A probe for detecting myrosinase and its preparation method and application

Abstract

The present invention discloses a probe for detecting myrosinase, a preparation method thereof and an application thereof, wherein the method synthesizes amino-thioglucosinolate precursors with different chain lengths by simulating the structure of plant secondary metabolites thioglucosinolates, and the precursor undergoes a nucleophilic substitution reaction with a halogenated active fluorescent parent and then undergoes deprotection to obtain a target compound. The synthesis method of the present invention has simple steps and mild reaction conditions. The probe for detecting myrosinase prepared by the present invention has good water solubility, high stability, good detection selectivity and high sensitivity; after the probe reacts with myrosinase, the absorption and fluorescence spectra are significantly red-shifted, and by monitoring the absorption and fluorescence changes, the detection of myrosinase can be achieved and the difference in enzyme activity can be reflected. The probe for detecting myrosinase of the present invention can be used not only for in vitro myrosinase detection, but also for detection and fluorescence imaging of endogenous myrosinase in plants or flora, and can also be used for myrosinase-related research such as myrosinase inhibitor screening.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a probe for detecting myrosinase and a preparation method and application thereof. Background Art

[0002] Myrosinase (MYR) is mainly found in cruciferous plants and is the only glucosinolate hydrolase found in nature so far. Myrosinase can catalyze the hydrolysis of glucosinolates to produce isothiocyanates (ITCs), thiocyanate, glucose and other products. Isothiocyanates have a pungent taste and are the source of flavor substances in many cruciferous vegetables (such as broccoli, radish, cabbage, kale, etc.). On the other hand, the glucosinolate-myrosinase system in edible vegetables has been shown to promote health and reduce the incidence of various diseases. There is increasing evidence that isothiocyanates produced by the hydrolysis of glucosinolates by myrosinase have a wide range of pharmacological effects, including anti-cancer, anti-inflammatory and anti-diabetic.

[0003] In order to detect myrosinase, there are currently a variety of methods for myrosinase detection, such as high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and pH-salt determination. These methods can all detect myrosinase indirectly, but usually require cumbersome sample preparation and the use of complex instruments and equipment, and cannot perform non-destructive analysis of myrosinase in living plants and flora, which has certain limitations. Fluorescence analysis refers to a technology that uses the luminescence characteristics of fluorescent substances for analysis and detection, and obtains information by detecting parameters such as the fluorescence intensity, fluorescence wavelength, and fluorescence lifetime of fluorescent substances. Due to its high sensitivity, visualization, good selectivity, rapid response, and the ability to achieve non-destructive detection of live imaging, fluorescence analysis has great advantages in the field of biochemistry. In order to solve the problems existing in existing myrosinase detection technology, it is of great advantage and research significance to develop a probe based on small molecule fluorescent dyes for the detection of myrosinase. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the current technology for detecting mustard enzymes, and to propose a fluorescent probe for detecting mustard enzymes based on small molecule fluorescent dyes, and a preparation method and application thereof, so as to solve the technical problems of cumbersome sample preparation, destructive analysis, poor visualization, and inability to detect live samples in the existing technology for detecting mustard enzymes. This type of probe is prepared by reacting different halogenated active fluorescent mothers with pre-prepared amino-thioglucosinolate precursors. The probe is hydrolyzed after binding to the active site of mustard enzymes to generate a molecule of glucose and an unstable aglycone intermediate. The intermediate will spontaneously undergo a Losen rearrangement reaction to quickly generate an isothiocyanate (R-NCS) with strong electrophilicity, and then attack the exposed secondary amine site of the fluorescent mother to undergo an intramolecular ring-closing reaction, generating a thiourea ring with an electron-withdrawing effect, which forms a strong push-pull electron effect with the fluorescent mother, causing its absorption and fluorescence emission spectra to be red-shifted, thereby realizing the detection of mustard enzymes.

[0005] The specific technical solution for achieving the purpose of the present invention is:

[0006] The present invention provides a probe for detecting myrosinase, which has the structures shown in the following formulas (I), (II), and (III):

[0007]

[0008] Where n = 2 or 3;

[0009] Among them, in the compound represented by formula (I):

[0010] R1 is an oxygen, carbon, silicon or germanium atom, wherein when R1 is oxygen, R2 and R3 do not exist;

[0011] R2 and R3 are selected from C1-C8 straight or branched saturated alkane groups, and R2 and R3 are the same or different;

[0012] R4 is selected from hydrogen, chlorine, bromine, iodine, cyano, nitro, carboxyl, aldehyde, azido, methyl, ethyl, amide, sulfonic acid, sulfonyl, ester (COOMe, COOEt, COOtBu).

[0013] R5 is nitrogen methyl, nitrogen ethyl, nitrogen hydrogen ethyl, nitrogen four-membered ring, nitrogen ethyl five-membered ring, nitrogen ethyl six-membered ring, nitrogen methyl pyrrole ring or quaternary ammonium piperazine ring.

[0014] Specifically, when n = 2, R1 is oxygen, R2 and R3 do not exist, and R4 is hydrogen, the specific form of the compound is:

[0015] ;

[0016] Among them, in the compound represented by formula (II):

[0017] R1 is an oxygen, carbon, silicon or germanium atom, wherein when R1 is oxygen, R2 and R3 do not exist.

[0018] R2 and R3 are selected from C1-C8 straight chain or branched saturated alkane groups respectively, and R2 and R3 are the same or different.

[0019] Specifically, when n = 2, R1 is oxygen, R2 and R3 do not exist; or when n = 2, R1 is carbon, R2 and R3 are both methyl; or when n = 2, R1 is silicon, R2 and R3 are both ethyl; the specific structure of the compound is as follows:

[0020] ;

[0021] Among them, in the compound represented by formula (III):

[0022] R1, R3, R4 and R6 are selected from chlorine, bromine, iodine, methyl, ethyl, cyano, mercapto, aldehyde, C1-C8 straight or branched saturated alkane, and R1, R3, R4 and R6 are the same or different.

[0023] R2 and R5 are selected from fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, cyano, nitro, amino, aldehyde, carboxyl, sulfonic acid, methoxy, acetyl, phenyl, benzyl, ester (COOMe, COOEt, COOtBu) and differently substituted pyrrole rings, and R2 and R5 are the same or different.

[0024] For example, when n=2, R1, R3, R4 and R6 are all hydrogen, and R2 and R5 are hydrogen, chlorine, ethyl, and ester group (COOEt), respectively, the specific form of the compound is:

[0025] .

[0026] The present invention also proposes a method for synthesizing a probe for detecting myrosinase, which simulates the structure of secondary metabolites of mustard oil glycosides in plants, first synthesizes amino-thioglucoside precursors with different chain length structures, then synthesizes a halogenated active fluorescent matrix, and then reacts the above-synthesized amino-thioglucoside precursor with the halogenated active fluorescent matrix in a solvent and a catalyst, and then performs a deprotection reaction to obtain the final compound.

[0027] In the present invention, the catalyst is selected from one or more of piperidine, pyridine, pyrrole, morpholine, imidazole, trimethylamine, diethylamine, triethylamine, N, N-diisopropylethylamine, etc.; preferably, it is one or two of triethylamine and N, N-diisopropylethylamine.

[0028] In the present invention, the solvent is one or more of tetrahydrofuran, dichloromethane, 1,2-dichloroethane, N, N-dimethylformamide, acetone, benzene, toluene, nitrobenzene, p-xylene, methanol, ethanol, 1,4-dioxane, chloroform, alkane, dimethyl sulfoxide, acetonitrile, water, etc.; preferably, it is one or two of dichloromethane and N, N-dimethylformamide.

[0029] In the present invention, the reaction temperature of the amino-thioglucoside precursor and the halogenated active fluorescent matrix is ​​0°C-160°C, and the reaction time is 10 min-48 h; the temperature of the deprotection reaction is -20°C-80°C, and the reaction time is 15 min-10 h; preferably, the reaction temperature of the amino-thioglucoside precursor and the halogenated active fluorescent matrix is ​​80°C, and the reaction time is 12 h; the temperature of the deprotection reaction is 20°C, and the reaction time is 5 h.

[0030] In the present invention, the molar equivalent ratio of the synthesized amino-thioglucoside, halogenated active fluorescent matrix, catalyst and solvent is: 1: (1.0-4.0): (2.0-10.0): (5-200) preferably 1: 2: 5: 50.

[0031] In the present invention, the amino-thioglucoside precursors with different chain length structures have the following structures:

[0032] ; where n = 2 or 3.

[0033] In the present invention, the synthetic reaction route of the amino-thioglucosinolate precursor is as follows:

[0034] ;

[0035] Where n = 2 or 3;

[0036] Specifically, when n=2 or 3, the synthetic reaction route of the amino-thioglucoside precursor is as follows:

[0037] ;

[0038] The specific reaction steps of the amino-thioglucosinolate precursor are as follows:

[0039] (1) dissolving 3-aminopropanal diethyl acetal / 4-aminobutyraldehyde dimethyl acetal and N, N-diisopropylethylamine in a first solvent, and then dissolving Fmoc-Cl (9-fluorenylmethyl chloroformate) in the first solvent at -15-10°C and adding the mixture to the above reaction vessel, stirring the reaction overnight and naturally warming to room temperature to obtain compounds 1 and 1';

[0040] Wherein, the first solvent is selected from one or more of dichloromethane or 1,2-dichloroethane, etc.; preferably, dichloromethane;

[0041] Wherein, the molar ratio of 3-aminopropanal diethyl acetal / 4-aminobutyraldehyde dimethyl acetal to N, N-diisopropylethylamine and Fmoc-Cl is 1.0: (1.5 -3.0): (1.0-1.5); preferably, 1.0: 2.0: 1.2;

[0042] Wherein, preferably, Fmoc-Cl (9-fluorenylmethyl chloroformate) is dissolved in the first solvent at 0°C;

[0043] (2) dissolving compound 1 / 1' in a second solvent, preparing a hydrochloric acid solution with a concentration of 3.0-5.0 mol / L, adding the solution to a reaction vessel, and stirring the reaction at room temperature to obtain compounds 2 (n=2) and 2' (n=3);

[0044] Wherein, the second solvent is selected from one or more of tetrahydrofuran or 1,4-dioxane, etc.; preferably, tetrahydrofuran;

[0045] Wherein, the concentration of the hydrochloric acid solution is preferably 4 mol / L;

[0046] (3) dissolving compound 2(n=2) / 2' (n=3) and hydroxylamine hydrochloride in a third solvent, then adding triethylamine under stirring, and reacting under stirring at room temperature to obtain compounds 3(n=2) and 3' (n=3);

[0047] Wherein, the third solvent is selected from one or more of dichloromethane or 1,2-dichloroethane, etc.; preferably, dichloromethane;

[0048] Wherein, the molar ratio of the compound 2 (n=2) / 2' (n=3), hydroxylamine hydrochloride and triethylamine is 1: (1.5-3.0): (3.0-5.0); preferably, 1.0: 2.0: 4.0;

[0049] (4) Add compound 3 (n = 2) / 3' (n = 3) to a reaction vessel, add a fourth solvent to dissolve it under argon protection and add pyridine, then use the fourth solvent to dissolve NCS (N-chlorosuccinimide) and add it to the reaction vessel. The reaction is stirred at room temperature, and then 1-thio-β-D-glucose tetraacetate dissolved in the fourth solvent is added and triethylamine is added. The reaction is stirred at room temperature to obtain compounds 4 (n = 2) and 4' (n = 3);

[0050] Wherein, the fourth solvent is selected from one or more of dichloromethane or 1,2-dichloroethane, etc.; preferably, dichloromethane;

[0051] Wherein, the molar ratio of the compound 3 (n=2) / 3' (n=3), NCS, 1-thio-β-D-glucose tetraacetate and triethylamine is 1.0: (1.0-2.0): (1.5-4.0): (2.0-5.0); preferably, 1.0: 1.2: 2.0: 3.0;

[0052] (5) Compound 4 (n=2) / 4' (n=3) and pyridine sulfur trioxide were dissolved in acetonitrile, and the mixture was refluxed and stirred at 65-85°C to obtain compounds 5 (n=2) and 5' (n=3);

[0053] Wherein, the molar ratio of the compound 4 (n=2) / 4' (n=3) and pyridine sulfur trioxide is 1.0: (3.0-6.0); preferably, 1.0: 5.0;

[0054] Wherein, the reaction is preferably carried out under reflux stirring at 70°C;

[0055] (6) Compound 5(n=2) / 5' (n=3) was dissolved in a fifth solvent and diethylamine was added. The reaction was stirred at room temperature to obtain compound 6(n=2) / 6' (n=3).

[0056] Wherein, the fifth solvent is selected from one or more of dichloromethane or 1,2-dichloroethane, etc.; preferably, dichloromethane;

[0057] Wherein, the volume ratio of the fifth solvent to diethylamine is (3.0-6.0):1.0; preferably, it is 5.0:1.0.

[0058] In the present invention, the halogenated active fluorescent matrix has the structure shown in the following formula (IV), formula (V), and formula (VI):

[0059]

[0060] Wherein, R1, R2, R3, R4, and R5 in the compound represented by formula (IV) are as defined in formula (I);

[0061] In the compound represented by formula (V), R1, R2, and R3 are defined the same as those in formula (II);

[0062] In the compound represented by formula (VI), R1, R2, R3, R4, R5 and R6 are defined the same as those in formula (III).

[0063] Specifically, the present invention also provides a method for preparing myrosinase detection probes Cou-GSL, Rh-GSL, and BDP-GSL, wherein an amino-thioglucosinolate (n = 2) ligand is first synthesized, and the synthesis reaction is as follows:

[0064] ;

[0065] The specific steps of the synthetic reaction are as follows:

[0066] (1) 3-aminopropanal diethyl acetal and N, N-diisopropylethylamine were dissolved in dichloromethane, and then Fmoc-Cl was dissolved in dichloromethane at 0°C and added to the above reaction vessel. The reaction was stirred overnight and naturally heated to room temperature. After the reaction was completed, the solvent was removed and purified to obtain compound 1;

[0067] (2) Compound 1 was dissolved in tetrahydrofuran, and a 4 mol / L hydrochloric acid solution was prepared and added to the reaction vessel. The reaction was stirred at room temperature. After the reaction was completed, the solvent was removed and purified to obtain Compound 2;

[0068] (3) Compound 2 and hydroxylamine hydrochloride are dissolved in dichloromethane, and then triethylamine is added under stirring conditions, and the reaction is stirred at room temperature. After the reaction is completed, the solvent is removed and purified to obtain compound 3;

[0069] (4) Compound 3 is added to a reaction vessel, and dichloromethane is added under argon protection to dissolve it and pyridine is added, and then NCS (N-chlorosuccinimide) is dissolved in dichloromethane and added to the reaction vessel. After stirring the reaction at room temperature for a period of time, 1-thio-β-D-glucose tetraacetate dissolved in dichloromethane is added and triethylamine is added. The reaction is stirred at room temperature. After the reaction is completed, the solvent is removed and purified to obtain compound 4;

[0070] (5) Compound 4 and pyridine sulfur trioxide were dissolved in acetonitrile, and the mixture was refluxed and stirred at 70 °C. After the reaction was completed, the solvent was removed and the mixture was purified to obtain compound 5;

[0071] (6) Compound 5 was dissolved in dichloromethane and diethylamine was added in a certain volume ratio. The reaction was stirred at room temperature. After the reaction was completed, the solvent was removed and the mixture was purified to obtain Compound 6.

[0072] Then, the amino-thioglucosinolate (n = 2) precursor synthesized by the above scheme was reacted with chlorocoumarin, chlororhodamine, and chloroBODIPY fluorescent matrix (all known compounds) and deacetylated to successfully obtain the target compounds Cou-GSL, Rh-GSL, and BDP-GSL. The general synthesis reaction formula is as follows:

[0073] The synthesis steps of the target compounds Cou-GSL, Rh-GSL and BDP-GSL are as follows:

[0074] (1) For the synthesis of compound Cou-GSL: the chlorocoumarin fluorescent matrix and the amino-thioglucoside precursor (n = 2) prepared in the above scheme, namely compound 6, were dissolved in DMF (N, N-dimethylformamide) and triethylamine was added. The reaction was refluxed and stirred at 80 °C. After the reaction was completed, the solvent was removed and purified to obtain an intermediate compound; the intermediate compound was dissolved in methanol and potassium methoxide was added. The reaction was stirred at room temperature for 5 h. After the reaction was completed, the solvent was removed and purified to obtain the target compound Cou-GSL.

[0075] (2) For the synthesis of compound Rh-GSL: the chlororhodamine fluorescent matrix and the amino-thioglucoside (n = 2) prepared in the above scheme, namely compound 6, were dissolved in dichloromethane and triethylamine was added. The reaction was stirred at 0 °C for 20 min. After the reaction, the solvent was removed and purified to obtain an intermediate compound; the intermediate compound was dissolved in methanol and potassium methoxide was added. The reaction was stirred at room temperature for 3 h. After the reaction, the solvent was removed and purified to obtain the target compound Rh-GSL.

[0076] (3) For the synthesis of compound BDP-GSL: the chloro-BODIPY fluorescent matrix and the amino-thioglucoside (n = 2) prepared in the above scheme, namely compound 6, were dissolved in dichloromethane and triethylamine was added. The reaction was stirred at room temperature for 2 h. After the reaction, the solvent was removed and purified to obtain an intermediate compound; the intermediate compound was dissolved in methanol and potassium methoxide was added. The reaction was stirred at room temperature for 2 h. After the reaction, the solvent was removed and purified to obtain the target compound BODIPY-GSL.

[0077] Furthermore, in the above technical solution, the method for removing the solvent includes reduced pressure evaporation, etc.; the method for purification includes column chromatography, extraction and recrystallization, etc.

[0078] Furthermore, in the above technical solution, during each step of the reaction, the reaction is monitored by real-time TLC (thin layer chromatography).

[0079] Furthermore, in the above technical solution, when the method for purifying the compound in each step is column chromatography, the elution solvent used includes petroleum ether, n-hexane, ethyl acetate, dichloromethane, 1,2-dichloroethane, methanol, etc.

[0080] The present invention also provides the application of the probe for detecting myrosinase, which includes the application in detecting myrosinase, etc. The probe for detecting myrosinase is applied to myrosinase metabolism-related analysis such as myrosinase detection in vitro or in plants and bacterial flora, and screening of myrosinase inhibitors.

[0081] Specifically, the in vitro or in plant and bacterial flora myrosinase detection includes qualitative and quantitative detection of myrosinase.

[0082] Specifically, the in vitro or in plant and bacterial myrosinase detection includes the detection of commercial myrosinase and endogenous myrosinase in plants and bacterial flora.

[0083] Specifically, the screening of the myrosinase inhibitor includes: screening the myrosinase inhibitor using the myrosinase detection probe as a substrate.

[0084] After the myrosinase detection probes Cou-GSL, Rh-GSL and BDP-GSL of the present invention enter the myrosinase cavity during incubation, the sulfonic acid group and glucose part of the probe molecule can form hydrogen bond interactions with specific amino acids of myrosinase and catalyze the cleavage of the glucosinolate bond, releasing a molecule of glucose, hydrogen ions, and unstable aglycone intermediates. The intermediate will spontaneously undergo a Losen rearrangement reaction to rapidly generate an isothiocyanate (R-NCS) with strong electrophilicity, and then undergo an intramolecular ring-closing reaction with the exposed secondary amine site of the fluorescent matrix to generate a thiourea ring with an electron-withdrawing effect, which forms a strong push-pull electron effect with the fluorescent matrix, namely, compounds Cou-thione, Rh-thione and BDP-thione, causing their absorption and fluorescence emission spectra to red-shift, thereby realizing sensitive detection of myrosinase. By monitoring the generation of new fluorescence peaks of enzymatic ring-closing products and changes in fluorescence intensity, the rate difference of the enzymatic hydrolysis process or the change in enzyme activity under different incubation test conditions can be revealed.

[0085] In the present invention, the reaction mechanism of the probes Cou-GSL, Rh-GSL, BDP-GSL and myrosinase is as follows:

[0086]

[0087] The use of the probe of the present invention for detecting changes in the activity of myrosinase and for long-term monitoring of the expression of the enzyme in plants and bacterial populations has the following outstanding advantages:

[0088] (1) Good selectivity: The glucosinolates in the probe molecular structure are specifically recognized by mustard enzymes and undergo enzymatic hydrolysis. The resulting enzymatic cyclization products produce new UV-visible absorption peaks and fluorescence emission peaks, but have no UV-visible absorption or fluorescence response changes to a variety of interfering substances other than mustard enzymes (such as various enzymes, proteins, amino acids, biomacromolecules, active substances, and ionic compounds);

[0089] (2) High sensitivity: Before the probe is recognized and enzymatically hydrolyzed by mustard enzyme, the UV-visible absorption wavelength and fluorescence emission wavelength of the probe itself are relatively short. However, after the probe is recognized and enzymatically hydrolyzed by mustard enzyme, the UV-visible absorption wavelength and fluorescence emission wavelength of the newly generated enzymatic cyclization product are significantly red-shifted. By monitoring the generation of the new fluorescence peak, sensitive detection of mustard enzyme can be achieved.

[0090] (3) Good water solubility: The probe molecular structure has a strong hydrophilic sulfonic acid group and a hydrophilic hydroxyl group on the glucose, which makes this series of probes have good water solubility. This can overcome the disadvantage that most dyes are too fat-soluble and easily form aggregates and cannot be dispersed in water. The strong hydrophilicity enables this type of probe to be better dispersed in the incubation medium, i.e., the aqueous phase, so that it can enter the cavity of the mustard enzyme protein as much as possible, form hydrogen bonds with the surrounding amino acids, and thus catalyze the enzymatic reaction.

[0091] (4) Wide range of applications: The probe can not only be used for in vitro detection and concentration calibration of myrosinase, but can also be used for non-invasive endogenous myrosinase detection in living plants and bacterial communities, which greatly avoids the serious damage and crushing of living plants and bacterial communities by traditional myrosinase detection methods. By observing the change of fluorescence color under ultraviolet light, the process of enzymatic hydrolysis reaction can be understood very intuitively. In addition, the probe is suitable for in situ fluorescence confocal imaging with living plant and bacterial community samples.

[0092] Beneficial effects of the present invention: The present invention can provide a probe for detecting mustard enzyme based on an organic small molecule dye. After this type of probe enters the mustard enzyme cavity and binds to the active site for hydrolysis, the generated enzymatic cyclization product thiourea ring has a certain electron-withdrawing effect, thereby forming a stronger push-pull electron structure with the fluorescent matrix, resulting in its ultraviolet-visible absorption and red shift of the fluorescence spectrum, and the detection of mustard enzyme can be achieved by monitoring the new peak fluorescence signal. This type of probe for detecting mustard enzyme can not only realize the detection of mustard enzyme in vitro, but also can perform in situ non-destructive detection and fluorescence imaging of endogenous mustard enzyme in living plant samples, creatively using fluorescence detection technology to achieve rapid, visual, and highly sensitive detection of mustard enzyme, effectively overcoming the shortcomings of cumbersome sample preparation, destructive analysis, and low visualization in the existing traditional mustard enzyme detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0094] Figure 1The UV-visible absorption and fluorescence spectra of the probe Cou-GSL and the enzymatic cyclization product Cou-thione.

[0095] Figure 2 The UV-visible absorption and fluorescence spectrum responses of the probe Cou-GSL to myrosinase.

[0096] Figure 3 This is a test of the selectivity of the probe Cou-GSL to myrosinase and other interferents.

[0097] Figure 4 This is the fluorescence response spectrum and linear relationship of the probe Cou-GSL to myrosinase.

[0098] Figure 5 The probe Cou-GSL was tested for its response to different incubation temperatures and pH values ​​for myrosinase.

[0099] Figure 6 Fluorescence confocal imaging after incubation of probe Cou-GSL with root tips of vegetable seedlings from different families.

[0100] Figure 7 Time-dependent fluorescence confocal imaging after co-incubation of the probe Cou-GSL with Arabidopsis root tips.

[0101] Figure 8 The UV-visible absorption and fluorescence spectrum responses of the probe Rh-GSL to myrosinase.

[0102] Fig. 9 Fluorescence response test of probe Rh-GSL and different vegetable lysates.

[0103] Fig.10 This is a test of the selectivity of the probe Rh-GSL to myrosinase and other interferents. DETAILED DESCRIPTION

[0104] The following specific examples explain the synthesis method and application of the probes Cou-GSL, Rh-GSL, and BDP-GSL for myrosinase detection based on coumarin, rhodamine, and BODIPY fluorescent matrix in the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0105] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0106] The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0107] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0108] The present invention discloses a probe for detecting myrosinase, a preparation method and an application thereof. The method synthesizes amino-thioglucoside precursors with different chain lengths by simulating the structure of plant secondary metabolites thioglucosides, and the precursors react with halogenated active fluorescent mothers under the action of a catalyst to undergo nucleophilic substitution reaction and then undergo deprotection to obtain target compounds. The synthesis method of the present invention has simple steps and mild reaction conditions. The probe for detecting myrosinase prepared by the present invention has the advantages of good water solubility, high stability, good detection selectivity and high sensitivity; after the probe reacts with myrosinase, the absorption and emission spectra are significantly red-shifted, and by monitoring the generation of the red-shifted emission peak and the change in fluorescence intensity, the detection of myrosinase can be achieved and the difference in enzyme activity can be reflected. The probe for detecting myrosinase of the present invention can be used not only for in vitro myrosinase detection, but also for in situ detection and fluorescence imaging of endogenous myrosinase in plants or flora, and can also be used for myrosinase-related research such as myrosinase inhibitor screening.

[0109] Unless otherwise specified, the test materials used in the examples are all conventional biochemical reagents.

[0110] Example 1: Synthesis of amino-thioglucosinolate precursor, n = 2

[0111]

[0112] Prepared in the following manner:

[0113] (1) Add 3-aminopropanal diethyl acetal (1.47 g, 10.0 mmol) and N,N-diisopropylethylamine (3.5 mL, 20.0 mmol) to a reaction vessel and add dichloromethane (30.0 mL) to dissolve and stir, keeping the reaction temperature at 0 ℃. Then, dissolve Fmoc-Cl (2.71 g, 10.5 mmol) in an appropriate amount of dichloromethane at 0 ℃ and add dropwise to the above reaction vessel. Stir the reaction overnight and naturally warm to room temperature. After the reaction is completed, wash the reaction mixture with ammonium chloride, sodium bicarbonate and brine respectively, and then combine the organic phases. Dry the organic phases with anhydrous sodium sulfate and transfer them to a flask to remove the solvent by vacuum rotary evaporation. Column chromatography (PE / EA = 5 / 1, V / V ) The crude product was further purified to obtain compound 1 (2.58 g, 7.0 mmol) as a white solid with a yield of 70%. HRMS-ESI (m / z): [M+Na] + Calculate for C 22 H 27 NNaO4 + :392.1832, found: 392.1842.

[0114] Compound 1 NMR: 1 H NMR (500 MHz, Chloroform-d) δ 7.76 (d, J = 7.5 Hz,2H), 7.60 (d, J = 7.5 Hz, 2H), 7.41-7.38 (m, 2H), 7.32-7.29 (m, 2H), 5.32 (s,1H), 4.57 (t, J = 5.3 Hz, 1H), 4.38 (d, J = 7.1 Hz, 2H), 4.22 (t, J = 7.1 Hz,1H), 3.72-3.65 (m, 2H), 3.54-3.48 (m, 2H), 3.33 (q, J = 6.1 Hz, 2H), 1.85 (q, J = 6.0 Hz, 2H), 1.23 (t, J = 7.0 Hz, 6H). 13C NMR (126 MHz, Chloroform-d) δ156.36, 144.06, 141.33, 127.65, 127.00, 125.09, 119.96, 102.25, 66.56, 61.84,47.30, 37.15, 33.30, 15.34.

[0115] (2) Compound 1 (2.40 g, 6.5 mmol) was transferred to a reaction vessel, tetrahydrofuran (30.0 mL) was added to dissolve the compound, 4.0 mol / L hydrochloric acid solution was prepared and 6.0 mL was added dropwise to the reaction vessel. The reaction mixture was stirred at room temperature for 6 h. After the reaction, the reaction mixture was extracted with ethyl acetate and saturated sodium chloride brine, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate and transferred to a flask to remove the solvent by vacuum rotary evaporation. The column chromatography method (PE / EA= 2 / 1, V / V ) The crude product was further purified to obtain compound 2 (1.52 g, 5.2 mmol) as a white solid with a yield of 80%. HRMS-ESI (m / z): [M+Na] + Calculate for C 18 H 17 NNaO3 + : 318.1101, found: 318.1115.

[0116] Compound 2 NMR: 1 H NMR (500 MHz, Chloroform- d ) δ 9.80 (s, 1H), 7.76 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 7.5 Hz, 2H), 7.42-7.39 (m, 2H), 7.33-7.30 (m,2H), 5.20 (s, 1H), 4.39 (d, J = 7.0 Hz, 2H), 4.2 (t, J = 7.1 Hz, 1H), 3.50-3.47 (m, 2H), 2.73 (t, J = 5.8 Hz, 2H). 13 C NMR (126 MHz, Chloroform- d) δ201.27, 156.36, 143.89, 141.34, 127.72, 127.06, 125.05, 120.01, 66.71, 47.25,44.06, 34.47.

[0117] (3) Compound 2 (1.33 g, 4.5 mmol) and hydroxylamine hydrochloride (0.63 g, 9.0 mmol) were transferred to a reaction vessel and dichloromethane (25.0 mL) was added to dissolve and stir. Then triethylamine (2.5 mL, 18.0 mmol) was added dropwise during stirring. The reaction was stirred at room temperature for 4 h. After the reaction was completed, the mixture was transferred to a flask and the solvent and triethylamine were removed by high-temperature vacuum rotary evaporation. The residue was washed and extracted with dichloromethane and saturated sodium chloride brine for multiple times. The organic phases were combined and dried over anhydrous sodium sulfate. The organic phases were transferred to a flask and the solvent was removed by vacuum rotary evaporation. Column chromatography (PE / EA = 1 / 1, V / V ) The crude product was further purified to obtain compound 3 (1.05 g, 3.4 mmol) as a white solid with a yield of 75%. HRMS-ESI (m / z): [M+Na] + Calculate for C 18 H 18 N2NaO3 + : 333.1210, found: 333.1226.

[0118] Compound 3 NMR: 1 H NMR (600 MHz, Methanol- d 4) δ 7.77 (d, J = 7.5 Hz, 2H),7.62 (d, J = 7.5 Hz, 2H), 7.38-7.36 (m, 2H), 7.31-7.28 (m, 2 H), 4.32 (t, J =6.4 Hz, 2H), 4.18 (t, J = 6.9 Hz, 1H), 3.28 (q, J =6.5 Hz, 2H), 2.54 (q, J =6.4 Hz, 1H), 2.34 (q, J = 6.5 Hz, 1H). 13 C NMR (151 MHz, Methanol- d4) δ 148.65,148.31, 143.92, 141.19, 127.37, 126.75, 124.78, 119.52, 66.32, 47.06, 37.77,37.23, 29.84, 25.48.

[0119] (4) Compound 3 (0.93 g, 3.0 mmol) was added to a dry reaction vessel and argon was replaced. Under argon protection, ultra-dry dichloromethane was added to dissolve the mixture and anhydrous pyridine (0.48 mL, 6.0 mmol) was added dropwise. NCS (0.48 g, 3.6 mmol) was then dissolved in ultra-dry dichloromethane and transferred to the above reaction vessel. The reaction was stirred at room temperature under argon protection for 4 h, and then 1-thio-β-D-glucose tetraacetate (2.19 g, 6.0 mmol) dissolved in ultra-dry dichloromethane was added. Then triethylamine (1.25 mL, 9.0 mmol) was added dropwise. The reaction was stirred at room temperature overnight. After the reaction, the mixture was transferred to a flask and the solvent and triethylamine were removed by high-temperature vacuum rotary evaporation. The residue was washed and extracted with dichloromethane, 2.0 mol / L hydrochloric acid aqueous solution and saturated sodium chloride brine. The organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was transferred to a flask and the solvent was removed by vacuum rotary evaporation. Column chromatography (PE / EA = 1 / 1, V / V ) The crude product was further purified to obtain compound 4 (1.61 g, 2.4 mmol) as a white solid. Yield: 80%. HRMS-ESI (m / z): [M+Na] + Calculate for C 32 H 36 N2NaO 12 S + : 695.1881, found: 695.1896.

[0120] Compound 4 NMR: 1 H NMR (500 MHz, Chloroform- d ) δ 7.76 (d, J = 7.6 Hz,2H), 7.58 (d, J = 7.5 Hz, 2H), 7.41-7.38 (m, 2H), 7.32-7.29 (m, 2H), 5.39 (t, J = 6.2 Hz, 1H), 5.29 (t, J = 9.3 Hz, 1H), 5.20 (d, J= 10.1 Hz, 1H), 5.10-5.05 (m, 2H), 4.41 (d, J = 6.9 Hz, 2H), 4.20 (t, J = 6.8 Hz, 1H), 4.12 (d, J = 3.8 Hz, 2H), 3.87-3.83 (m, 1H), 3.47 (q, J = 6.5 Hz, 2H), 2.80-7.70 (m,2H), 2.04-2.00 (m, 12H). 13 C NMR (126 MHz, Chloroform- d ) δ 170.69 170.21,169.52, 169.35, 156.69, 143.83, 141.32, 127.76, 127.09, 125.01, 120.04, 79.5875.70, 73.78, 70.12, 68.03 66.84, 62.11 47.18, 38.55, 32.78, 20.63, 20.61,20.58

[0121] (5) Compound 4 (1.43 g, 2.0 mmol) and pyridine sulfur trioxide (1.59 g, 10.0 mmol) were added to a reaction vessel, anhydrous acetonitrile (20.0 mL) was added and dissolved in 70 °C, and the mixture was refluxed and stirred for 4 h. After the reaction was completed, the mixture was vacuum evaporated to remove the solvent, and the residue was washed and extracted with ethyl acetate and saturated sodium chloride brine for multiple times. The organic phases were combined and dried over anhydrous sodium sulfate, and the organic phases were transferred to a flask and vacuum evaporated to remove the solvent. Column chromatography (DCM / MeOH = 10 / 1, V / V ) The crude product was further purified to obtain compound 5 (1.10 g, 1.6 mmol) as a white solid with a yield of 80%. HRMS-ESI (m / z): [M] - Calculate for C 32 H 35 N2O 15 S2 - : 751.1484; found: 751.1487.

[0122] Compound 5 NMR: 1 H NMR (500 MHz, Methanol- d 4) δ 7.78 (d, J= 7.5 Hz, 2H),7.66 (d, J = 7.4 Hz, 2H), 7.40-7.37 (m, 2H), 7.33-7.30 (m, 2H), 5.50 (d, J =10.2 Hz, 1H), 5.38 (t, J = 9.3 Hz, 1H), 5.04-4.98 (m, 2H), 4.40-4.31 (m, 2H), 4.22 (t, J = 7.0 Hz,1 H), 4.13 (d, J = 3.9 Hz,2H), 4.08-4.04 (m, 1H), 3.48(t, J = 6.9 Hz, 2H), 2.92-2.85 (m, 2H), 2.01 (s, 3H), 2.00 (s, 3H), 1.97 (s,3H), 1.95 (s, 3H). 13 C NMR (126 MHz, Methanol- d 4) δ 170.88, 170.14, 169.87,169.60, 157.53, 155.98, 143.99, 143.91, 141.19, 127.40, 126.83, 124.92,119.54, 79.07, 75.24, 73.68, 69.88, 68.14, 66.50, 61.99, 47.05, 38.04, 32.45,19.25, 19.17, 19.15.

[0123] (6) Compound 5 (0.90 g, 1.2 mmol) was transferred to a reaction vessel, and dichloromethane (20.0 mL) and ethylenediamine (4.0 mL) were added and stirred at room temperature for 3 h. After the reaction was completed, the mixture was transferred to a flask and vacuum evaporated to remove the solvent and ethylenediamine. The residue was washed and extracted with dichloromethane and saturated sodium chloride brine for multiple times. The organic phases were combined and dried with anhydrous sodium sulfate, and the organic phases were transferred to a flask and vacuum evaporated to remove the solvent. Column chromatography (PE / EA = 3 / 1, V / V ) The crude product was further purified to obtain compound 6 (0.19 g, 0.36 mmol) as a light yellow solid. Yield: 30%. HRMS-ESI (m / z): [M] - Calculate for C 17 H25 N2O 13 S2 - : 529.0804; found:529.0823.

[0124] Example 2: Synthesis of coumarin-based fluorescent parent myrosinase detection probe Cou-GSL

[0125]

[0126] (1) 3-Diethylaminophenol (3.3 g, 20 mmol) and diphenyl malonate (5.12 g, 20 mmol) were transferred to a reaction vessel, toluene (150 mL) was added and stirred at 110 °C for 12 h, and then the reaction mixture was filtered through diatomaceous earth to remove the reaction precipitate, and the remaining reaction solution was transferred to a flask and the solvent was removed by vacuum rotary evaporation. Column chromatography (DCM / MeOH = 3 / 1, V / V ) The crude product was further purified to obtain compound 7 (1.16 g, 5.0 mmol) as a white solid. Yield: 25%. HRMS-ESI (m / z): [M] - Calculate for C 13 H 14 NO3 - : 232.0979; found:232.0981.

[0127] (2) Compound 7 (1.16 g, 5.0 mmol) was transferred to a reaction vessel, phosphorus oxychloride (15 mL) was added and stirred at 85 °C for 6 h. After the reaction was completed, the reaction mixture was poured into a stirred ice-water bath. After the exotherm was completed, the aqueous phase was extracted several times with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the organic phases were transferred to a flask and vacuum evaporated to remove the solvent. Column chromatography (PE / EA = 5 / 1, V / V ) The crude product was further purified to obtain compound 8 (0.37 g, 1.5 mmol) as a yellow solid. Yield: 30%. HRMS-ESI (m / z): [M] + Calculate for C 13 H 14 ClNO2: 251.0713; found: 251.0726.

[0128] (3) Compound 8 (50.0 mg, 0.20 mmol) and the amino-thioglucoside precursor (n = 2) prepared in Example 1 of the present invention, namely compound 6 (0.11 g, 0.2 mmol), were transferred to a reaction vessel and dissolved with DMF solvent (4.0 mL), and then triethylamine (0.14 mL, 1.0 mmol) was added dropwise. The reaction mixture was refluxed and stirred at 80 °C for 48 h. After the reaction was completed, the reaction mixture was repeatedly washed with ethyl acetate and saturated sodium chloride brine, the organic phases were combined and dried over anhydrous sodium sulfate, and the organic phases were transferred to a flask and vacuum evaporated to remove the solvent. Column chromatography (DCM / MeOH = 10 / 1, V / V ) The crude product was further purified to obtain compound 9 (75.0 mg, 0.10 mmol) as a light brown solid. Yield: 50%. HRMS-ESI (m / z): [M] - Calculate for C 30 H 38 N3O 15 S2 - : 744.1750; found: 744.1733.

[0129] (4) Compound 9 (73.0 mg, 0.10 mmol) was transferred to a reaction vessel, methanol (2.0 mL) was added, and potassium methoxide (35.0 mg, 0.50 mmol) was slowly added under stirring. The reaction was stirred at room temperature for 5 h. After the reaction was completed, the mixture was transferred to a flask and vacuum evaporated to remove the solvent. The reaction was performed by high performance liquid chromatography (30-90% MeCN / H2O, 0-40 min linear gradient, 0.1% V / V TFA, t = 6.5 min) to further purify the crude product to obtain the target compound Cou-GSL (5.7 mg, 0.01 mmol) as a white solid. Yield: 10%. HRMS-ESI (m / z): [M] - Calculate for C 22 H 30 N3O 11 S2 - : 576.13270; found: 576.1311.

[0130] Compound Cou-GSL NMR: 1 H NMR (600 MHz, DMSO- d 6) δ 7.70 (d, J= 9.0 Hz,1H), 7.21 (s, 1H), 6.63 (d, J = 9.1 Hz, 1H), 6.43 (s, 1H), 4.90 (s, 1H), 4.82(d, J = 9.8 Hz, 1H), 3.41 (q, J = 6.6 Hz, 8H), 3.28-3.22 (m, 3H), 3.13-3.05(m, 3H), 2.92 (q, J = 6.2 Hz, 2H), 2.07 (s, 1H), 1.11 (t, J = 7.0 Hz, 6H).

[0131] Example 3: Synthesis of Rh-GSL, a rhodamine fluorescent parent myrosinase detection probe

[0132]

[0133] (1) Dissolve 3-bromo-N,N-dimethylaniline (2.40 g, 12.0 mmol) in 37% formaldehyde solution (1.90 mL) and acetic acid (17.0 mL) and stir at 60 °C for 2 hours. After the reaction is completed, remove the solvent by vacuum rotary evaporation, add saturated sodium bicarbonate aqueous solution and extract the aqueous phase with ethyl acetate. Wash the reaction mixture repeatedly with ethyl acetate and saturated sodium chloride brine, combine the organic phases and dry them with anhydrous sodium sulfate, transfer the organic phase to a flask and remove the solvent by vacuum rotary evaporation. Column chromatography (PE / EA = 5 / 1, V / V ) The crude product was further purified to obtain compound 10 (3.45 g, 8.4 mmol) as a white solid. Yield: 70%. HRMS-ESI (m / z): [M] + Calculate for C 17 H 21 Br2N 2: 413.0046; found:413.0025.

[0134] (2) Compound 10 (2.5 g, 6.0 mmol) was transferred to a reaction vessel and argon was replaced. Then, anhydrous tetrahydrofuran (30 mL) was added as solvent and n-butyl lithium (2.5 mol / L cyclohexane solution, 4.00 mL, 10.0 mmol) was slowly added dropwise at -78°C and maintained at -78°C for 1.5 h. Dichlorodimethylsilane (0.97 mL, 10.0 mmol) was added dropwise to the reaction vessel at -78°C. The mixture was naturally warmed to room temperature and stirred for 2 h. After the reaction, HCl aqueous solution (10 mL, 2 mol / L) was added to quench the reaction mixture and the solution was neutralized to pH = 7 with saturated sodium bicarbonate aqueous solution. The reaction mixture was repeatedly washed with ethyl acetate and saturated sodium chloride brine, the organic phases were combined and dried over anhydrous sodium sulfate, and the organic phases were transferred to a flask and vacuum evaporated to remove the solvent. The reaction mixture was purified by column chromatography (PE / EA = 5 / 1, V / V ) The crude product was further purified to obtain compound 11 (0.93 g, 3.0 mmol) as a light yellow solid. Yield: 50%. HRMS-ESI (m / z): [M] calculated for C 20 H 26 N2Si : 311.1938; found: 311.1929.

[0135] (3) Powdered potassium permanganate (0.71 g, 4.5 mmol) was slowly added to a solution of compound 11 (0.93 g, 3.0 mmol) in acetone (25 mL) at 0 °C, and the reaction mixture was stirred at 0 °C for 2 h. After the reaction, the insoluble residue was removed with diatomaceous earth, and the remaining solution was transferred to a flask and removed by vacuum rotary evaporation. The product was purified by column chromatography (PE / EA = 4 / 1, V / V ) The crude product was further purified to obtain compound 12 (0.58 g, 1.8 mmol) as a yellow solid. Yield: 40%. HRMS-ESI (m / z): [M] + Calculate for C 19 H 25 N2OS : 325.1731; found: 325.1714.

[0136] (4) Compound 12 (0.58 g, 1.8 mmol) was transferred to a reaction vessel and argon was replaced. Ultra-dry dichloromethane (15 mL) was added to dissolve the compound, and then oxalyl chloride (0.76 mL, 9.0 mmol) was slowly added at 0 °C for 20 min. After the reaction, the reaction solvent and oxalyl chloride were pumped away with a water pump while stirring the mixture. The remaining trace solvent in the reaction vessel was then pumped dry with an oil pump to obtain compound 13 (0.58 g, 0.90 mmol), a green solid, with a yield of 50%. HRMS-ESI (m / z): [M] calcd for C 19 H 24 ClN2Si : 343.1392; found:343.1378.

[0137] (5) The nitrogen atmosphere in the reaction vessel of compound 13 (102.9 mg, 0.30 mmol) was replaced, and then the amino-thioglucoside (n = 2) prepared in Example 1 of the present invention, namely compound 6 (0.16 g, 0.30 mmol), was dissolved in ultra-dry dichloromethane solvent (6.0 mL) and transferred to the above reaction vessel, and then triethylamine (0.21 mL, 1.50 mmol) was added dropwise. The reaction mixture was stirred at 0 ° C for 20 min. After the reaction was completed, the reaction mixture was repeatedly washed with dichloromethane and saturated sodium chloride brine, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was transferred to a flask and the solvent was removed by vacuum rotary evaporation. Column chromatography (DCM / MeOH = 10 / 1, V / V ) The crude product was further purified to obtain compound 14 (167.2 mg, 0.20 mmol) as an orange solid with a yield of 67%. HRMS-ESI (m / z): [M] calculated for C 36 H 48 N4O 13 S2S - : 836.2429; found: 836.2413.

[0138] (6) Compound 14 (167.2 mg, 0.20 mmol) was transferred to a reaction vessel, methanol (5.0 mL) was added, and potassium methoxide (70.1 mg, 1.0 mmol) was slowly added under stirring. The reaction was stirred at room temperature for 3 h. After the reaction was completed, the mixture was transferred to a flask and vacuum evaporated to remove the solvent. Column chromatography (DCM / MeOH = 3 / 1, V / V) The crude product was further purified to obtain compound Rh-GSL (20.4 mg, 0.03 mmol) as an orange solid with a yield of 15%. HRMS-ESI (m / z): [M] - Calculate for C 28 H 40 N4O9S2Si - : 668.2006; found: 668.2017.

[0139] Example 4: Synthesis of BODIPY-based fluorescent parent myrosinase detection probe BDP-GSL

[0140]

[0141] (1) Pyrrole (0.7 mL, 10 mmol) and N,N-diisopropylethylamine (1.76 mL, 10 mmol) were transferred to a reaction vessel and 1,2-dichloroethane (15 mL) was added to dissolve the mixture. The mixture was stirred in an ice bath under a nitrogen atmosphere for 20 minutes. Triphosgene (0.9 g, 3 mmol) dissolved in 1,2-dichloroethane (15 mL) was then added dropwise to the mixture and stirred at room temperature for 2 h. After the reaction was completed, the reaction mixture was repeatedly washed with 1,2-dichloroethane and saturated sodium chloride brine. The organic phases were combined and dried over anhydrous sodium sulfate. The organic phases were transferred to a flask and the solvent was removed by vacuum rotary evaporation. The reaction mixture was purified by column chromatography (DCM / EA = 10 / 1, V / V ) The crude product was further purified to obtain compound 15 (0.43 g, 2.7 mmol) as a white solid. Yield: 90%. HRMS-ESI (m / z): [M] + calcd for C9H8N2ONa: 183.0534; found:183.0530.

[0142] (2) Compound 15 (0.43 g, 2.7 mmol) was transferred to a reaction vessel and 1,2-dichloroethane (10 mL) was added to dissolve it, and then phosphorus oxychloride (0.50 mL, 5.4 mmol) was added and heated to reflux for 3 h. After the reaction time was up, the reactants were cooled and triethylamine (1.8 mL, 13.5 mmol) was added at 0 °C and stirred for 5 min, and then boron trifluoride etherate (1.7 mL, 13.5 mmol) was added dropwise and continued to stir for 2 h and warmed to room temperature. After the reaction was completed, the reaction mixture was repeatedly washed with 1,2-dichloroethane and saturated sodium chloride brine, the organic phases were combined and dried over anhydrous sodium sulfate, and the organic phases were transferred to a flask and vacuum evaporated to remove the solvent. The reaction mixture was purified by column chromatography (DCM / EA = 5 / 1, V / V ) The crude product was further purified to obtain compound 16 (0.30 g, 1.35 mmol) as a yellow solid. Yield: 50%. HRMS-ESI (m / z): [M] + calcd for C9H7BClF2N2: 227.0359; found: 227.0341.

[0143] (3) Compound 16 (67.8 mg, 0.30 mmol) and the amino-thioglucoside (n = 2) synthesized in Example 1 of the present invention, namely compound 6 (0.16 g, 0.3 mmol), were transferred to a reaction vessel and dissolved with dichloromethane solvent (5.0 mL), and then triethylamine (0.21 mL, 1.5 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 4 h. After the reaction was completed, the reaction mixture was repeatedly washed with dichloromethane and saturated sodium chloride brine, the organic phases were combined and dried over anhydrous sodium sulfate, and the organic phases were transferred to a flask and vacuum evaporated to remove the solvent. Column chromatography (DCM / MeOH = 5 / 1, V / V ) The crude product was further purified to obtain compound 17 (107.8 mg, 0.15 mmol) as an orange solid. Yield: 50%. HRMS-ESI (m / z): [M] - Calculate for C 26 H 30 BF2N4O 13 S2 - : 719.1317; found: 719.1302.

[0144] (4) Compound 17 (107.8 mg, 0.15 mmol) was transferred to a reaction vessel, methanol (4.0 mL) was added, and potassium methoxide (52.6 mg, 0.75 mmol) was slowly added under stirring. The reaction was stirred at room temperature for 5 h. After the reaction was completed, the mixture was transferred to a flask and the solvent was removed by vacuum rotary evaporation. Column chromatography (DCM / MeOH = 2 / 1, V / V ) The crude product was further purified to obtain compound BDP-GSL (16.53 mg, 0.03 mmol) as an orange solid with a yield of 20%. HRMS-ESI (m / z): [M] - Calculate for C 18 H 22 BF2N4O9S2 - : 551.0895; found: 551.0877.

[0145] Example 5: Changes in UV-visible absorption and fluorescence spectrum response of the probe Cou-GSL reacting with myrosinase

[0146] Incubation operation and conditions: 1.0 μL of the probe Cou-GSL prepared in Example 2 of the present invention with a concentration of 1.0 mM and 4.0 μL of commercial mustard enzyme (purchased from Sigma-Aldrich, derived from white mustard) prepared with a concentration of 7000 U / L were aspirated into PBS buffer (20 mM, pH=7.4) to make a total volume of 400 μL, and the concentration of the probe Cou-GSL prepared in Example 2 of the present invention was kept at 2.5 μM and the concentration of mustard enzyme was kept at 70 mU / mL. The above mixture was mixed evenly and transferred to a PE tube, and the PE tube was placed in a water bath at 37°C and incubated for different times (0-40 min). The reaction solution in the PE tube was subjected to UV-visible absorption and fluorescence tests every 10 min of incubation.

[0147] like Figure 1 As shown in FIG. 1 , the UV-visible absorption spectrum and fluorescence spectrum of the probe Cou-GSL prepared in Example 2 of the present invention and the enzymatic cyclization product Cou-thione are shown. Figure 2 As shown in (a), the UV-visible absorption spectrum of the reaction solution after the probe Cou-GSL prepared in Example 2 of the present invention was incubated with mustard enzyme for different time periods. As the incubation time increased, the UV-visible absorption of the probe Cou-GSL at 352 nm gradually decreased, and at the same time, an obvious new absorption peak was generated at 405 nm. The above indicates that as the incubation time increased, the enzymatic hydrolysis process was improved, and the absorption spectrum of the enzymatic hydrolysis ring-closing product Cou-thione was red-shifted by nearly 50 nm compared with the probe Cou-GSL itself; at the same time, Figure 2 As shown in (b), with the extension of incubation time, when the excitation wavelength is 420 nm, an obvious new fluorescence peak can be observed at 500 nm and the growth intensity is synchronized with the extension of incubation time.

[0148] Example 6: Selectivity test of probe Cou-GSL against myrosinase and other interferents

[0149] Incubation operation and conditions: 1.0 μL of the probe Cou-GSL prepared in Example 2 of the present invention with a concentration of 1.0 mM and 4.0 μL of commercial mustard enzyme (purchased from Sigma-Aldrich, derived from white mustard) with a concentration of 7000 U / L were taken into PBS buffer (20 mM, pH = 7.4) to make the total volume 400 μL, and the probe concentration was kept at 2.5 μM and the mustard enzyme concentration was kept at 70 mU / mL. The above mixture was mixed evenly and transferred to a PE tube, and the PE tube was placed in a water bath at 37°C and incubated for 40 minutes. After the incubation, the reaction solution in the PE tube was tested for fluorescence.

[0150] In the response test involving other interfering substances (various enzymes, proteins, amino acids, biomacromolecules, ionic compounds) except mustard enzyme, each interfering substance (50 times equivalent concentration) was incubated with 2.5 μM probe in PE tube at 37 °C for 40 min. After the incubation, the reaction solution was sent for fluorescence analysis with an excitation wavelength of 420 nm. The fluorescence intensity at the optimal emission wavelength of 500 nm was measured. At the same time, the same concentration of probe PBS solution without mustard enzyme and various interfering substances was measured as the background fluorescence intensity. Figure 3 It can be seen that the fluorescence measured in the experimental group incubated with mustard enzyme increased by nearly 33 times compared with the control group without any interfering substances, while the other experimental groups incubated with various interfering substances showed almost no fluorescence increase compared with the control group. The probe Cou-GSL prepared in Example 2 of the present invention has a high selectivity for mustard enzyme.

[0151] Example 7: Concentration-dependent fluorescence response spectrum and linear relationship of probe Cou-GSL to myrosinase

[0152] In the same incubation system as Example 5 of the present invention, the concentration of myrosinase was changed (10 mU / mL-110 mU / mL) for incubation, and the incubation reaction solution was subjected to fluorescence analysis after the incubation. The fluorescence intensity of the enzymatic hydrolysis product at 500 nm and the protein concentration were used as a standard curve, and the minimum detection limit of the probe for myrosinase was calculated by the formula DL= 3σ / k, where σ represents the standard deviation of the blank sample, and k represents the linear coefficient between the fluorescence intensity at 500 nm and the enzyme concentration after the probe Cou-GSL prepared in Example 2 of the present invention reacts with myrosinase, i.e., the slope. Figure 4 As shown, Figure 4 (a) is the fluorescence intensity curve measured after adding different concentrations of mustard enzyme to the reaction solution. Figure 4 (b) shows the linear relationship between the concentration of myrosinase protein and the fluorescence intensity, and the minimum detection limit of myrosinase is calculated; the excitation wavelength is 420 nm. Figure 4 As shown in (b), within the range of 10-70 mU / mL of myrosinase protein concentration, the fluorescence peak intensity at 500 nm gradually increased after the enzymatic reaction and showed a good linear relationship with the myrosinase concentration. When the myrosinase concentration was higher than 70 mU / mL, the fluorescence was close to saturation. According to the linear relationship, the detection limit of myrosinase was as low as 0.8 mU / mL. The above results prove that the probe Cou-GSL prepared in Example 2 of the present invention can achieve sensitive detection of myrosinase.

[0153] Example 8: Test of the different reaction temperatures and pH responses of the probe Cou-GSL to myrosinase

[0154] In the same incubation system as in Example 5 of the present invention, the incubation temperature was changed and the incubation time was different. After the incubation, the fluorescence test was performed on the incubation reaction solution. Figure 5 As shown in (a), the fluorescence intensity of the enzymatic hydrolysis product at 500 nm shows very different growth trends over time under incubation conditions at different temperatures: the time required for the fluorescence to reach saturation under low temperature conditions is greatly prolonged, while the time required for the fluorescence to reach saturation under high temperature conditions of 55°C is only about 15 minutes. The above shows that temperature has a great influence on enzyme activity, and appropriately increasing the incubation temperature can greatly increase enzyme activity. Taking into account that subsequent practical applications involve living plant samples, the relatively mild condition of 37°C was finally selected as the incubation temperature for the application of this system. After testing, the time required for the fluorescence of the enzymatic hydrolysis product to approach saturation was 40 minutes. At the same time, in the same incubation system as Example 5, the pH value of the incubation medium buffer was changed to maintain the incubation temperature and incubation time unchanged. After the incubation, the fluorescence of the incubation reaction solution was measured. As Figure 5 As shown in (b), the fluorescence intensity of the enzymatic hydrolysis product at 500 nm also changes under different pH buffer conditions. In general, the fluorescence intensity measured in the incubation solution in the pH = 6-10 range is stronger, and the light intensity measured in the incubation solution in the pH < 6 strong acidic range and pH > 10 strong alkaline range is weaker. The three-dimensional structure of the enzyme may be destroyed under strong acid and strong alkaline conditions, and the enzyme activity will also decrease accordingly.

[0155] Example 9: Fluorescence confocal imaging of the probe Cou-GSL after incubation with living root tissues of vegetable seedlings from different families

[0156] According to relevant literature reports, myrosinase is mainly distributed in cruciferous plants, such as cabbage, white radish, cauliflower, broccoli, etc. The myrosinase-glucosinolate system together constitutes the defense system of cruciferous plants to deal with aphids, while myrosinase is almost non-existent in non-cruciferous plants. In order to verify the literature reports, several cruciferous vegetable seeds (such as cabbage, white radish, cabbage, cauliflower, broccoli) and two non-cruciferous vegetable seeds such as lettuce (Asteraceae) and cucumber (Cucurbitaceae) were purchased from the local farmers' market. The above different vegetable seeds were sown in the soil in appropriate amounts, watered and fertilized regularly every day, and 5-day-old seedlings were uniformly collected as experimental samples; further, in order to prevent the plant's own chlorophyll from interfering with fluorescence detection, the stems and leaves of the above-collected seedlings were removed, and the remaining roots of different vegetables were co-incubated with 5.0 μL of the probe Cou-GSL prepared in Example 2 of the present invention at 37 ° C for 5 h. After the incubation time was up, the roots of the seedlings were washed three times with clean water and placed on a glass slide for fluorescence confocal imaging (excitation wavelength: 440 nm). Figure 6 As shown, under the same excitation light intensity and other parameter settings, the root tips of several cruciferous vegetable seedlings all showed strong fluorescence distribution; while no fluorescence distribution appeared in the root tips of non-cruciferous vegetable lettuce and cucumber seedlings. The above experimental phenomena can explain that when the probe Cou-GSL prepared in Example 2 of the present invention diffuses into the root tip cells, the mustard enzyme in the root tip of the cruciferous vegetable seedlings catalyzes the hydrolysis of the probe to generate the enzymatic cyclization product Cou-thione, so under the excitation of 440 nm wavelength, obvious fluorescence accumulation can be observed in the root tip; while the non-cruciferous vegetable seedlings, cucumber and lettuce, do not express mustard enzymes, and the probes are metabolized into the plant body and cannot be enzymatically hydrolyzed, so the fluorescence distribution in the root tip cannot be observed. It can be seen that the probe Cou-GSL prepared in Example 2 of the present invention can perform non-invasive in situ detection of mustard enzymes on real living plant samples.

[0157] Example 10: Time-dependent fluorescence confocal imaging of the probe Cou-GSL after co-incubation with living Arabidopsis root tissue

[0158] After disinfection, an appropriate amount of Arabidopsis seeds (Colombia wild type Col-0) were planted in 1 / 2 MS culture medium in a clean bench. The culture medium was sealed and dark and placed in a 4 ℃ refrigerator for vernalization for 48 hours. After that, it was transferred to an intelligent artificial climate box for cultivation. The culture conditions were: 22 ℃, 65% humidity, light (6600 lx) for 16 hours; dark for 8 hours under the same temperature and humidity conditions, with two sections for a total of 24 hours of reciprocating cycles. Five-day-old Arabidopsis seedlings were taken as experimental samples. The stems and leaves of the seedlings were removed, and the remaining Arabidopsis roots were incubated with 5.0 μM of the probe Cou-GSL prepared in Example 2 of the present invention at 37 ℃ for different times (0-20 hours), with a group set up every 5 hours. After the incubation time of each group was up, the roots of the Arabidopsis seedlings were washed three times with clean water and placed on a glass slide for fluorescence confocal imaging (excitation wavelength: 440 nm). Figure 7 As shown, as the incubation time of the probe Cou-GSL prepared in Example 2 of the present invention with the Arabidopsis root tip increases, the fluorescence accumulation of the Arabidopsis root tip is also significantly enhanced, showing a time-dependent fluorescence growth. It can be seen that it takes a certain amount of time for the probe Cou-GSL prepared in Example 2 of the present invention to be metabolized into the root tip cells, and then undergoes an enzymatic hydrolysis reaction with the myrosinase in the root tip cells. Therefore, the fluorescence growth of the probe Cou-GSL prepared in Example 2 of the present invention incubated with the Arabidopsis root tip has a certain time dependence.

[0159] Example 11: Changes in UV-visible absorption and fluorescence spectrum response of the probe Rh-GSL reacting with myrosinase

[0160] Incubation operation and conditions: 4.0 μL of the probe Rh-GSL prepared in Example 3 of the present invention with a concentration of 1.0 mM and 8.0 μL of commercial mustard enzyme (purchased from Sigma-Aldrich, derived from white mustard) prepared with a concentration of 7000 U / L were aspirated into PBS buffer (20 mM, pH=7.4) to make a total volume of 400 μL, and the concentration of the probe prepared in Example 3 of the present invention was kept at 10.0 μM and the concentration of mustard enzyme was kept at 140 mU / mL. The above mixture was mixed evenly and transferred to a PE tube, and the PE tube was placed in a water bath at 37°C and incubated for different times (0 h, 36 h, 60 h), and then the reaction solution in the PE tube was subjected to UV-visible absorption and fluorescence tests.

[0161] like Figure 8As shown in (a), the UV-visible absorption spectrum of the reaction solution after the probe Rh-GSL was incubated with mustard enzyme for 36 h and 60 h, with the extension of the incubation time, the UV-visible absorption of the probe Rh-GSL prepared in Example 3 of the present invention at 468 nm gradually decreased, and at the same time, an obvious new UV-visible absorption peak was generated at 677 nm, which indicated that with the extension of the incubation time, the enzymatic hydrolysis process was improved, and the absorption spectrum of the enzymatic hydrolysis ring-closing product Rh-thione was significantly red-shifted by nearly 210 nm compared with the probe Rh-GSL prepared in Example 3 of the present invention itself; at the same time, as shown in Figure 8 As shown in (b), with the extension of incubation time, when the excitation wavelength is 660 nm, an obvious new fluorescence peak is generated at 693 nm and the increasing intensity is synchronized with the extension of incubation time. In general, the enzymatic hydrolysis rate of the commercial mustard enzyme (purchased from Sigma-Aldrich, derived from white mustard) on the probe Rh-GSL prepared in Example 3 of the present invention is slower than that of Cou-GSL.

[0162] Example 12: Fluorescence response measurement of probe Rh-GSL after incubation with different vegetable lysates

[0163] In view of the slow enzymatic hydrolysis rate of the commercial mustard enzyme (purchased from Sigma-Aldrich, derived from white mustard) in Example 11 for the probe Rh-GSL prepared in Example 3 of the present invention, according to relevant literature reports, the present invention considers that mustard enzymes from different sources may have large differences in response selectivity to the same substrate. The present invention purchased different cruciferous vegetables (baby cabbage, cabbage, white radish, cauliflower, broccoli) at the local farmers' market to verify whether the mustard enzymes expressed by different varieties may have different enzymatic hydrolysis rates for the probe Rh-GSL prepared in Example 3 of the present invention. At the same time, the present invention selects a non-cruciferous vegetable (lettuce) as a control group. The present invention weighs different vegetables of the same mass and thoroughly crushes and lyses them, maintaining the final lysate concentration of 5 mg / mL, and then centrifuges the lysates of different vegetables for 20 min (6000r / min) to remove vegetable residues and retain the supernatant. The present invention absorbs 4.0 μL of the probe Rh-GSL prepared in Example 3 of the present invention with a concentration of 1.0 mM and the supernatant of different vegetable lysates to make the total volume 400 μL, and maintains the concentration of the probe Rh-GSL prepared in Example 3 of the present invention at 10.0 μM. The above mixture is mixed evenly and transferred to a PE tube, and the PE tube is placed in a water bath at 55°C for incubation for 30 minutes. After the incubation, the reaction solution in the PE tube is subjected to fluorescence test.

[0164] like Fig. 9 (a) and Fig. 9(b) shows the fluorescence intensity of the reaction solution measured at a wavelength of 693 nm (excitation wavelength = 660 nm) after the probe Rh-GSL prepared in Example 3 of the present invention was incubated with different vegetable lysates. It can be seen that the enzymatic hydrolysis rates of the probe Rh-GSL prepared in Example 3 of the present invention by the mustard enzymes expressed in different cruciferous vegetables are quite different, resulting in different fluorescence intensities of the enzymatic hydrolysis cyclization products measured at 693 nm. In general, the enzymatic hydrolysis rate of the probe Rh-GSL prepared in Example 3 of the present invention by the mustard enzyme expressed by baby cabbage, white radish, and cabbage is relatively high, and the background fluorescence at 693 nm of the probe Rh-GSL prepared in Example 3 of the present invention itself has a high increase multiple, while the enzymatic hydrolysis rate of the probe Rh-GSL by the mustard enzyme of cauliflower and broccoli leaves is relatively low, and the fluorescence increase multiple is also lower than that of the former; and the lettuce used as the control group is a plant of the Asteraceae family and does not belong to the Cruciferae family and cannot express mustard enzyme, nor can it enzymatically hydrolyze the probe Rh-GSL, and the fluorescence of the enzymatic hydrolysis cyclized product measured at 693 nm is so low that it can be ignored.

[0165] Example 13: Selective testing of probe Rh-GSL against myrosinase and other interferents

[0166] Incubation operation and conditions: 4.0 μL of the probe Rh-GSL prepared in Example 3 of the present invention with a concentration of 1.0 mM and 8.0 μL of commercial mustard enzyme (purchased from Sigma-Aldrich, derived from white mustard) with a concentration of 7000 U / L were aspirated into PBS buffer (20 mM, pH = 7.4) to make the total volume 400 μL, and the concentration of the probe Rh-GSL prepared in Example 3 of the present invention was kept at 10.0 μM and the concentration of mustard enzyme was kept at 140 mU / mL. The above mixture was mixed evenly and transferred to a PE tube, and the PE tube was placed in a water bath at 55°C for incubation for 30 min. After the incubation, the reaction solution in the PE tube was subjected to fluorescence test.

[0167] In the response test involving other interfering substances (various enzymes, proteins, amino acids, biomacromolecules, ionic compounds) except mustard enzyme, each interfering substance (50 times equivalent concentration) was incubated with 10.0 μM probe in PE tube at 55°C for 30 min in PBS buffer. After the incubation, the reaction solution was sent for fluorescence analysis with an excitation wavelength of 660 nm. The fluorescence intensity at the optimal emission wavelength of 693 nm was measured. At the same time, the same concentration of probe PBS solution without mustard enzyme and various interfering substances was measured as the background fluorescence intensity. Fig.10It can be seen that the fluorescence measured in the experimental group incubated with mustard enzyme increased by nearly 23 times compared with the control group without any interfering substances, while the other experimental groups incubated with various interfering substances showed almost no fluorescence increase compared with the control group. It can be seen that the probe Rh-GSL prepared in Example 3 of the present invention has a high selectivity for mustard enzyme.

[0168] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0169] As used in the present invention, the term "comprising" is an open expression, that is, including the contents specified in the present invention but not excluding other contents.

[0170] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0171] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A probe for detecting myrosinase, characterized in that: It has the structure shown in the following formula (I) and (II): ； Where n = 2 or 3; Among them, in the compound represented by formula (I): R1 is oxygen, R2 and R3 do not exist; R4 is selected from hydrogen, chlorine, bromine, iodine, cyano, nitro, carboxyl, aldehyde, azido, methyl, ethyl, amide, sulfonic acid, COOMe, COOEt, COOtBu; R5 is a nitrogen methyl group, a nitrogen ethyl group, a nitrogen four-membered ring group, a nitrogen ethyl five-membered ring group, a nitrogen ethyl six-membered ring group, a nitrogen methyl pyrrole ring group or a quaternary ammonium piperazine ring group; Among them, in the compound represented by formula (II): R1 is silicon; R2 and R3 are selected from C1-C8 straight chain or branched saturated alkane groups respectively, and R2 and R3 are the same or different.

2. The probe for detecting myrosinase according to claim 1, characterized in that: In the compound represented by formula (I), when n = 2, R1 is oxygen, R2 and R3 do not exist, and R4 is hydrogen, the specific form of the compound is: ； Or, in the compound represented by formula (II), when n = 2, R1 is silicon, and R2 and R3 are both ethyl; the specific form of the compound is: 。 3. A method for synthesizing a probe for detecting myrosinase as claimed in claim 1, characterized in that: The method simulates the structure of the secondary metabolite mustard oil glycoside in plants, first synthesizes amino-thioglucoside precursors with different chain length structures, then synthesizes a halogenated active fluorescent matrix, and then reacts the synthesized amino-thioglucoside precursor with the halogenated active fluorescent matrix in a solvent and a catalyst, and then performs a deprotection reaction to obtain the probe for detecting myrosinase; The amino-thioglucosinolate precursors with different chain length structures have the structures shown below: ; where n = 2 or 3; The halogenated active fluorescent matrix has the structures shown in the following formula (IV), formula (V), and formula (VI): ； Wherein, the definitions of R1, R2, R3, R4 and R5 in the compound represented by formula (IV) are the same as those of the compound represented by formula (I) described in claim 1; The definitions of R1, R2 and R3 in the compound represented by formula (V) are the same as those of the compound represented by formula (II) described in claim 1; The definitions of R1, R2, R3, R4, R5 and R6 in the compound represented by formula (VI) are the same as those of the compound represented by formula (III) described in claim 1.

4. The synthesis method according to claim 3, characterized in that The catalyst is selected from one or more of piperidine, pyridine, pyrrole, morpholine, imidazole, trimethylamine, diethylamine, triethylamine, and N, N-diisopropylethylamine; and / or, the solvent is one or more of tetrahydrofuran, dichloromethane, 1,2-dichloroethane, N, N-dimethylformamide, acetone, benzene, toluene, nitrobenzene, p-xylene, methanol, ethanol, 1,4-dioxane, chloroform, alkane, dimethyl sulfoxide, acetonitrile, and water; And / or, the reaction temperature of the amino-thioglucoside precursor and the halogenated active fluorescent matrix is ​​0°C-160°C, and the reaction time is 10 min-48 h; And / or, the temperature of the deprotection reaction is -20°C-80°C, and the reaction time is 15 min-10 h; And / or, the molar equivalent ratio of the synthesized amino-thioglucoside precursor, halogenated active fluorescent matrix, catalyst, and solvent is: 1: (1.0-4.0): (2.0-10.0): (5-200).

5. The synthesis method according to claim 3, characterized in that The specific reaction steps of the amino-thioglucosinolate precursor are as follows: (1) dissolving 3-aminopropanal diethyl acetal and N, N-diisopropylethylamine in a first solvent in a reaction container, and then dissolving 9-fluorenylmethyl chloroformate Fmoc-Cl in the first solvent at -15-10°C and adding the solution to the reaction container, stirring the reaction overnight and naturally warming to room temperature to obtain compound 1; or, dissolving 4-aminobutyraldehyde dimethyl acetal and N, N-diisopropylethylamine in a first solvent in a reaction container, and then dissolving 9-fluorenylmethyl chloroformate Fmoc-Cl in the first solvent at -15-10°C and adding the solution to the reaction container, stirring the reaction overnight and naturally warming to room temperature to obtain compound 1'; (2) dissolving the compound 1 / 1' in a second solvent, preparing a hydrochloric acid solution with a concentration of 3.0-5.0 mol / L, adding the solution to the reaction vessel, and stirring the reaction at room temperature to obtain compounds 2 (n=2) and 2' (n=3); (3) dissolving the compound 2(n=2) / 2' (n=3) and hydroxylamine hydrochloride in a third solvent, and then adding triethylamine under stirring conditions, and reacting and stirring at room temperature to obtain compounds 3(n=2) and 3' (n=3); (4) Add the compound 3(n=2) / 3' (n=3) to a reaction container, add a fourth solvent to dissolve it under argon protection and add pyridine, then use the fourth solvent to dissolve N-chlorosuccinimide NCS and add it to the reaction container, react and stir at room temperature, then add 1-thio-β-D-glucose tetraacetate dissolved in the fourth solvent and add triethylamine, react and stir at room temperature to obtain compounds 4(n=2) and 4' (n=3); (5) dissolving the compound 4 (n=2) / 4' (n=3) and pyridine sulfur trioxide in acetonitrile, and reacting under reflux stirring at 65-85°C to obtain compounds 5 (n=2) and 5' (n=3); (6) dissolving the compound 5(n=2) / 5' (n=3) in a fifth solvent and adding diethylamine, and reacting and stirring at room temperature to obtain a compound 6(n=2) / 6' (n=3); The synthetic reaction route of the amino-thioglucosinolate precursor is as follows: ； Where n = 2 or 3.

6. The synthesis method according to claim 5, characterized in that In step (1), the first solvent is selected from one or more of dichloromethane and 1,2-dichloroethane; and / or the molar ratio of 3-aminopropionaldehyde diethyl acetal / 4-aminobutyraldehyde dimethyl acetal to N, N-diisopropylethylamine and 9-fluorenylmethyl chloroformate Fmoc-Cl is 1.0: (1.5-3.0): (1.0-1.5); and / or, In step (2), the second solvent is selected from one or both of tetrahydrofuran and 1,4-dioxane; and / or, In step (3), the third solvent is selected from one or more of dichloromethane and 1,2-dichloroethane; and / or the molar ratio of the compound 2(n=2) / 2' (n=3), hydroxylamine hydrochloride and triethylamine is 1:(1.5-3.0):(3.0-5.0); and / or, In step (4), the fourth solvent is selected from one or more of dichloromethane and 1,2-dichloroethane; and / or the molar ratio of the compound 3 (n=2) / 3' (n=3), N-chlorosuccinimide NCS, 1-thio-β-D-glucose tetraacetate and triethylamine is 1.0: (1.0-2.0): (1.5-4.0): (2.0-5.0); and / or, In step (5), the molar ratio of the compound 4 (n=2) / 4' (n=3) to pyridine sulfur trioxide is 1.0: (3.0-6.0); and / or, In step (6), the fifth solvent is selected from one or both of dichloromethane and 1,2-dichloroethane; and / or the volume ratio of the fifth solvent to diethylamine is (3.0-6.0):1.

0.

7. A use of the probe for detecting myrosinase as claimed in claim 1 for non-disease treatment and diagnosis purposes, characterized in that: The probe for detecting myrosinase is applied to myrosinase metabolism-related analysis and screening of myrosinase inhibitors in vitro or in plants and in bacterial flora.

8. The use according to claim 7, characterized in that The in vitro or plant and bacterial mustard enzyme detection includes qualitative and quantitative detection of mustard enzyme; and / or, the in vitro or plant and bacterial mustard enzyme detection includes detection of commercial mustard enzyme and endogenous mustard enzyme of plants and bacterial flora.

9. The use according to claim 7, characterized in that The screening of the myrosinase inhibitor comprises: screening the myrosinase inhibitor using the myrosinase detection probe as a substrate.

Images