; 成凯; 刘晓黎; 姜凌; 刘买利
Original assignee: Institute of Precision Measurement Science and Technology Innovation of CAS
Current assignee: Institute of Precision Measurement Science and Technology Innovation of CAS
Priority date: 2020-04-18
Filing date: 2020-04-18
Publication date: 2022-06-10
Anticipated expiration: 2040-04-18
Also published as: CN113527193A

Abstract

The invention belongs to the field of magnetic resonance detection, and particularly relates to a probe for nuclear magnetic fluorine spectrum detection and application thereof in detecting mercapto compounds, wherein the probe A or B can be used for detecting in a mixed system of an organic solvent and water aiming at a complex system, such as a complex system containing a fat-soluble substance to be detected, wherein the organic solvent can be acetonitrile or dimethyl sulfoxide, and the volume ratio of the organic solvent to the dimethyl sulfoxide is 25-70%; for a water-soluble object to be detected or a biological material, the probe B can be used for detection in a water phase; the probe provided by the invention can be used for realizing the detection of the content of mercaptan in a medicament or the recognition and metabolism observation of mercaptan in living cells, and can also be mixed with gamma-glutamic acid-cysteine to detect the activity of gamma-glutamyl transpeptidase.

Description

Probe for nuclear magnetic fluorine spectrum detection and application of probe in detection of sulfhydryl compounds

Technical Field

The invention belongs to the field of magnetic resonance detection, and particularly relates to a probe for nuclear magnetic fluorine spectrum detection and application thereof in detection of mercapto compounds.

Technical Field

Thiols are a class of compounds that are widely found in biological systems, pharmaceutical engineering and synthetic fields, and may be abbreviated as RSH. In biological systems, biological thiols are involved in signaling, oxidative stress, metabolic regulation, etc., and abnormal thiol levels are often accompanied by disease. Thiol drugs such as penicillamine, tiopronin and methimazole also exhibit different therapeutic effects due to their different structures. However, some thiol contamination can also be physically unpleasant and even cause serious medical conditions. Therefore, the development of a thiol detection method has important significance for clinical diagnosis, drug metabolic process monitoring and pollution assessment. However, due to the chemical similarity of these compounds, many techniques have difficulty responding characteristically to multiple thiols simultaneously.

Nuclear magnetic resonance technology can provide structural and kinetic information at the atomic level, and is a potential non-invasive detection method. Wherein¹⁹Nuclear magnetic resonance technique of F (¹⁹F NMR) due to the ratio of magnetic spin to¹The characteristics of similar H (83.4%), wide spectrum peak range (400 ppm), high natural abundance (100%), no biological background interference and the like are used for researching biological processes. But are currently used in complex systems¹⁹F NMR probes are still quite rare and do not achieve endogenous thiol recognition in biological systems.

Disclosure of Invention

In view of the defects in the prior art, the first object of the present invention is to provide a probe for nuclear magnetic fluorine spectrum detection, which has the following structural formula:

the invention also aims to provide a preparation method of the probe for nuclear magnetic fluorine spectrum detection.

The last purpose of the invention is to provide the application of the probe for nuclear magnetic fluorine spectrum detection in the detection of sulfhydryl compounds.

In order to achieve the purpose, the invention adopts the following technical measures:

the invention utilizes the rapid specific combination of the fluorine-containing probe and the mercaptan and the sensitivity of fluorine to the surrounding chemical environment to ensure that each mercaptan to be detected generates unique distinguishable¹⁹F, NMR spectrum and establishing a corresponding spectrum library.

Under the condition of being close to neutral or alkaline (pH: 6.0-9.0), the fluorine-containing sulfuryl compound and sulfydryl are subjected to specific reaction to obtain corresponding fluorine-containing thioester, and a corresponding fluorine spectrum is collected. Aiming at a complex system, such as a complex system containing a fat-soluble substance to be detected, a probe A or a probe B can be used for detecting in a mixed system of an organic solvent and water, wherein the organic solvent can be acetonitrile or dimethyl sulfoxide, and the volume percentage is 25-70%; for water-soluble analytes or biological materials, probe B can be used for detection in the aqueous phase. Wherein the fluorine-containing probe structure is:

the preparation method of the probe for nuclear magnetic fluorine spectrum detection comprises the following steps:

(1) the preparation method of the probe A comprises the following steps:

the method comprises the following steps: dispersing pentafluoropyridine and sodium benzene sulfinate in N, N-dimethylacetamide (or N, N-dimethylformamide), adding a phase transfer catalyst tetrabutylammonium bromide (or tetrabutylammonium chloride) to improve the solubility of sulfinate, reacting at 90-120 ℃ for 6-24 hours, cooling to room temperature, adding water, and filtering to obtain a white solid probe A;

(2) preparation method of probe B

Step 1: dispersing pentafluoropyridine and 4-methyl benzene sulfinic acid sodium in N, N-dimethyl acetamide (or N, N-dimethyl formamide), adding phase transfer catalyst tetrabutyl ammonium bromide (or tetrabutyl ammonium chloride) to raise the solubility of sulfinate, reaction at 90-120 deg.c for 6-24 hr, cooling to room temperature, adding water, filtering to obtain white solid C with structure as shown

Step 2: dissolving the solid C obtained in the step 1, N-bromosuccinimide and dibenzoyl peroxide in carbon tetrachloride, reacting for 6-24 hours at 50-80 ℃, cooling to room temperature, evaporating the solvent, and purifying to obtain a white solid compound D with the structure of

And 3, step 3: and (3) dissolving the solid D obtained in the step (2) and pyridine in acetonitrile, reacting for 2-24 hours at the temperature of 60-90 ℃, cooling to room temperature, filtering, and purifying to obtain a light yellow solid probe B.

The application of the probe for nuclear magnetic fluorine spectrum detection in the detection of sulfhydryl compounds comprises the following steps: each mercaptan to be detected generates unique distinguishable property by utilizing rapid specific combination of fluorine-containing probe and mercaptan and sensitivity of fluorine to surrounding chemical environment¹⁹F, NMR spectrum and establishing a corresponding spectrum library. Aiming at a complex system, such as a complex system containing a fat-soluble substance to be detected, a probe A or a probe B can be used for detecting in a mixed system of an organic solvent and water, wherein the organic solvent can be acetonitrile or dimethyl sulfoxide, and the volume percentage is 25-70%; for water-soluble analytes or biological materials, probe B can be used for detection in the aqueous phase. The method comprises the detection of the content of thiol in the medicine or the recognition and metabolic observation of thiol in living cells, and the probe provided by the invention can also be mixed with gamma-glutamic acid-cysteine to detect the activity of gamma-glutamyltranspeptidase.

Compared with the prior art, the invention has the following advantages:

the invention provides a probe for detecting mercapto compounds by nuclear magnetic fluorine spectrometry for the first time, the probe can be used for detecting fat-soluble and water-soluble mercapto compounds, can realize the detection of thiol groups in a complex system, and can finish the identification and metabolic observation of endogenous thiols in a biological system.

Drawings

Fig. 1 probe a illustrates the recognition of lipid soluble thiols.

FIG. 2 probe B illustrates the recognition of water-soluble thiols.

FIG. 3 is a schematic diagram of content detection of probe B on N-acetylcysteine effervescent tablets.

FIG. 4 is a schematic diagram showing the real-time detection of small molecule thiol in HeLa cells by probe B.

FIG. 5 is a schematic diagram showing the detection of gamma-glutamyl transpeptidase under different environments after thiol reaction of probe B;

wherein: a is a schematic diagram of detection of different activity gamma-glutamyl transpeptidase by modified probe B in dilute solution;

b is a schematic diagram of detection of gamma-glutamyl transpeptidase in Hela and A2780 cells by the modified probe B.

Detailed Description

For a better understanding of the present invention, the following examples are given in order to illustrate the present invention, but the present invention is not limited to the following examples.

The starting materials used in the examples of the present invention may be commercially available or may be synthesized by methods known in the art.

Example 1:

synthesis of Probe A

The synthetic route is as follows:

sodium benzenesulfinate (1.94g,11.83mmol), pentafluoropyridine (2.00g,11.83mmol) and tetrabutylammonium bromide (1.14g,3.54mmol) were dissolved in 10mL of N, N-dimethylacetamide under nitrogen, and stirred (600 rpm) at 100 ℃ for 20 hours. And cooling the reaction liquid to room temperature, slowly adding 30mL of deionized water, carrying out suction filtration, and washing with water for 3 times to obtain the product. The product was further purified by silica gel chromatography and dried under vacuum to give probe A (2.70g, 78.37%). The characterization data are as follows:¹H NMR(CDCl₃,500MHz)δ(ppm):8.13(d,2H,J＝8.0Hz),7.78(t,1H,J＝7.5Hz),7.65(t,2H,J＝7.5Hz).¹³C NMR(CDCl₃,126MHz)δ(ppm):145.24(dm,J_CF＝250.6Hz),139.33,138.95(dm,J_CF＝272.9Hz),135.84,133.26(t,J_CF＝13.0Hz),130.07,128.64.¹⁹F NMR(CDCl₃,471MHz)δ(ppm):-85.76(m,2F),-137.01(m,2F).HRMS(ESI,m/z):[M+H]⁺calcd forC₁₁H₆F₄NO₂S:292.0055；Found,256.0220.

example 2:

synthesis of Probe B

The synthetic route is as follows:

compound C sodium 4-methylbenzenesulfonate (1.11g,6.21mmol), pentafluoropyridine (1.00g,5.92mmol) and tetrabutylammonium bromide (0.57g,1.78mmol) were dissolved in 10mL of N, N-dimethylacetamide under nitrogen and stirred (600 rpm) at 100 ℃ for 20 hours. And cooling the reaction liquid to room temperature, slowly adding 15mL of deionized water, performing suction filtration, and washing with water for 3 times to obtain the product. The product was further purified by silica gel chromatography and dried in vacuo to give Compound C (1.55g, 85.8%). The characterization data are as follows:¹H NMR(CDCl₃,500MHz)δ(ppm):7.99(d,2H,J＝7.5Hz),7.43(d,2H,J＝8.5Hz).¹³C NMR(CDCl₃,126MHz)δ(ppm):147.50,144.41(dm,J_CF＝250.5Hz),138.75(dm,J_CF＝273.7Hz),136.839,133.60(t,J_CF＝13.0Hz),130.68,128.69.¹⁹F NMR(CDCl₃,471MHz)δ(ppm):-86.01(m,2F),-137.18(m,2F).HRMS(ESI,m/z):[M+H]⁺calcd forC₁₂H₈F₄NO₂S:306.0212；Found,306.0475.

compound D: under nitrogen protection, compound C (500mg,1.64mmol), N-bromosuccinimide (306mg,1.72 mmol) and dibenzoyl peroxide (20mg,0.082mmol) were dissolved in 15mL of carbon tetrachloride and stirred (600 rpm) at reflux overnight, and after completion of the reaction, saturated brine was added to the reaction solution, extracted with dichloromethane, and the organic phase was collected and dried over anhydrous sodium sulfate. The product was subjected to silica gel chromatography, separation and purification, and vacuum drying to obtain compound D (240mg, 38.1%). The characterization data are as follows:¹H NMR(CDCl₃,500MHz)δ(ppm):8.09(d,2H,J＝8.0Hz),7.66(d,2H,J＝8.0Hz),4.51(s,2H).¹³C NMR(CDCl₃,126MHz)δ(ppm):146.15,144.30(dm,J_CF＝241.9Hz),138.96,138.81(dm,J_CF＝268.4Hz),133.01(t,J_CF＝12.6Hz),130.59,129.23,30.87.¹⁹F NMR(CDCl₃,471MHz)δ(ppm):85.46(m,2F),136.87(m,2F).HRMS(ESI,m/z):[M+H]⁺calcd for C₁₂H₇BrF₄NO₂S:383.9317；Found,383.9592.

and probe B: under the protection of nitrogen, the compound is mixedD (105mg,0.27mmol) and anhydrous pyridine (26mg,0.33mmol) were dissolved in 3mL acetonitrile and stirred (600 rpm) at reflux overnight. After the reaction was complete, it was cooled to room temperature, filtered with suction and washed several times with dichloromethane to give product B as a pale yellow product (100mg, 78.8%). The characterization data are as follows:¹H NMR(MeOD-d₄,600MHz)δ(ppm):9.12(d,2H,J＝6.0Hz),8.67(t,1H,J＝7.8Hz),8.20(m,4H),7.81(d,2H,J＝8.4Hz),6.03(s,2H).¹³C NMR(MeOD-d₄,126MHz)δ(ppm):147.83,146.52,145.52(dm,J_CF＝248.1Hz),142.47,141.91,140.38(dm,J_CF＝271.2Hz),133.31(t,J_CF＝12.6Hz),131.40,130.65,130.01,64.49.¹⁹F NMR(MeOD-d₄,564MHz)δ(ppm):89.62(m,2F),139.61(m,2F).HRMS(ESI,m/z):[M-Br]⁺calcd for C₁₇H₁₁F₄N₂O₂S:383.0472；Found,383.0619.

example 3:

the application of the probe A and the probe B in the detection of the sulfhydryl compound:

the example comprises probe A for detecting fat-soluble thiol molecules, probe B for detecting water-soluble thiol molecules, probe B for detecting thiol drugs, probe B for monitoring the dynamic change of intracellular thiol molecules, and probe B and thiol reaction products for detecting gamma-glutamyl transferase activity.

1. The probe A is used as a detection probe of fat-soluble thiol molecules:

the fat-soluble thiol is exemplified by 4-methylphenylthiol, t-butylthiol, 2-mercaptobutane, 1-mercaptobutane, 2-mercaptopropane, 1, 2-ethanedithiol, ethanethiol; the solvent was phosphate buffer (100mM, pH 7.40, 30% acetonitrile, 5% water).

Each thiol (10mM) was mixed with probe A (100. mu.M) in a nuclear magnetic tube and then left at room temperature for 1h, and its nuclear magnetic fluorine spectrum (564MHz) was collected with sodium trifluoroacetate (-75.46ppm) as an internal standard, and the results of the measurement are shown in FIG. 1. After the compound A reacts with fat-soluble mercaptan, obvious chemical shift changes occur (the compound A and tert-butyl mercaptan do not completely react), and differences are also shown among different mercaptans, which indicates that the compound A has good mercaptan recognition capability in a mixed solvent system and can be further used for complex system detection.

2. The probe B is used as a detection probe of a water-soluble thiol molecule:

the thiols are exemplified by 6-mercaptopurine, methimazole, azulamine, tiopronin, N-acetylcysteine, 1, 2-ethanedithiol, 2-mercaptopropane, 1-mercaptopropane, ethanethiol, 3-mercaptopropionic acid, 2-mercaptoethanol, sodium thiomethoxide, dithiothreitol, glutathione, homocysteine, cysteine; the solvent was phosphate buffer (100mM, pH 7.40, 5% by weight water).

Wherein the ethanethiol, 1-mercaptopropane, 2-mercaptopropane and 1, 2-ethanedithiol which are slightly soluble in water contain 2% of DMSO.

Each thiol (10mM) was mixed with probe B (100. mu.M) in a nuclear magnetic tube, and the nuclear magnetic spectrum (564MHz) was collected by allowing 6-mercaptopurine (low solubility, saturated solution) having low mercapto activity and methimazole to stand at room temperature for 72 hours, and the results were measured using sodium trifluoroacetate (-75.46ppm) as an internal standard, as shown in FIG. 2. After the compound B reacts with thiol, obvious chemical shift changes (the compound A is not completely reacted with methimazole and 6-mercaptopurine) appear, and differences are shown among different thiols, so that the probe B has good thiol recognition capability and good water solubility, and can be further used for detecting biological materials.

3. Probe B for detecting thiol drugs

The thiol-group drug content of N-acetylcysteine effervescent Tablet (600mg/Tablet) was measured as an example. 1mM of standard N-acetylcysteine and phosphate buffer (100mM, pH 7.40) were prepared in each effervescent tablet. After dilution, a mixed solution (100mM, pH 7.40, 5% by weight of water) of the test substance (100 μ M) and probe B (400 μ M) was left at room temperature for 20 minutes, and the nuclear magnetic fluorine spectrum of the standard substance and the drug was measured. And finally determining the content of the sulfhydryl drug in the drug according to the comparison of the fluorine signal integral intensities of the standard substance and the drug to be detected. The test results are shown in fig. 3 and table 1. The result obtained by the method is very small in difference with the HPLC test result, which indicates that the method can be used for measuring the content of the thiol drugs.

TABLE 1 comparison of the content detection results of probe B on N-acetylcysteine effervescent tablets with the results of High Performance Liquid Chromatography (HPLC)

4. Probe B for dynamic change detection of thiol molecules in Hela cells

Cells (approximately 4 x 10 in number) harvested by pancreatin/EDTA (0.25%/0.02%) separation and centrifugation (200 xg)⁷) The sample was dispersed again in 0.3mL of DEME culture medium (90mM glucose, 5mM HEPES, 5% weight water), 2mM probe B was added, and the nuclear magnetic fluorine spectrum was continuously collected at room temperature, and the results are shown in FIG. 4. In the process of detection for 24h, the probe B gradually disappears initially; characteristic peaks for cysteine, glycine-cysteine and glutathione appear in the initial stage, and finally glycine-cysteine and glutathione are all converted into cysteine with the time. It was shown that probe B can be used for real-time dynamic monitoring of intracellular thiols.

5. The probe B reacts with biological thiol glycine-cysteine to detect the activity of the intracellular gamma-glutamyl transferase

Thiol-containing γ -glutamic acid-cysteine (3.0mM) was reacted with probe B (2.0mM) in phosphate buffer (100mM, pH 7.40) to give the compound γ -Glu-Cys-Py_4FHas a structure of

The compound is degraded into Cys-Py under the action of gamma-glutamyl transpeptidase_4FHas a structure of

The F signals in the two compounds show different chemical shifts and can be used to detect gamma-glutamyltranspeptidase activity in cells.

At room temperature, in the presence of gamma-Glu-Cys-Py_4FTo the solution (100. mu.M), 25, 50, 100, 200, 500U/L of gamma-glutamyl transglutaminate was addedThe nuclear magnetic fluorine spectrum of the enzyme was continuously measured, and the result is shown as A in FIG. 5, wherein the probe gamma-Glu-Cys-Py_4FThe degradation rate is accelerated along with the increase of the activity of the gamma-glutamyl transferase, which shows that the probe can reflect different activities of the gamma-glutamyl transferase; adding the probe gamma-Glu-Cys-Py into different cell lines Hela and A2780 (the cell processing method is the same as 4)_4F(170. mu.M) and the nuclear magnetic fluorine spectrum was continuously measured at room temperature, and the result is shown as B in FIG. 5, in which the probe γ -Glu-Cys-Py_4FThe degradation rate in Hela cells is obviously higher, which indicates that the method can reflect the activity of gamma-glutamyl transferase in different cells.

Claims

1. A probe for nuclear magnetic fluorine spectrum detection, wherein the structural formula of the probe is as follows:

2. the method for preparing the probe for nuclear magnetic fluorine spectrum detection according to claim 1:

(1) the method for preparing a probe according to claim 1, wherein R is H:

the method comprises the following steps: dispersing pentafluoropyridine and sodium benzene sulfinate in N, N-dimethylacetamide or N, N-dimethylformamide, adding a phase transfer catalyst tetrabutylammonium bromide or tetrabutylammonium chloride to improve the solubility of sulfinate, reacting at 90-120 ℃ for 6-24 hours, cooling to room temperature, adding water, and filtering to obtain a white solid probe A;

(2) r in claim 1 is

The preparation method of the probe comprises the following steps:

step 1: dispersing pentafluoropyridine and 4-methyl benzene sulfinic acid sodium in N, N-dimethyl acetamide or N, N-dimethyl formamide, adding phase transfer catalyst tetrabutyl ammonium bromide or tetrabutyl ammonium chloride to raise the solubility of sulfinate, reaction at 90-120 deg.c for 6-24 hr, cooling to room temperatureAdding water at room temperature, and filtering to obtain white solid C with structure of

And step 3: and (3) dissolving the solid D obtained in the step (2) and pyridine in acetonitrile, reacting at 60-90 ℃ for 2-24 hours, cooling to room temperature, filtering, and purifying to obtain a light yellow solid probe B.

3. Use of the nuclear magnetic fluorine spectrometry detection probe of claim 1 for preparing a probe for detecting a mercapto compound.

4. The use according to claim 3, wherein said sulfhydryl compounds comprise fat-soluble sulfhydryl compounds and water-soluble sulfhydryl compounds.

5. The use according to claim 3 for detecting the thiol content of a drug.

6. The use according to claim 3, for the recognition and metabolic observation of thiols in living cells.

7. The use according to claim 3 for the detection of gamma-glutamyl transpeptidase in admixture with gamma-glutamic acid-cysteine.