Water-soluble fluorine-containing palladium complex analysis reagent

Abstract

The invention provides a water-soluble fluorine-containing palladium complex probe. Specifically, the invention provides a fluorine-containing reagent which has a structure shown in the specification and can be used for distinguishing and identifying water-soluble nitrogen-containing biological small molecule metabolites and a preparation method of the reagent. The reagent can be combined with a nuclear magnetic resonance fluorine spectrum technology to realize rapid detection of various biological metabolites, and an object to be analyzed does not need to have ultraviolet absorption or crystallinity. The reagent can be used for selectively complexing nitrogen-containing heterocyclic ring and amino acid water-soluble analytes in a water phase and other high-polarity solvents, so that accurate detection of various biological metabolism micromolecules and a biological metabolism process in a complex system is realized, and the detection efficiency of multi-component and complex component samples is greatly improved. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry. Specifically, the present invention provides a water-soluble fluorine-containing palladium complex analysis, wherein the reagent can realize the rapid detection of multiple amino acid enantiomers and the analysis and detection of multiple biological metabolites and biological metabolic processes, without requiring the sample to have ultraviolet absorption or crystallinity. The method can also be used for the qualitative and quantitative analysis of amino acid enantiomers and biological metabolites in complex systems, especially under physiological conditions. Background Art

[0002] Amino acid enantiomers play an extremely important role in the fields of life sciences, chemistry, and pharmaceuticals. The universal importance and wide application of amino acids have led researchers to put forward new requirements for the accuracy and speed of methods for detecting amino acids. At present, the methods for identifying amino acid enantiomers mainly include chiral high performance liquid chromatography, fluorescence, circular dichroism, and X-ray single crystal diffraction. These methods usually require purified samples and have certain requirements on the light absorption properties or crystallinity of the samples. At the same time, the cultivation of single crystals and the screening of chromatographic separation conditions are relatively time-consuming and difficult to use for the rapid differentiation and detection of a large number of samples. In addition, amino acids are widely present in aqueous solutions, and the detection of amino acids using the above methods usually needs to be carried out in aqueous solutions or highly polar organic solvents.

[0003] In addition to amino acids, the precise detection of other biological metabolites containing nitrogen heterocycles is also a great challenge. Methods and technologies for the precise detection of complex biological samples and metabolic processes are of great significance for the rapid development of biological mechanism research, disease diagnosis and other fields. Biological metabolism is a complex dynamic process and is the basis for maintaining life and physiological functions. Metabolic abnormalities are often closely related to diseases, and differences in the types and contents of small molecule metabolites can provide important references for early diagnosis of diseases. Studies on enzyme metabolic kinetics can also provide a path for drug screening.

[0004] Nuclear magnetic resonance spectroscopy is a non-separation-based test method. Since its detection relies only on low-energy radio frequency radiation and is non-destructive, it has been widely used in the fields of biological mechanism research and disease diagnosis. However, when using nuclear magnetic resonance to directly analyze biological metabolites, there are problems such as complex spectra, low sensitivity, and susceptibility to matrix effects. In addition, in highly polar organic solvents and water, chiral chemical shift reagents are difficult to apply to the analysis of chiral substrates such as amino acids.

[0005] In summary, existing detection reagents and methods are difficult to achieve accurate in-situ detection of chiral amino acids, multiple biological metabolites, or simultaneous detection. Therefore, it is very necessary to develop a probe that can perform quantitative and qualitative analysis of chiral amino acids and biological metabolites by nuclear magnetic resonance spectroscopy in water-soluble and highly polar solvents when the sample does not have ultraviolet absorption or crystallinity. Summary of the invention

[0006] The purpose of the present invention is to provide a method for quickly and accurately detecting biological metabolites and amino acid enantiomers.

[0007] Another object of the present invention is to provide a method for synthesizing the probe molecule of the present invention and its precursor molecule.

[0008] In a first aspect of the present invention, a water-soluble chiral fluorine-containing palladium complex probe is provided, wherein the complex has a structure shown in the following formula A:

[0009]

[0010] Specifically, a water-soluble chiral fluorine-containing palladium complex probe is provided, wherein the complex has a structure shown in the following formula I:

[0011]

[0012] Wherein, n is an integer of 1 or 0.

[0013] R 1 Selected from the following group: substituted or unsubstituted C1-C10 alkyl; wherein the substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the following group: halogen, C1~C4 haloalkyl.

[0014] R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 A group independently selected from the following group: hydrogen atom, halogen, sulfonic acid group, trialkylamino group, substituted or unsubstituted C1-C20 alkyl group, substituted or unsubstituted C1-C10 alkoxy group, substituted or unsubstituted C6-C20 aryl group, substituted or unsubstituted C4-C20 heteroaryl group, or a water-soluble group selected from the following group: O(CH2CH2O) p CH3, or two groups located on adjacent carbon atoms form -O (CH2CH2O) p, wherein p is selected from the following group 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein the heteroaryl skeleton has 1 to 3 heteroatoms selected from O or S; wherein the substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the following group: halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl.

[0015] And when n is an integer 1, The structure is chiral, and its absolute configuration is R or S.

[0016] And when R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 There are no two groups on adjacent carbon atoms forming -O(CH2CH2O) p In this case, there is at least one O(CH2CH2O) in the above group. p CH3.

[0017] In a preferred embodiment, the probe according to the first aspect of the present invention has a structure as shown in the following formula Ia:

[0018]

[0019] Where R 1 , R 4 , R 9 and R 10 Among them, at least one is selected from a fluorine atom or a fluorine-substituted group;

[0020] Or the probe has a structure as shown in the following formula Ib:

[0021]

[0022] As described in the first aspect of the present invention, the complex wherein said R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11A group independently selected from the following group: hydrogen atom, halogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy; or a water-soluble group selected from the following group: O(CH2CH2O) p CH3, or two groups on adjacent carbon atoms form -O (CH2CH2O) p , wherein p is selected from 3, 4, 5, 6, 7, 8, 9 or 10.

[0023] In another preferred embodiment, the complex according to the first aspect of the present invention has a structure selected from the following group:

[0024]

[0025] The second aspect of the present invention provides a water-soluble chiral fluorine-containing palladium complex probe, wherein the complex has a structure shown in the following formula II:

[0026]

[0027] Among them, n, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 As described in the first aspect of the present invention respectively.

[0028] L is a non-acetonitrile compound or anion that can complex with palladium;

[0029] Specifically, L is a molecule or anion selected from the group consisting of: C1 to C 20 Chain or cyclic amines, C3~C 20 Chain or cyclic nitriles, C1~C 20 Chain or cyclic thiols, C1~C 20 Chain or cyclic sulfide, C1~C 20 Chain or cyclic amides, C3-~C 30 Nitrogen-containing heterocyclic ring; C3~C 30 sulfur-containing heterocyclic rings; chloride ions (Cl-), bromide ions (Br - ), iodide ion (I-), azide (N3 - ), thiocyanate (SCN - ), cyanate (CN - )、C1~C 20 of carboxylate ions.

[0030] In a preferred embodiment, the probe has a structure shown in the following formula II-a or formula II-b:

[0031]

[0032] In another preferred embodiment, the probe is formed by complexing the complex described in the first aspect of the present invention with L.

[0033] The third aspect of the present invention provides a ligand for preparing the compound of formula I as described in the first aspect of the present invention, wherein the ligand has a structure shown in the following formula III:

[0034]

[0035] Among them, n, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 As described in the first aspect of the present invention respectively.

[0036] Preferably, the ligand has a structure shown in the following formula III-a or III-b:

[0037]

[0038] In another preferred embodiment, the ligand has a structure selected from the following group:

[0039]

[0040] The fourth aspect of the present invention provides a method for preparing the compound of formula I as described in the first aspect of the present invention, wherein the method comprises the steps of:

[0041]

[0042] The compound of formula III described in the third aspect of the present invention and palladium acetate are added to a reaction bottle, acetonitrile is added to dissolve, and stirred at a temperature of 20-80° C. for 2 to 24 hours to obtain the compound of formula I described in the first aspect of the present invention.

[0043] In another preferred embodiment, the reaction temperature may be 40-80°C.

[0044] The fifth aspect of the present invention provides a method for synthesizing the compound of formula II as described in the second aspect of the present invention, wherein the method comprises the steps of:

[0045]

[0046] In a solvent, the compound of formula I as described in the first aspect of the present invention is mixed with L to obtain a compound of formula II;

[0047] Wherein, L is a non-acetonitrile compound or anion that can be complexed with palladium.

[0048] Specifically, L is a molecule or anion selected from the group consisting of: C1 to C 20 Chain or cyclic amines, C3~C 20 Chain or cyclic nitriles, C1~C 20 Chain or cyclic thiols, C1~C 20 Chain or cyclic sulfide, C1~C 20 Chain or cyclic amides, C3~C 30 Nitrogen-containing heterocyclic ring; C3~C 30 sulfur-containing heterocyclic rings; chloride ions (Cl - ), bromide ion (Br - ), iodide ion (I - ), azidoate (N3 - ), thiocyanate (SCN - ), cyanate (CN - )、C1~C 20 of carboxylate ions.

[0049] In another preferred embodiment, the organic solvent is selected from the following group: C1-C6 alkane solvents, aromatic solvents, C1-C6 alcohol solvents, C1-C6 ether solvents, C1-C6 nitrile solvents, sulfone solvents, C1-C6 ester solvents, or a combination thereof.

[0050] In another preferred embodiment, the organic solvent is selected from the following group: chloroform, dichloromethane, toluene, n-hexane, benzene, chlorobenzene, petroleum ether, carbon tetrachloride, acetonitrile, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, acetone, methanol, ethanol, isopropanol, dimethyl sulfoxide, water, or a combination thereof.

[0051] In another preferred embodiment, the organic solvent is a deuterated solvent or a non-deuterated solvent.

[0052] The sixth aspect of the present invention provides a method for using the compound of formula I as described in the first aspect of the present invention to analyze the chiral composition of amino acids and other biological metabolites.

[0053] Specifically, the present invention provides a method for distinguishing and identifying amino acid enantiomers, wherein the method comprises the steps of:

[0054] (1) In a highly polar solvent, the compound of formula I (n is an integer of 1) as described in the first aspect of the present invention is mixed with the chiral amino acid enantiomer to be detected, and a certain amount of base is added to obtain a complex solution of the probe molecule and the amino acid;

[0055] (2) performing fluorine spectrum nuclear magnetic resonance detection on the complex solution of the probe molecule and the amino acid to obtain two groups of characteristic fluorine spectrum nuclear magnetic resonance signals, which correspond to amino acids with different absolute configurations;

[0056] (3) By integrating and calculating the two sets of fluorine spectrum NMR signals, the enantiomeric composition information of the chiral amino acids in the sample can be obtained.

[0057] In another preferred embodiment, the amino acid to be tested may be a natural amino acid or a non-natural amino acid.

[0058] In another preferred embodiment, the amino acid to be tested contains a structural fragment selected from the following group: hydroxyl, amide, carboxylic acid, sulfoxide, ester, ether, nitrogen oxide, amino, nitro, and oxazoline ring.

[0059] In another preferred embodiment, the highly polar solvent is selected from the group consisting of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, acetone, methanol, ethanol, and water.

[0060] In another preferred embodiment, the solvent may be a deuterated solvent or a non-deuterated solvent.

[0061] In another preferred embodiment, the base is selected from the following group: sodium hydroxide, sodium hydride, sodium carbonate, sodium bicarbonate, sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium hydroxide, potassium carbonate, potassium bicarbonate, lithium hydroxide, lithium carbonate, magnesium hydroxide, calcium hydroxide, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, or a combination thereof.

[0062] Specifically, the present invention provides an analytical method for in situ detection of enzyme metabolic processes in a nuclear magnetic resonance tube, comprising the steps of:

[0063] (1) mixing the compound of formula I according to the first aspect of the present invention with a reaction substrate for enzyme metabolism in a prepared buffer to obtain a mixed solution of the probe and the substrate;

[0064] (2) adding an enzyme solution of a specific concentration to a mixed solution of the probe and substrate, and recording the time of enzyme addition;

[0065] (3) performing fluorine nuclear magnetic resonance detection on the sample to which the enzyme is added, and recording the time of each spectrum acquisition;

[0066] (4) By determining and analyzing the fluorine spectrum signal obtained in step (3), information on the changes in substrates and products over time during enzyme metabolism can be obtained.

[0067] In another preferred embodiment, the enzyme may be ATP hydrolase, glucose dehydrogenase, hexokinase, or fucosyltransferase.

[0068] In another preferred embodiment, the reaction substrate of the enzyme metabolism is selected from the following group: adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, nicotinamide adenine dinucleoside phosphate, reduced nicotinamide adenine dinucleotide phosphate, nicotinamide adenine nucleotide, reduced nicotinamide adenine dinucleotide, guanosine-5'-triphosphate, guanosine-5'-diphosphate, guanosine-5'-phosphate, thymidine-5'-phosphate, uridine-5'-phosphate, deoxyadenylic acid, deoxythymidylic acid, deoxyguanosine monophosphate, deoxycytidylic acid.

[0069] In another preferred embodiment, the buffer solution can be prepared using deuterated water, non-deuterated water, or a mixed solution of the two.

[0070] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The schematic diagram of the probe molecule of the present invention for identifying and detecting amino acids of different absolute configurations. After the amino acid enantiomers bind to the probe molecule, a signal peak corresponding to each analyte can appear at a specific chemical position, and the detection of amino acid enantiomers can be achieved without separation. For example, the red and blue fluorine spectrum signals in the figure are generated by amino acids of different configurations, respectively.

[0072] Figure 2 This is a diagram showing the effect of probe molecule I-a3 on distinguishing and recognizing amino acid enantiomers in the presence of different bases.

[0073] Figure 3 This is a diagram showing the effect of probe molecule I-a3 in distinguishing and recognizing different amino acids.

[0074] Figure 4 This is a diagram showing the effect of probe molecule I-a1 in distinguishing and recognizing different amino acids.

[0075] Figure 5 This is a diagram showing the effect of probe molecule I-a2 in distinguishing and recognizing leucine.

[0076] Figure 6 This is a diagram showing the effect of probe molecule I-b1 in distinguishing and identifying different biological metabolic molecules.

[0077] Figure 7 This is a diagram showing the effect of probe molecule I-b2 in distinguishing and identifying different biological metabolic molecules.

[0078] Figure 8 This is a diagram showing the effect of probe molecule I-b3 in distinguishing and identifying different biological metabolic molecules.

[0079] Fig. 9 This is a diagram showing the effect of probe molecule I-b4 in distinguishing and identifying different biological metabolic molecules.

[0080] Fig.10 This is a diagram showing the effect of probe molecule I-b5 in distinguishing and identifying different biological metabolic molecules.

[0081] Fig.11 This is a diagram showing the effect of probe molecule I-b6 in distinguishing and identifying different biological metabolic molecules.

[0082] Fig.12 This is a diagram showing the effect of probe molecule I-b7 in distinguishing and identifying different biological metabolic molecules.

[0083] Fig.13 This is a diagram showing the detection effect of probe molecule I-b1 on glucose dehydrogenase activity.

[0084] Fig.14 This is a diagram showing the detection effect of probe molecule I-b1 on ATP hydrolase activity.

[0085] Fig.15 This is a diagram showing the detection effect of probe molecule I-b1 on type I hexokinase activity. DETAILED DESCRIPTION

[0086] After long-term and in-depth research, the inventors have designed and prepared a series of water-soluble fluorine-containing palladium complex nuclear magnetic probe molecules. After this type of probe molecule is complexed with a specific analyte in a solution, its nuclear magnetic resonance fluorine spectrum is measured, and by analyzing the specific fluorine spectrum chemical shift signal, the absolute configuration and type of the analyte can be distinguished and detected. In addition, by integration and calculation, information such as the content of various analytes in the measured sample and the enantiomeric composition of chiral analytes can also be obtained. After introducing a suitable chiral group into this type of probe, it can be used as a reagent to distinguish and identify amino acid enantiomers. In addition, this type of probe can also be used for the identification of biological metabolites and real-time detection of enzyme reaction kinetics. The preparation conditions of this type of reagent are mature, and the dosage is very low during each analysis, which is suitable for mass production and commercial promotion and application. On this basis, the inventors have completed the present invention.

[0087] the term

[0088] As used herein, the term "C1-C20 alkyl" refers to a straight or branched alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

[0089] The term "C6-C20 aryl" refers to an aryl group having 6 to 20 carbon atoms, including monocyclic or polycyclic aryl groups, such as phenyl, naphthyl, or similar groups. In a preferred embodiment, the aryl group may be a C6-C10 aryl group.

[0090] The term "5-20 membered heteroaryl" refers to a heteroaryl group having 5 to 20 ring atoms (having 1 to 8 heteroatoms selected from N, O or S in the backbone), such as pyrrolyl, pyridyl, furanyl, or the like.

[0091] The term "substituted" means that one or more hydrogen atoms on the group are replaced by substituents selected from the following groups: C1~C10 alkyl, C3~C10 cycloalkyl, C1~C20 alkoxy, halogen, hydroxyl, carboxyl (~COOH), C1~C20 aldehyde, C2~C10 acyl, C2~C10 ester, amino, phenyl; the phenyl group includes unsubstituted phenyl or substituted phenyl with 1-3 substituents, and the substituents are selected from: halogen, C1~C10 alkyl, cyano, OH, nitro, C3~C10 cycloalkyl, C1~C20 alkoxy, amino.

[0092] The term "halogen" refers to F, Cl, Br and I.

[0093] The term "water-soluble group" refers to O(CH2CH2O) p CH3, or two groups located on adjacent carbon atoms form -O (CH2CH2O) p , wherein p is selected from the following group 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0094] Water-soluble probes

[0095] The present invention provides a water-soluble fluorine-containing palladium complex analysis. After the fluorine-containing metal complex probe is complexed with biological metabolites and chiral amino acids, a fluorine signal can be generated at a specific chemical shift, thereby realizing the detection of metabolites and amino acid enantiomers without separation.

[0096] Specifically, the chiral complex has a structure shown in the following formula II:

[0097]

[0098] Among them, n, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11As described in the first aspect of the present invention respectively;

[0099] L is a non-acetonitrile compound or anion that can complex with palladium;

[0100] Specifically, L is a molecule or anion selected from the group consisting of: C1 to C 20 Chain or cyclic amines, C3~C 20 Chain or cyclic nitriles, C1~C 20 Chain or cyclic thiols, C1~C 20 Chain or cyclic sulfide, C1~C 20 Chain or cyclic amides, C3-~C 30 Nitrogen-containing heterocyclic ring; C3~C 30 sulfur-containing heterocyclic rings; chloride ions (Cl - ), bromide ion (Br - ), iodide ion (I - ), azidoate (N3 - ), thiocyanate (SCN - ), cyanate (CN - )、C1~C 20 of carboxylate ions;

[0101] In a preferred embodiment, the probe has a structure shown in the following formula II-a or formula II-b:

[0102]

[0103] Synthesis method of water-soluble fluorine-containing probe

[0104] The fluorine-containing chiral metal complex probe of the present invention, i.e., the compound of formula I, can be synthesized by the following design route:

[0105] Route 1:

[0106]

[0107] In a first type of solvent, the compound of formula III is reacted with a palladium complex to obtain a compound I; wherein acetonitrile is an organic solvent coordination molecule;

[0108] The reaction is carried out between 20 degrees Celsius and 80 degrees Celsius.

[0109] Preferably, the palladium complex is palladium acetate or tetraacetonitrile palladium chloride.

[0110] The above route is to obtain a complex by the reaction of metal and ligand, and the final fluorine-containing metal complex probe will be complexed with solvent molecules to obtain a stable complex.

[0111] In addition, the probe molecule shown in formula II can also be obtained by complexing or ligand exchange between the compound shown in formula I and a solvent molecule or analyte molecule L that can be complexed.

[0112] For example, route 2:

[0113]

[0114] In a coordination solvent or in the presence of a coordination molecule L, a complex of formula I is added, and a corresponding complex of formula II is obtained by ligand exchange;

[0115] Wherein, L is a non-acetonitrile compound or anion capable of complexing with palladium. Specifically, L is a molecule or anion selected from the group consisting of: C1 to C 20 Chain or cyclic amines, C3~C 20 Chain or cyclic nitriles, C1~C 20 Chain or cyclic thiols, C1~C 20 Chain or cyclic sulfide, C1~C 20 Chain or cyclic amides, C3~C 30 Nitrogen-containing heterocyclic ring; C3~C 30 sulfur-containing heterocyclic rings; chloride ions (Cl - ), bromide ion (Br), iodide ion (I - ), azidoate (N3 - ), thiocyanate (SCN - ), cyanate (CN - )、C1~C 20 of carboxylate ions.

[0116] In another preferred embodiment, the organic solvent is selected from the following group: C1-C6 alkane solvents, aromatic solvents, C1-C6 alcohol solvents, C1-C6 ether solvents, C1-C6 nitrile solvents, sulfone solvents, C1-C6 ester solvents, or a combination thereof.

[0117] In another preferred embodiment, the organic solvent is selected from the following group: chloroform, dichloromethane, toluene, n-hexane, benzene, chlorobenzene, petroleum ether, carbon tetrachloride, acetonitrile, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, acetone, methanol, ethanol, isopropanol, dimethyl sulfoxide, water, or a combination thereof.

[0118] In another preferred embodiment, the organic solvent is a deuterated solvent or a non-deuterated solvent.

[0119] A method for distinguishing and detecting water-soluble metabolites and chiral amino acids in biological systems

[0120] The rapid and accurate detection of complex biological samples has extremely important applications in biological mechanism research, disease diagnosis, etc. In view of the key problem that existing detection reagents and methods are difficult to achieve simultaneous in-situ accurate detection of multiple biological metabolites, the inventors have developed a class of water-soluble fluorine-containing probes, combined with nuclear magnetic resonance technology to achieve simultaneous accurate tracking and detection of multiple metabolites in biological metabolic reactions, providing a more accurate detection method for enzyme inhibitor screening, metabolic pathway research, and disease screening.

[0121] Based on the above probe design, the inventors also provide a method for analyzing water-soluble fluorine-containing palladium complexes. The analysis principle of the method is: after the water-soluble fluorine-containing metal complex probe is complexed with biological metabolites and chiral amino acids, a fluorine signal can be generated at a specific chemical shift. These nuclear magnetic signals do not change with the change of the analyte content and ratio, and are characteristic signals. When the chiral probe molecule is complexed with amino acids of different configurations, different characteristic signals will be generated, thereby realizing the distinction and identification of amino acid enantiomers. When the probe reversibly interacts with the biological metabolite, it will cause the change of the chemical shift of the fluorine atoms around the cavity, and produce a series of fluorine spectrum signals with specific chemical shifts, i.e., characteristic signals. These signals correspond one to one with the biological metabolites in the complex system, and the detection of metabolites can be realized without separation. Because after the analyte is complexed with the probe molecule, the integral area ratio of the characteristic nuclear magnetic signal obtained has a linear relationship with the ratio of the enantiomers in the chiral sample, so that the composition of the chiral sample can be quantitatively analyzed by nuclear magnetic integration, and then the composition of the chiral sample can be determined. Because after the complexation of different metabolite probe molecules, the integral area ratio of the characteristic NMR signal obtained has a linear relationship with the metabolite content ratio, the NMR integration can be used to quantitatively analyze the composition of different metabolites, and then determine the composition of the metabolite sample and the changes in the metabolic process.

[0122] Specifically, the present invention provides a method for quickly and conveniently determining water-soluble chiral amino acids, the method comprising:

[0123] (1) In an inert solvent, mixing the compound of formula I (n is an integer of 1) according to the first aspect of the present invention with the chiral enantiomer to be tested and a trace amount of base to obtain a second complex;

[0124] (2) treating the second complex 19 F NMR, respectively, obtained new characteristic fluorine spectrum signals I (left) and I (right) ; Among them, I (left) Refers to the integral value of the fluorine spectrum on the left side of the measurement, I (right) Refers to the integral value of the fluorine spectrum on the right side of the measurement;

[0125] (3) Determining the configuration of the chiral enantiomer to be measured by the peak area ratio of the fluorine spectrum signal obtained in step (2).

[0126] Specifically, the present invention provides a method for quickly and conveniently determining water-soluble metabolites in a biological system, the method comprising:

[0127] (1) mixing the compound of formula I according to the first aspect of the present invention with a metabolic analyte to be detected in a buffer solution to obtain a second complex;

[0128] (2) treating the second complex 19 F NMR, obtain new characteristic fluorine spectrum signals;

[0129] (3) Comparing the fluorine spectrum signal with the standard test signal in the database can determine the type of the metabolite;

[0130] (4) A specific amount of a known water-soluble analyte that can complex with the probe is added to the test solution, and the concentration of the biological analyte to be tested is determined by integrating the fluorine spectrum signal.

[0131] In another preferred embodiment, the chiral amino acid enantiomer to be detected contains a structural fragment selected from the group consisting of: C1 to C 20 Chain or cyclic amines, C3~C 20 Chain or cyclic nitriles, C1~C 20 Chain or cyclic thiols, C1~C 20 Chain or cyclic sulfide, C1~C 20 Chain or cyclic amides, C3~C 30 Nitrogen-containing heterocyclic ring; C3~C 30 sulfur-containing heterocyclic rings; chloride ions (Cl - ), bromide ion (Br - ), iodide ion (I - ), azidoate (N3 - ), thiocyanate (SCN - ), cyanate (CN - )、C1~C 20 of carboxylate ions.

[0132] In another preferred embodiment, the metabolic analyte to be detected contains a structural fragment or ion selected from the following groups: hydroxyl, amide, carboxylic acid, sulfoxide, ester, ether, nitrogen oxide, amino, nitro, oxazoline ring; chloride ion (Cl - ), bromide ion (Br), iodide ion (I - ), azidoate (N3 - ), thiocyanate (SCN - ), cyanate (CN - )、C1~C 20 of carboxylate ions.

[0133] In another preferred embodiment, the inert solvent is selected from the following group: C1-C6 alkane solvents, aromatic solvents, C1-C6 alcohol solvents, C2-C6 ether solvents, C1-C6 nitrile solvents, sulfone solvents, C2-C6 ester solvents, or a combination thereof; preferably chloroform, dichloromethane, toluene, n-hexane, benzene, chlorobenzene, petroleum ether, carbon tetrachloride, acetonitrile, ethyl acetate, tetrahydrofuran, methanol, ethanol, isopropanol, water, dimethyl sulfoxide, or a combination thereof.

[0134] In another preferred embodiment, the solvent may be a deuterated solvent or a non-deuterated solvent.

[0135] In the above method, the mixing ratio of the fluorine-containing chiral probe complex represented by formula II and the chiral analyte is not limited, and is preferably 0.01-10.

[0136] In the above method, the test temperature of nuclear magnetic resonance is -78-80℃.

[0137] Specifically, the present invention provides an analytical method for in situ detection of enzyme metabolic processes in a nuclear magnetic resonance tube, the method comprising:

[0138] (1) Mixing the compound of formula I according to the first invention of the present invention with a reaction substrate for enzyme metabolism in a prepared buffer to obtain a mixed solution of the probe and the substrate;

[0139] (2) adding an enzyme solution of a specific concentration to a mixed solution of the probe and substrate, and recording the time of enzyme addition;

[0140] (3) performing fluorine nuclear magnetic resonance detection on the sample to which the enzyme is added, and recording the time of each spectrum acquisition;

[0141] (4) By determining and analyzing the fluorine spectrum signal obtained in step (3), information on the changes in substrates and products over time during enzyme metabolism can be obtained.

[0142] In another preferred embodiment, the enzyme may be ATP hydrolase, glucose dehydrogenase, hexokinase, or fucosyltransferase.

[0143] In another preferred embodiment, the reaction substrate of the enzyme metabolism can be adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, nicotinamide adenine dinucleoside phosphate, reduced nicotinamide adenine dinucleotide phosphate, nicotinamide adenine nucleotide, reduced nicotinamide adenine dinucleotide, guanosine-5'-triphosphate, guanosine-5'-diphosphate, guanosine-5'-phosphate, thymidine-5'-phosphate, uridine-5'-phosphate, deoxyadenylic acid, deoxythymidylic acid, deoxyguanosine monophosphate, and deoxycytidylic acid.

[0144] In another preferred embodiment, the buffer solution can be prepared using deuterated water, non-deuterated water, or a mixed solution of the two.

[0145] In the above method, the test temperature of nuclear magnetic resonance is -78-80℃.

[0146] 19 F NMR method for detecting target analytes

[0147] Nuclear magnetic resonance fluorine spectroscopy has the characteristics of high detection sensitivity, simple spectral analysis, and quantitative analysis; the method based on dynamic derivatization has the characteristics of rapid response and simple operation.

[0148] The present invention provides a fluorine-containing reagent that can be used to distinguish and identify water-soluble nitrogen-containing biological small molecule metabolites. Specifically, the present invention provides a water-soluble fluorine-containing palladium complex probe. Through nuclear magnetic resonance fluorine spectroscopy technology, the reagent can achieve rapid detection of multiple biological metabolites without requiring the analyte to have ultraviolet absorption or crystallinity. This method can selectively complex nitrogen-containing heterocyclic and amino acid water-soluble analytes in aqueous phase and other highly polar solvents, thereby achieving accurate detection of multiple biological metabolic small molecules and biological metabolic processes in complex systems, greatly improving the detection efficiency of multi-component and complex component samples.

[0149] Rapid and accurate detection of complex biological samples has extremely important applications in biological mechanism research, disease diagnosis, etc. The series of water-soluble fluorine-containing probes provided by the present invention can realize the simultaneous and accurate tracking and detection of multiple metabolites in biological metabolic reactions, providing a more accurate detection method for enzyme inhibitor screening, metabolic pathway research, and disease screening.

[0150] Compared with the prior art, the main advantages of the present invention are:

[0151] (1) The reagent of the present invention can detect amino acid enantiomers quickly and accurately, and the characterization signal is simple and easy to distinguish.

[0152] (2) The reagents of the present invention can realize the in situ accurate detection of multiple biological metabolites.

[0153] (3) The reagent of the present invention can be detected in an aqueous system.

[0154] (4) The reagent of the present invention can realize the detection of multiple amino acid enantiomers in a mixed system without the need to separate the amino acids after derivatization.

[0155] (5) The reagent of the present invention can realize the in situ accurate detection of multiple biological metabolites in a mixed system.

[0156] (6) The reagent of the present invention can realize in situ detection of various biological metabolic processes.

[0157] (7) The present invention has no requirements on the ultraviolet absorption properties and crystallinity of the analyte amino acid.

[0158] (8) The reagent preparation method of the present invention is mature and suitable for large-scale preparation.

[0159] (9) The method of the present invention can be widely applied to the analysis of various types of compounds, which can make up for the shortcomings of traditional chromatography technology to a certain extent, and has great application value in large-scale catalyst screening, drug development, quality control and biological metabolic process research.

[0160] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0161] Synthesis routes of the compounds involved:

[0162] There are two synthetic routes for the chiral acetonitrile-coordinated palladium complex probe.

[0163] Route 1:

[0164]

[0165] Route 2:

[0166]

[0167] The synthetic route of the achiral acetonitrile-coordinated palladium complex probe is as follows:

[0168]

[0169] Example 1: Introduction of water-soluble functional groups

[0170]

[0171] Weigh p-hydroxyphenol (3.0 g, 24.57 mmol) and Cs2CO3 (21.83 g, 67 mmol) in a 250 mL round-bottom flask and add a magnetic particle. Then add 150 mL of tetrahydrofuran, stir at room temperature for 10 min, add p-toluenesulfonyl-substituted polytetraethylene glycol monomethyl ether (8.09 g, 22.33 mmol) dissolved in THF to the round-bottom flask, and heat to 60 ° C. After reacting for 2 h, monitor by LC-MS or TLC. After the reaction is complete, wait for the reaction system to return to room temperature, filter the reaction solution with diatomaceous earth, and wash with dichloromethane (300 mL) 3 times. Remove the solvent at reduced pressure, purify by column chromatography (n-hexane / ethyl acetate = 1:1), and dry to obtain 4.24 g of a light yellow transparent liquid with a yield of 61%. 1H NMR(600MHz,Chloroform-d)δ9.88(s,1H),7.84-7.80(m,2H),7.04-7.00(m,2H),4.23-4.20(m,2H),3.89(dd ,J=5.5,4.1Hz,2H),3.73(dd,J=6.0,3.6Hz,2H),3.71-3.62(m,8H),3.56-3.52(m,2H),3.37(d,J=1.1Hz,3H).

[0172] Embodiment 2:

[0173]

[0174] The preparation method is the same as above, except that the compound Replaced raw material Va. Yellow oily liquid, yield 2.41g, 71%. 1 H NMR(400MHz, CDCl3) δ6.84(d,J=8.7Hz,1H),6.60(dd,J=2.7,1.4Hz,1H),6.55(dd,J=8.7,2.7Hz,1H),4.07 (t,2H),3.81(dd,J=5.7,4.2Hz,2H),3.74-3.70(m,2H),3.66-3.62(m,8H),3.56-3.51(m,2H),3.36(s,3H). 19 F NMR (376MHz,CDCl3)δ-57.94.

[0175] Embodiment 3:

[0176]

[0177] The preparation method is the same as above, except that the compound Replaced raw material Va. Yellow oily liquid, yield 2.98g, 85%. 1 H NMR (400MHz, CDCl3) δ6.89(d,J=2.8Hz,1H),6.85(d,J=8.7Hz,1H),6.79(dd,J=8.7,2.8Hz,1H),4.10(t,J =5.0Hz,2H),3.83(t,J=4.9Hz,2H),3.75-3.71(m,2H),3.68-3.61(m,8H),3.56-3.51(m,2H),3.36(s,3H); 19 F NMR (376MHz, CDCl3) δ-62.12.

[0178] Embodiment 4:

[0179]

[0180] The preparation method is the same as above, except that the compound Replaced raw material Va. Yellow oily liquid, yield 1.30g, 69%. 1 H NMR (400MHz, CDCl3) δ6.25-6.16(m,2H),4.12(t,J=5.6,4.0Hz,2H),3.77(t ,2H),3.72-3.68(m,2H),3.67-3.62(m,8H),3.56-3.52(m,2H),3.37(s,3H); 19 F NMR(376MHz, CDCl3)δ-127.96--128.07(m).

[0181] Example 5: Reaction with tert-butylsulfenamide

[0182]

[0183] Weigh S-tert-butylsulfenamide (1.89 g, 15.58 mmol) in a 100 mL round-bottom flask, add 50 mL of tetrahydrofuran, then add VI-1 (3.24 g, 10.39 mmol) dissolved in THF and tetraethyl titanate (7.11 g, 31.16 mmol) to the reaction flask, heat to 60 ° C, the reaction system is yellow. After 3 hours of reaction, monitor with LC-MS or TLC, the reaction is complete. After the system returns to room temperature, add 30 mL of water to produce a large amount of white flocculent solids, filter with diatomaceous earth, and wash with dichloromethane (150 mL). Remove the solvent under reduced pressure, purify by column chromatography (n-hexane / ethyl acetate = 1:1), and dry to obtain 3.64 g of yellow liquid with a yield of 88%. 1 H NMR(600MHz,Chloroform-d)δ8.49(dd,J=9.5,3.7Hz,1H),7.79(dp,J=9.2,3.0Hz,2H),6.98(dp,J=8.9,2.9Hz,2H),4.18(dq,J=8.3,4.3,3 .8Hz,2H),3.88(dt,J=8.9,4.9Hz,2H),3.68(ddddd,J=32.0,19.5,10.2,6.4,3.4Hz,10H),3.53(tq,J=7.0,3.2Hz,2H),3.38-3.32(m,3H).

[0184] Example 6: Trifluoromethylation followed by deprotection to obtain an ammonium salt

[0185]

[0186] Add a magnetic bar to a 200mL Schlenk reaction bottle, bake in an electric oven under vacuum for 2 minutes, cool to room temperature, replace nitrogen with vacuum 3 times, weigh TBAT (9.75g, 14.44mmol) in a reaction bottle under nitrogen protection, and add 60mL of ultra-dry tetrahydrofuran. Add 3-3 (3.0g, 7.22mmol) dissolved in THF to the reaction bottle, then place the reaction system in a cold bath and cool to -20°C, and add TMSCF3 (1.54g, 10.83mmol) dropwise. After a period of reaction, the reaction solution turns orange. After 24h of reaction, monitor with LC-MS or TLC, the reaction is complete. Add 50mL of saturated NH4Cl aqueous solution to the reaction system, remove the solvent under reduced pressure, and obtain a solid. The solid was then washed with 500 mL of DCM and purified by column chromatography (n-hexane / ethyl acetate = 1:1) to obtain a light yellow liquid. The liquid was dissolved in a methanol solution of HCl and stirred for 1 h. The solvent was removed by vacuum evaporation and dried to obtain 2.44 g of a light yellow transparent liquid with a yield of 70%. 1 H NMR(600MHz,Chloroform-d)δ6.78-6.69(m,2H),6.64-6.58(m,2H),4.04(t,J=5.0Hz,2H) ,3.80(t,J=5.0Hz,2H),3.74-3.60(m,11H),3.54(t,J=4.8Hz,2H),3.37(d,J=1.6Hz,3H); 19 F NMR (376MHz, Chloroform-d) δ-77.03 (d, J=7.5Hz).

[0187] Example 7: Catalytic Hydrogenation Synthesis of Amines

[0188]

[0189] Weigh 3-2 (3.18 g, 9.67 mmol) and palladium carbon (320 mg, about 10% of the mass of 3-2) in a 200 mL round-bottom flask, add a magnetic bar and methanol (80 mL), and stir overnight at 40 ° C under a hydrogen atmosphere. Monitor the reaction by LC-MS or TLC until the reaction is complete, filter the reaction solution with diatomaceous earth, purify by column chromatography (EA), and dry to obtain 2.72 g of a light yellow liquid with a yield of 94%. 1HNMR(600MHz,Chloroform-d)δ6.78-6.69(m,2H),6.64-6.58(m,2H),4.04(t,J=5.0Hz,2H ),3.80(t,J=5.0Hz,2H),3.74-3.60(m,11H),3.54(t,J=4.8Hz,2H),3.37(d,J=1.6Hz,3H).

[0190] Embodiment 8:

[0191]

[0192] The preparation method is the same as VII-4, except that the raw material VI-4 is replaced by compound VI-1, red oily liquid, 550 mg, 94%. 1 H NMR(400MHz, CDCl3) δ6.84(d,J=8.7Hz,1H),6.60(dd,J=2.7,1.4Hz,1H),6.55(dd,J=8.7,2.7Hz,1H),4.07 (t,2H),3.81(dd,J=5.7,4.2Hz,2H),3.74-3.70(m,2H),3.66-3.62(m,8H),3.56-3.51(m,2H),3.36(s,3H); 19 F NMR (376MHz,CDCl3)δ-57.94.

[0193] Embodiment 9:

[0194]

[0195] The preparation method is the same as VII-4, except that the raw material VI-4 is replaced by compound VI-2, red oil, yield 450mg, 89%. 1 H NMR (400MHz, CDCl3) δ6.89(d,J=2.8Hz,1H),6.85(d,J=8.7Hz,1H),6.79(dd,J=8.7,2.8Hz,1H),4.10(t,J =5.0Hz,2H),3.83(t,J=4.9Hz,2H),3.75-3.71(m,2H),3.68-3.61(m,8H),3.56-3.51(m,2H),3.36(s,3H); 19 F NMR (376MHz, CDCl3) δ-62.12.

[0196] Embodiment 10:

[0197]

[0198] The preparation method is the same as VII-4, except that the raw material VI-4 is replaced by compound VI-3, red oil, yield 400 mg, 79%. 1 H NMR (400MHz, CDCl3) δ6.25-6.16(m,2H),4.12(t,J=5.6,4.0Hz,2H),3.77(t ,2H),3.72-3.68(m,2H),3.67-3.62(m,8H),3.56-3.52(m,2H),3.37(s,3H); 19 F NMR(376MHz, CDCl3)δ-127.96--128.07(m).

[0199] Example 11: Synthesis of ligand

[0200]

[0201] Add a magnet to a 50mL Schlenk reaction tube, bake in an electric oven under vacuum for 2 minutes, and cool to room temperature. Weigh pyridine dichloride (350mg, 1.72mmol) in a glove box and add ultra-dry toluene to the reaction tube under nitrogen protection, weigh b (498.41mg, 1.72mmol) in the reaction tube, heat to 120°C, stir for 1h, and monitor the complete reaction of the monosubstituted product by LC-MS. Lower the reaction temperature to room temperature, add 3-5 (576.15mg, 1.72mmol), heat to 120°C, stir for 2h, and monitor the complete reaction by LC-MS. After the reaction system is cooled to room temperature, purify by column chromatography (n-hexane / ethyl acetate = 1:2), and dry to obtain 541.58mg of a light yellow oily liquid with a yield of 46%. 1 H NMR(600MHz,Chloroform-d)δ9.46(s,1H),8.57(d,J=9.5Hz,1H),8.44(dd,J=7.7,1.1 Hz,1H),8.36(dd,J=7.7,1.2Hz,1H),8.08(t,J=7.8Hz,1H),7.56-7.53(m,2H),7.53-7 .50(m,2H),7.40(d,J=8.3Hz,2H),6.91-6.85(m,2H),5.92-5.84(m,1H),3.86(dd,J=5 .6,4.0Hz,2H),3.75-3.71(m,2H),3.71-3.62(m,10H),3.56-3.52(m,2H),3.36(s,3H). 19 F NMR (376MHz, Chloroform-d) δ-73.38 (d, J=7.8Hz).

[0202] Embodiment 12:

[0203]

[0204] The preparation method is the same as III-a3, except that the raw material is replaced by compound VI-2, white solid, yield 586 mg, 93%. 1 H NMR (400MHz, CD3CN) δ10.09(s,2H),8.42(d,J=7.8Hz,2H),8.21(t,J=7.8Hz,1H),7.99(s,2H),7.80(dd,J=9.0,2.6Hz,2H),7.22(d,J =9.0Hz,2H),4.24-4.18(m,4H),3.85-3.78(m,4H),3.65(dd,J=5.8,3.1Hz,4H),3.60-3.51(m,16H),3.47-3.42(m,4H),3.27(s,6H); 19 F NMR (376MHz, CD3CN)δ-58.76.

[0205] Embodiment 13:

[0206]

[0207] The preparation method is the same as III-a3, except that the raw material is replaced by compound VI-2, white solid, yield 475 mg, 91%. 1 H NMR(400MHz,CD3CN)δ10.13(s,2H),8.43(d,J=7.8Hz,2H),8.26-8.18(m,3H),8.07(dd,J=9.0,2.7Hz,2H),7.25(d,J=9.0Hz,2H), 4.27-4.22(m,4H),3.86-3.80(m,4H),3.65(dd,J=5.8,3.3Hz,4H),3.59-3.51(m,16H),3.45(dd,J=5.8,3.3Hz,4H),3.27(s,6H); 19 F NMR (376MHz, CD3CN)δ-62.54.

[0208] Embodiment 14:

[0209]

[0210] The preparation method is the same as III-a3, except that the raw material is replaced by compound VI-2, white solid, yield 212 mg, 77%. 1H NMR(400MHz,CD3CN)δ8.20(d,J=7.7Hz,2H),8.09-8.01(m,1H),7.71-7.61(m,4H),4.23-4.16(m,4H),3.7 7-3.71(m,4H),3.62(dd,J=5.8,3.0Hz,4H),3.59-3.50(m,16H),3.47-3.42(m,4H),3.26(d,J=0.7Hz,6H); 19 F NMR(376MHz,CD3CN)δ-128.89--129.01(m).

[0211] Embodiment 15:

[0212]

[0213] The preparation method is the same as III-a3, except that the raw material is replaced by compound VI-2, white solid, yield 165 mg, 55%. 1 H NMR (400MHz, CD3CN) δ10.18 (s, 1H), 9.78 (s, 1H), 8.39 (dd, J = 7.8, 2.5Hz, 1H), 8. 27(dd,J=8.4,1.7Hz,1H),8.19(t,J=7.8Hz,1H),7.75-7.66(m,2H),7.50-7.38(m ,2H),7.31(td,J=7.8,1.7Hz,1H),7.00-6.92(m,2H),4.15-4.06(m,2H),3.83-3. 74(m,2H),3.68-3.61(m,2H),3.60-3.50(m,8H),3.47-3.43(m,2H),3.28(s,3H); 19 F NMR (376MHz, CD3CN)δ-58.09.

[0214] Embodiment 16:

[0215]

[0216] The preparation method is the same as III-a3, except that the raw material is replaced by compound VI-2, white solid, yield 153 mg, 51%. 1H NMR (400MHz, CD3CN) δ10.23(s,1H),10.02(s,1H),8.38(d,J=7.8Hz,2H),8.17(dd,J=8 .1,7.4Hz,1H),8.01(q,J=1.1Hz,1H),7.85(ddd,J=8.2,2.1,0.9Hz,1H),7.77-7.68(m, 2H),7.47(t,J=8.2Hz,1H),7.14-7.05(m,1H),7.00-6.88(m,2H),4.12-4.05(m,2H),3. 82-3.74(m,2H),3.66-3.61(m,2H),3.61-3.51(m,8H),3.47-3.43(m,2H),3.28(s,3H); 19 FNMR (376MHz, CD3CN)δ-58.45.

[0217] Embodiment 17:

[0218]

[0219] The preparation method is the same as III-a3, except that the raw material is replaced by compound VI-2, white solid, yield 133 mg, 45%. 1 H NMR (400MHz, CD3CN) δ10.23(s,1H),10.05(s,1H),8.39(d,J=7.8Hz,2H),8.17(t,J=7.8Hz,1H),8.04-7.91(m,2H),7.79-7.71(m,2H),7.3 3(d,J=8.6Hz,2H),7.01-6.89(m,2H),4.15-4.05(m,2H),3.84-3.75(m,2H),3.68-3.51(m,10H),3.46(dd,J=5.8,3.3Hz,2H),3.28(s,3H); 13 CNMR(126MHz,CD3CN)δ162.85,162.36,156.81,150.37,149.83,146.18-145.93(m),140.67,138.08,132 .06,126.30,126.18,123.78,123.31,122.55,115.54,72.59,71.34,71.18,71.15,71.12,70.97,58.88; 19 F NMR (376MHz, CD3CN)δ-58.82.

[0220] Embodiment 18:

[0221]

[0222] The preparation method is the same as III-a3, except that the raw material is replaced by compound VI-2, colorless oil, yield 378 mg, 46%. 1 H NMR (400MHz, CD3CN) δ8.20(t,J=7.6Hz,1H),7.74(d,J=7.7Hz,2H),7.29-7.14(m,4H),7.03(d,J=8.8Hz,1H),6.84( d,J=8.3Hz,2H),4.15(t,J=4.6Hz,2H),4.08(t,J=4.6Hz,2H),3.83-3.70(m,4H),3.62-3.44(m,24H),3.27(s,6H); 19 F NMR (376MHz, CD3CN)δ-58.62.

[0223] Embodiment 19:

[0224]

[0225] The preparation method is the same as III-a3, except that the raw material is replaced by compound VI-2, colorless oil, yield 121 mg, 46%. 1 H NMR (400MHz, CD3CN) δ9.94(s,1H),8.99(d,J=9.6Hz,1H),8.41(d,J=7.8Hz,1H),8.33(d,J=7.7Hz,1H),8.16(t,J=7.8Hz,1H),7.68-7.58( m,4H),7.50(d,J=2.4Hz,1H),7.28(dd,J=8.7,2.4Hz,1H),6.09(p,J=8.7Hz,1H),4.16-4.03(m,4H),3.87-3.77(m,4H),3.72-3.59(m,8H); 19 FNMR(376MHz,CD3CN)δ-73.08(d,J=8.3Hz).

[0226] Example 20: Formation of ligand in steps

[0227]

[0228] Add a magnet to a 50mL Schlenk reaction tube, bake in an electric oven under vacuum for 2 minutes, and cool to room temperature. Weigh pyridine dichloride (367.21mg, 1.8mmol) in a glove box and put it in a reaction tube. Then add 5mL of ultra-dry toluene under nitrogen protection. After stirring for 2min, add 3-5 (590mg, 1.8mmol) to the reaction system and heat to 60°C. After 10min of reaction, the system turns orange. Continue heating and stirring for 1h. After LC-MS or TLC monitoring until the reaction is complete, purify by column chromatography (n-hexane / ethyl acetate = 3:7), and dry to obtain 729.8mg of orange solid with a yield of 90%. 1 H NMR(600MHz,Chloroform-d)δ10.14(d,J=15.1Hz,1H),8.56-8.25(m,2H),8.09(s,1H),7.52(d,J=8.2Hz,2H),6. 74(d,J=9.2Hz,2H),4.02(s,2H),3.80(s,2H),3.75-3.57(m,10H),3.52(d,J=5.6Hz,2H),3.32(d,J=9.1Hz,3H).

[0229] Example 21: Formation of ligand in steps

[0230]

[0231] Add a magnetic bar to a 50mL Schlenk reaction tube, bake in an electric oven for 2 minutes under vacuum, and cool to room temperature. Replace nitrogen with vacuum 3 times, add 3-6 (729.8mg, 1.63mmol), DMAP (19.88mg, 1.79mmol) and 5mL of ultra-dry dichloromethane to the reaction tube under nitrogen protection, and then add 3-4 (620.64mg, 1.63mmol) dissolved in ultra-dry dichloromethane to the reaction system. Cool the reaction system to 0℃, add EDCI (343.15mg, 1.79mmol) to the system, return to room temperature and stir. After 2h of reaction, monitor with LC-MS. The reaction is complete. Add water to the reaction system to quench the reaction, extract, remove the solvent under reduced pressure, and purify by reverse phase column chromatography to obtain 1.16g of light yellow liquid with a yield of 88%. 1H NMR(600MHz,Chloroform-d)δ9.65(s,1H),8.76-8.67(m,1H),8.43(dd,J=7.8,1.1Hz,1H),8 .37(dd,J=7.8,1.2Hz,1H),8.06(t,J=7.8Hz,1H),7.57-7.52(m,2H),7.42(d,J=8.5Hz,2H), 6.95-6.90(m,2H),6.86(d,J=8.5Hz,2H),5.88(p,J=8.2Hz,1H),4.11(q,J=4.9Hz,4H),3.84 (ddd,J=8.3,5.6,4.1Hz,4H),3.77-3.58(m,21H),3.57-3.48(m,4H),3.34(d,J=19.7Hz,6H). 19 F NMR (376MHz, Chloroform-d) δ-73.74 (d, J=8.1Hz).

[0232] Example 22: Synthesis of Palladium Complex

[0233]

[0234] Weigh 3-7 (1.16 g, 1.42 mmol) and Pd(OAc)2 (383.32 mg, 1.71 mmol) into a 100 mL reaction bottle, add 30 mL of acetonitrile, heat to 40 °C and stir for 2 hours, monitor by LC-MS and NMR until the reaction is complete. Remove the solvent under reduced pressure, wash with n-hexane, and dry to obtain 1.32 g of brown-black viscous liquid, with a yield of 88%. 1 H NMR(400MHz,Chloroform-d)δ8.11(t,J=7.8Hz,1H),7.91(d,J=7.7Hz,1H),7.86(d,J=7.6Hz,1H), 7.42(d,J=8.4Hz,2H),7.06(d,J=8.6Hz,2H),6.87(d,J=8.7Hz,2H),6.78(d,J=8.7Hz,2H),6.17(q, J=8.7Hz,1H),4.08(dt,J=22.2,5.2Hz,4H),3.81(dt,J=12.8,4.7Hz,4H),3.73-3.59(m,20H),3.5 2(dt,J=6.0,3.0Hz,4H),3.35(dd,J=1.9,0.8Hz,6H),2.15(d,J=0.8Hz,3H),1.99(d,J=0.8Hz,5H). 19F NMR (376MHz, Chloroform-d) δ-68.95 (d, J=8.9Hz).

[0235] Embodiment 23:

[0236]

[0237] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-a2, orange-yellow solid, 485.3 mg, yield 89%. 1 H NMR(600MHz,Chloroform-d)δ8.15(t,J=7.8Hz,1H),7.93(dd,J=7.8,1.4Hz, 1H),7.90-7.86(m,1H),7.50-7.43(m,4H),7.11-7.07(m,2H),6.83-6.79(m,2 H),6.21(q,J=8.6Hz,1H),4.07(dd,J=5.7,4.0Hz,2H),3.84-3.80(m,2H),3. 70(dt,J=5.2,2.0Hz,2H),3.68-3.62(m,8H),3.56-3.52(m,2H),3.37(s,3H). 19 F NMR (376MHz, Acetonitrile-d3) δ-69.52 (d, J=9.0Hz).

[0238] Embodiment 24:

[0239]

[0240] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-b1, orange-yellow solid, 99 mg, 85%. 1 H NMR (600MHz, CD3CN) δ8.21(t,J=7.8Hz,1H),7.75(d,J=7.8Hz,2H),7.26(dt,J=2.6,1.4Hz,2H),7.20(dd,J=8.8,2.5Hz,2H),7.0 4(d,J=8.8Hz,2H),4.18-4.14(m,4H),3.81-3.77(m,4H),3.66-3.62(m,4H),3.57-3.51(m,16H),3.46-3.43(m,4H),3.28(s,6H); 19 F NMR (565MHz, CD3CN)δ-58.59.

[0241] Embodiment 25:

[0242]

[0243] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-a1, orange-yellow solid, yield 125 mg, 89%. 1 H NMR (400MHz, CD3CN) δ8.22(t,J=7.8Hz,1H),7.83(d,J=7.8Hz,1H),7.74(d,J=7.8Hz,1H),7.54(d,J=8.2Hz,2H),7.42(d,J=8.2Hz,2H), 6.77(d,J=8.5Hz,1H),6.71(s,1H),6.59(d,J=8.5Hz,1H),6.15(q,J=9.1Hz,1H),4.05-3.94(m,4H),3.74(q,J=4.6Hz,4H),3.59(s,8H); 19 F NMR (376MHz, CD3CN) δ-69.51 (d, J=9.0Hz).

[0244] Embodiment 26:

[0245]

[0246] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-b2, orange-yellow solid, yield 100 mg, 87%. 1 H NMR (600MHz, CD3CN) δ8.24(t,J=7.8Hz,1H),7.79(d,J=7.8Hz,2H),7.50(d,J=2.6Hz,2H),7.47(dd,J=8.8,2.6Hz,2H),7.09(d ,J=8.8Hz,2H),4.22-4.18(m,4H),3.83-3.77(m,4H),3.65-3.61(m,4H),3.57-3.52(m,16H),3.46-3.43(m,4H),3.28(s,6H); 19 F NMR (565MHz, CD3CN)δ-62.08.

[0247] Embodiment 27:

[0248]

[0249] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-b3, orange-yellow solid, yield 122 mg, 78%. 1H NMR (400MHz, CD3CN) δ8.27-8.14(m,1H),7.75(d,J=7.8Hz,1H),7.03-6.91(m,4H),4.20(t ,J=4.5Hz,4H),3.73(t,J=4.5Hz,4H),3.64-3.49(m,20H),3.49-3.41(m,4H),3.27(s,6H); 13 C NMR(126MHz,CD3CN)δ169.54,156.13(dd,J=244.6,7.7Hz),152.61,143.26,143.71-132.12(m), 127.17,111.89-111.23(m),74.88-74.63(m),72.56,71.20,71.13,71.08,70.97,70.94,58.88. 19 F NMR (376MHz, CD3CN) δ-130.89 (d, J = 10.2Hz).

[0250] Embodiment 28:

[0251]

[0252] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-b4, orange-yellow solid, yield 98 mg, 80%. 1 H NMR (400MHz, CD3CN) δ8.22(t,J=7.8Hz,1H),7.76(t,J=7.1Hz,2H),7.38-7.20(m,4H),7.19-7.09(m,2H),6.81(d,J=8.4Hz,2H),4.06(t,J= 4.5Hz,2H),3.74(t,J=4.6Hz,2H),3.60(dd,J=6.1,3.1Hz,2H),3.54(d,J=14.1Hz,8H),3.43(dd,J=6.0,3.4Hz,2H),3.26(d,J=1.2Hz,3H); 19 FNMR (376MHz, CD3CN)δ-57.48.

[0253] Embodiment 29:

[0254]

[0255] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-b5, orange-yellow solid, yield 21 mg, 68%. 1H NMR (400MHz, CD3CN) δ8.24(t,J=7.7Hz,1H),7.78(t,J=7.6Hz,2H),7.35(t,J=8.0Hz,1H),7.27(d,J=8.1Hz,1H),7.23(s,1H),7.17(d,J=8.3Hz,2 H),7.02(d,J=8.4Hz,1H),6.85(d,J=8.4Hz,2H),4.11-4.06(m,2H),3.76 (t,J=4.5Hz,2H),3.70-3.48(m,8H),3.45(d,J=5.1Hz,2H),3.28(s,3H); 19 F NMR (376MHz, CD3CN)δ-58.41.

[0256] Embodiment 30:

[0257]

[0258] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-b6, orange-yellow solid, yield 53 mg, 68%. 1 H NMR (400MHz, CD3CN) δ8.20(t,J=7.8Hz,1H),7.74(dd,J=7.9,3.7Hz,2H),7.34(d,J=8.5Hz,2H),7.18(dd,J=8.9,2.7Hz,4H),6. 83(d,J=8.4Hz,2H),4.08(t,J=4.6Hz,2H),3.75(t,J=4.6Hz,2H),3.66-3.50(m,11H),3.45(dd,J=5.9,3.4Hz,2H),3.27(s,3H); 19 F NMR (376MHz, CD3CN)δ-58.74.

[0259] Embodiment 31:

[0260]

[0261] The preparation method is the same as I-a3, except that the raw material is replaced by compound III-b7, orange-yellow solid, yield 98 mg, 83%. 1H NMR (400MHz, CD3CN) δ8.20(t,J=7.6Hz,1H),7.74(d,J=7.7Hz,2H),7.29-7.14(m,4H),7.03(d,J=8.8Hz,1H),6.84( d,J=8.3Hz,2H),4.15(t,J=4.6Hz,2H),4.08(t,J=4.6Hz,2H),3.83-3.70(m,4H),3.62-3.44(m,24H),3.27(s,6H); 19 F NMR (376MHz, CD3CN)δ-58.62.

[0262] Test Example 1: Effect of probe molecules distinguishing and identifying amino acid enantiomers

[0263] Dissolve 1.2 mg of I-a3 probe molecule, 0.4-0.9 mg of analyte and about 0.2 mg of NaOH in 0.5 mL of water, transfer them to a 0.5 mm NMR tube, and perform NMR fluorine spectrum test or hydrogen decoupled hydrogen spectrum detection.

[0264] The results show that:

[0265] like Figure 1 , probe I-a3 has different signals for amino acids with different configurations.

[0266] like Figure 2 , the peaks of probe I-a3 for the same amino acid in the presence of different bases. Sodium acetate will complex with the probe when present in large amounts.

[0267] like Figure 3 , the detection effect of probe I-a3 on different amino acid enantiomers. When amino acid enantiomers of different configurations are complexed with probe I-a3, fluorine spectrum signals are generated at different chemical shifts, and this signal does not change with the change of analyte and probe concentration. The fluorine signal of amino acids with D configuration often appears in a low-field position relative to amino acids with L configuration, which can be used to determine the absolute configuration of amino acid enantiomers.

[0268] like Figure 4 The results showed that probe I-a1 can effectively distinguish different amino acid enantiomers.

[0269] like Figure 5 The results of the detection and differentiation of leucine enantiomers by probe I-a2 showed that probe I-a1 can effectively distinguish different leucine enantiomers.

[0270] Test Example 2: Effect diagram of probe molecule distinguishing and identifying metabolites

[0271] Take the probe molecule and use 100mM potassium dihydrogen phosphate-potassium hydrogen phosphate (KPi) buffer to prepare 2mM probe and 20mM dAMP, dCMP, dGMP, dTMP, UMP, AMP, CMP, and GMP analyte solutions. Take 0.3mL of the probe solution and 0.3mL of the analyte solution, mix and add to a 0.5mm nuclear magnetic resonance tube for nuclear magnetic resonance fluorine spectrum detection to obtain the spectrum of each analyte detected separately. Take 1.5 times the volume of dAMP, dGMP, AMP, GMP and 1 times the volume of dCMP, dTMP, UMP, and CMP analyte solutions to obtain a mixed analyte solution with a total concentration of 20mM. Take 0.3mL of the probe solution and 0.03mL of the mixed analyte solution, add 0.27mL of KPi buffer, add to the nuclear magnetic resonance tube and perform nuclear magnetic resonance fluorine spectrum detection to obtain the spectrum of the mixed detection of eight analytes. The results are as follows. Figure 6 shown.

[0272] Figure 6 The detection of different (deoxy)ribonucleotides by the probe molecule is shown in Figure 1. When analytes with different structures are complexed with probe I-b1, fluorine spectrum signals are generated at different chemical shifts, and this signal does not change with changes in the concentration of the analyte and the probe.

[0273] like Figure 7 The I-b2 probe has good detection effect on ATP, ADP and AMP. For the three analytical objects, after the probe of the present invention is combined with the detection object, strong signals can be observed under the fluorine spectrum, and the characteristic peaks of different analytes can be clearly distinguished.

[0274] like Figure 8 The I-b3 probe has the effect of distinguishing and detecting ATP, ADP and AMP. For the three analytical objects, after the probe of the present invention is combined with the detection object, strong signals can be observed under the fluorine spectrum, and the characteristic peaks of different analytes can be clearly distinguished.

[0275] like Fig. 9 The I-b4 probe has the effect of distinguishing and detecting ATP, ADP and AMP. For the three analytical objects, after the probe of the present invention is combined with the detection object, strong signals can be observed under the fluorine spectrum, and the characteristic peaks of different analytes can be clearly distinguished.

[0276] like Fig.10 The I-b5 probe has the effect of distinguishing and detecting ATP, ADP and AMP. For the three analytical objects, after the probe of the present invention is combined with the detection object, strong signals can be observed under the fluorine spectrum, and the characteristic peaks of different analytes can be clearly distinguished.

[0277] like Fig.11 The I-b6 probe has the effect of distinguishing and detecting ATP, ADP and AMP. For the three analytical objects, after the probe of the present invention is combined with the detection object, strong signals can be observed under the fluorine spectrum, and the characteristic peaks of different analytes can be clearly distinguished.

[0278] like Fig.12 The I-b7 probe has the distinguishing detection effect on NADP, cAMP, ATP, ADP, AMP, deoxyadenosine and adenosine. For the seven analytical objects, after the probe of the present invention is combined with the detection object, a strong signal can be observed under the fluorine spectrum, and the characteristic peaks of different analytes can be clearly distinguished.

[0279] Test Example 3 Probe molecule I-b1 Enzyme reaction effect diagram for distinguishing and identifying metabolites

[0280] Take 0.6 mg of the probe and analyte (2-3 mg) and dissolve them in buffer, add the corresponding enzyme, take 0.6 mL and add it to a 0.5 mm NMR tube, and perform NMR fluorine spectrum detection at different time points. The results are as follows: Fig.13 It is shown that for the glucose dehydrogenase-catalyzed reaction, the NMR spectra corresponding to NAD and NADH are integrated and calculated after correction using the correction coefficient to obtain the changes in metabolites, thereby judging the process changes of the enzymatic reaction and the changes in the content of each component in real time and in situ.

[0281] As shown in the case of ATP phosphatase, it can be detected that ATP generates ADP and then further removes phosphorylation to generate AMP. Fig.14 As shown, after integrating the NMR spectra corresponding to ATP, ADP, and AMP and correcting them with the correction coefficient, the changes in metabolites can be obtained, thereby determining the process changes of the enzymatic reaction and the content changes of each component in real time and in situ.

[0282] Test Example 4: Detection effect diagram of the inhibition of the enzymatic reaction of metabolites by probe molecule I-b1

[0283] Take 0.6 mg of the probe and the analyte (2-3 mg) and dissolve them in buffer, add the corresponding enzyme, add inhibitors to the control group, take 0.6 mL and add it to a 0.5 mm NMR tube, and perform NMR fluorine spectrum detection at different time points.

[0284] The results are as follows Fig.15 As shown, for hexokinase, it can be detected that ATP generates ADP, and the addition of inhibitors significantly reduces the enzymatic reaction. After integrating the NMR spectra corresponding to ATP and ADP, the correction coefficient is used for calculation to obtain the effect of the inhibitor on the metabolic process, thereby judging the process changes of the enzymatic reaction and the content changes of each component in real time in situ.

Claims

1. A water-soluble chiral fluorine-containing palladium complex probe, characterized in that: The complex has a structure shown in the following formula I: Where n is an integer 1 or 0; R 1 Selected from the following group: substituted or unsubstituted C1-C10 alkyl; wherein the substitution refers to one or more hydrogen atoms on the group being replaced by a substituent selected from the following group: halogen, C1-C4 haloalkyl; R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 A group independently selected from the following group: hydrogen atom, halogen, sulfonic acid group, trialkylamino group, substituted or unsubstituted C1-C20 alkyl group, substituted or unsubstituted C1-C10 alkoxy group, substituted or unsubstituted C6-C20 aryl group, substituted or unsubstituted C4-C20 heteroaryl group, or a water-soluble group selected from the following group: O(CH2CH2O) p CH3, or two groups located on adjacent carbon atoms form -O (CH2CH2O) p , wherein p is selected from the following group 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein the heteroaryl skeleton has 1 to 3 heteroatoms selected from O or S; wherein the substitution refers to one or more hydrogen atoms on the group being replaced by a substituent selected from the following group: halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl; And when n is an integer 1, The structure is chiral, and its absolute configuration is R configuration or S configuration; And when R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 There are no two groups on adjacent carbon atoms forming -O(CH2CH2O) p In this case, there is at least one O(CH2CH2O) in the above group. p CH3.

2. The complex according to claim 1, characterized in that The probe has a structure as shown in the following formula Ia: Among them, the R 1 , R 4 , R 9 and R 10 Among them, at least one is selected from a fluorine atom or a fluorine-substituted group; Or the probe has a structure shown in the following formula Ib:

3. The complex according to claim 1, characterized in that The R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 A group independently selected from the following group: hydrogen atom, halogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy; or a water-soluble group selected from the following group: O(CH2CH2O) p CH3, or two groups located on adjacent carbon atoms form -O (CH2CH2O) p , wherein p is selected from 3, 4, 5, 6, 7, 8, 9 or 10.

4. The complex according to any one of claims 1 to 3, characterized in that The complex has a structure selected from the group consisting of:

5. A water-soluble chiral fluorine-containing palladium complex probe, characterized in that: The complex has a structure shown in the following formula II: Among them, n, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 As claimed in claim 1 respectively; L is a non-acetonitrile compound or anion that can complex with palladium; preferably, L is a molecule or anion selected from the group consisting of: C1-C 20 Chain or cyclic amines, C3~C 20 Chain or cyclic nitriles, C1~C 20 Chain or cyclic thiols, C1~C 20 Chain or cyclic sulfide, C1~C 20 Chain or cyclic amides, C3-~C 30 Nitrogen-containing heterocyclic ring; C3~C 30 sulfur-containing heterocyclic rings; chloride ions (Cl - ), bromide ion (Br - ), iodide ion (I - ), azidoate (N3 - ), thiocyanate (SCN - ), cyanate (CN - )、C1~C 20 of carboxylate ions; Preferably, the probe has a structure shown in the following formula II-a or formula II-b:

6. A ligand for synthesizing a water-soluble chiral palladium complex, characterized in that: The ligand has a structure shown in the following formula III: Among them, n, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 As claimed in claim 1 respectively; Preferably, the ligand has a structure shown in the following formula III-a or III-b: More preferably, the ligand has a structure selected from the group consisting of:

7. A method for synthesizing the complex of formula I as claimed in claim 1, characterized in that: The method comprises the steps of: Add the ligand III described in claim 4 and palladium acetate into a reaction bottle, add acetonitrile to dissolve, and stir at a temperature of 20-80° C. for 2-24 hours to obtain the complex I described in claim 1.

8. A method for synthesizing the complex of formula II as claimed in claim 5, characterized in that: The method comprises the steps of: In a solvent, the complex I described in claim 1 is mixed with L to obtain the complex II described in claim 5; Wherein, L is a non-acetonitrile compound or anion that can be complexed with palladium; Specifically, L is a molecule or anion selected from the group consisting of: C1 to C 20 Chain or cyclic amines, C3~C 20 Chain or cyclic nitriles, C1~C 20 Chain or cyclic thiols, C1~C 20 Chain or cyclic sulfide, C1~C 20 Chain or cyclic amides, C3~C 30 Nitrogen-containing heterocyclic ring; C3~C 30 sulfur-containing heterocyclic rings; chloride ions (Cl - ), bromide ion (Br - ), iodide ion (I - ), azidoate (N3 - ), thiocyanate (SCN - ), cyanate (CN - )、C1~C 20 of carboxylate ions.

9. A method for distinguishing and identifying amino acid enantiomers, characterized in that: The method comprises the steps of: (1) In a highly polar solvent, the complex of formula I as claimed in claim 1 is mixed with the chiral amino acid enantiomer to be detected, and a certain amount of base is added to obtain a complex solution of the probe molecule and the amino acid; (2) performing fluorine spectrum nuclear magnetic resonance detection on the complex solution of the probe molecule and the amino acid to obtain two groups of characteristic fluorine spectrum nuclear magnetic resonance signals, which correspond to amino acids with different absolute configurations; (3) By integrating and calculating the two sets of fluorine spectrum NMR signals, the enantiomeric composition information of the chiral amino acids in the sample can be obtained; In the formula I, n is an integer of 1.

10. A method for in situ detection of enzyme metabolites in a nuclear magnetic resonance tube, characterized in that: Includes steps: (1) Mixing the compound of formula I as claimed in claim 1 with a reaction substrate for enzyme metabolism in a prepared buffer to obtain a mixed solution of the probe and the substrate; (2) adding an enzyme solution of a specific concentration to a mixed solution of the probe and substrate, and recording the time of enzyme addition; (3) performing fluorine nuclear magnetic resonance detection on the sample to which the enzyme is added, and recording the time of each spectrum acquisition; (4) By determining and analyzing the fluorine spectrum signal obtained in step (3), information on the changes in substrates and products over time during enzyme metabolism can be obtained.

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