BODIPY fluorescent probe for rapidly detecting myrosinase as well as preparation method and application of BODIPY fluorescent probe

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a BODIPY fluorescent probe for rapidly detecting myrosinase as well as a preparation method and application of the BODIPY fluorescent probe. When the fluorescent probe (formula I) provided by the invention is used for detecting the myrosinase, the fluorescent probe enters an active cavity of the myrosinase and then is subjected to enzymatic reaction to generate a ring closing product, so that the photo-induced electron transfer (PET) effect between aryl and fluorophore is inhibited, a fluorescent signal is recovered, and a lightening detection effect from nothing to the existence is realized. The fluorescent probe is not only suitable for quantitative detection and analysis of in-vitro myrosinase, but also can be used for in-situ and nondestructive detection and fluorescence imaging of endogenous myrosinase in plant living bodies and microorganism samples. The method has the advantages of rapidness, visualization, high sensitivity, high throughput screening and the like in the aspect of myrosinase detection, and effectively overcomes the technical limitations of complex sample pretreatment, destructiveness, difficulty in real-time and rapid observation and the like in a traditional detection method. Formula I.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a rapid detection of myrosinase BODIPY-type fluorescent probe, its preparation method, and its application. Background Technology

[0002] Myrosinase (MYR) is a glucosinolate hydrolase widely found in cruciferous plants. It catalyzes the hydrolysis of glucosinolates (GSLs) to produce bioactive products such as isothiocyanates (ITCs). Isothiocyanates play a crucial role in plant defense systems due to their toxicity to insects; however, they also possess significant nutritional and health benefits in humans, including anticancer, anti-inflammatory, and antioxidant activities. For example, sulforaphane, produced by the hydrolysis of glucosinolates by myrosinase, has shown effective therapeutic effects in numerous preclinical and clinical studies. Generally, isothiocyanates can be obtained through chemical synthesis or biotransformation. Chemical synthesis typically requires harsh conditions, and the resulting products are complex and often accompanied by numerous byproducts, resulting in limited purity. In contrast, biotransformation via myrosinase-catalyzed hydrolysis of glucosinolates offers high specificity, enabling the in-situ, precise, and efficient production of specific isothiocyanates. However, plant-derived myrosinases often exhibit low catalytic efficiency, limiting the large-scale biosynthesis of isothiocyanates. Therefore, exploring microbial myrosinases as more efficient and bioactive biotransformation tools is particularly important. Although research on bacterial myrosinases remains limited, recent reports on bacterial myrosinases have deepened our understanding of their biotransformation capabilities. However, current methods for screening highly active myrosinase strains rely on cumbersome, slow, and low-throughput processes, including enzyme purification and in vitro assays. This bottleneck highlights the urgent need for rapid assays of myrosinase activity and high-throughput screening methods for strains of myrosinase.

[0003] Myrosinase is typically detected using two methods: one is by detecting isothiocyanates and their derivatives using ultraviolet spectrophotometry, gas chromatography-mass spectrometry (GC-MS), or high-performance liquid chromatography (HPLC); the other is by determining immobilized enzymatic hydrolysis products, such as hydrogen ions, glucose, or sulfate, using pH-salt assays, electrophoresis, or enzyme-coupled assays. However, these methods require complex sample processing and advanced equipment, limiting their use in real-time, convenient detection, especially in in vivo systems.

[0004] Chinese patent CN118852290A developed a class of myrosinase detection probes based on wavelength redshift changes, pioneering the sensitive detection of myrosinase through a combination of organic small molecule probes and fluorescence analysis technology. However, it still suffers from slow detection speed (>40 min) and insufficient fluorescence signal from the probe itself. To improve the response speed of myrosinase detection and make it suitable for high-throughput rapid detection applications, it is essential to develop a class of fluorescent probes for rapid detection of myrosinase based on the PET response mechanism (the fluorescence "lighting up" process from zero to positive). Summary of the Invention

[0005] In view of this, the present invention provides a BODIPY-type fluorescent probe for rapid detection of myrosinase, its preparation method and application. When the fluorescent probe provided by the present invention is used for myrosinase detection, it can solve the technical defects of existing myrosinase detection technology, such as cumbersome sample preparation steps, destructive sample analysis, large data errors, slow detection speed and inability to detect live samples.

[0006] This invention provides a BODIPY-type fluorescent probe for rapid detection of myrosinase, having the structure shown in Formula I: Formula I; In Equation I, n = 2 or 3; R1, R3, R4 and R6 are independently hydrogen, halogroup, cyanogroup, mercaptogroup, or C1-C8 straight-chain or branched saturated alkane group; R2 and R5 are independently hydrogen, ethyl, propyl, isopropyl, halogen, cyano, nitro, carboxyl, sulfonic acid, methoxy, acetyl, phenyl, benzyl, ester, or pyrrole rings with different substitutions; R7, R8, R9 and R 10 It can be hydrogen, cyano, nitro, aldehyde, carboxyl, sulfonic acid, methoxy, acetyl, or ester group independently.

[0007] Preferably, the BODIPY-type fluorescent probe has a structure shown in any one of formulas BDP-GSL1 to BDP-GSL8: .

[0008] This invention also provides a method for preparing a fluorescent probe of formula BDP-GSL1 or BDP-GSL5, comprising the following steps: (1) Dissolve compound 1, cuprous iodide, L-proline, potassium carbonate and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal in the first solvent, and then carry out the first nucleophilic substitution reaction to obtain compound 2-1 or compound 2-2. ; (2) The compound 2-1 or compound 2-2, 9-fluorenyl chloroformate and the second solvent are mixed and subjected to a second nucleophilic substitution reaction and an acid-catalyzed hydrolysis reaction in sequence to obtain compound 3-1 or compound 3-2; The structural formulas of compounds 2-1, 2-2, 3-1, and 3-2 are as follows: ; ; ; (3) The compound 3-1 or compound 3-2, hydroxylamine hydrochloride, triethylamine and a third solvent are mixed and oxime reaction is carried out to obtain compound 4-1 or compound 4-2; (4) Mix the compound 4-1 or compound 4-2, the fourth solvent, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate and triethylamine, and carry out the third nucleophilic substitution reaction to obtain compound 5-1 or compound 5-2; The structural formulas of compounds 4-1, 4-2, 5-1, or 5-2 are as follows: ; ; (5) The compound 5-1 or 5-2, pyridine sulfur trioxide, and acetonitrile are mixed and subjected to an electrophilic substitution reaction to obtain compound 6-1 or 6-2; the structural formula of compound 6-1 or 6-2 is as follows: ; (6) Mix the compound 6-1 or compound 6-2, potassium methoxide and the fifth solvent, and carry out an alkaline-catalyzed hydrolysis reaction to obtain compound BDP-GSL1 or BDP-GSL5.

[0009] Preferably, the first solvent is dimethyl sulfoxide and / or N,N-dimethylformamide; The molar ratio of compound 1, cuprous iodide, L-proline, potassium carbonate, and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal is 1.0:(0.05-0.4):(0.2-0.6):(1.0-3.0). The temperature of the first nucleophilic substitution reaction is 50~100℃, and the reaction time is 1.5~3 hours.

[0010] Preferably, the second solvent is 1,4-dioxane and / or tetrahydrofuran; the molar ratio of compound 2-1 or compound 2-2 to fluorenyl chloroformate is 1:(1.0~2.0). The temperature for the second nucleophilic substitution reaction is 0~25℃, and the reaction time is 30~60 minutes; The acid-catalyzed hydrolysis reaction is carried out by adding hydrochloric acid aqueous solution and a second solvent to the second nucleophilic substitution reaction solution; the acid-catalyzed hydrolysis reaction is carried out at room temperature for 6 to 9 hours.

[0011] Preferably, the third solvent is dichloromethane and / or 1,2-dichloroethane; the molar ratio of compound 3-1 or compound 3-2, hydroxylamine hydrochloride and triethylamine is 1:(1.5~3.5):(3.0~6.0); the oxime reaction is carried out at room temperature for 3~5 hours.

[0012] Preferably, the fourth solvent is dichloromethane and / or 1,2-dichloroethane; the molar ratio of compound 4-1 or compound 4-2, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate, and triethylamine is 1.0:(2.0~5.0):(1.0~2.5):(2.0~4.5):(3.0~6.0); the third nucleophilic substitution reaction is carried out at room temperature for 4~8 hours.

[0013] Preferably, the molar ratio of compound 5-1 and compound 5-2 to pyridine sulfur trioxide is 1.0:(3.5~6.5); the electrophilic substitution reaction is carried out at a temperature of 60~90℃ for 5~8 hours. The fifth solvent is methanol and / or ethanol; the molar ratio of compound 6-1 or compound 6-2 to potassium methoxide is 1.0:(5.0~12.0); the alkaline-catalyzed hydrolysis reaction is carried out at room temperature for 2~4 hours.

[0014] The application of the fluorescent probe described in this invention or the fluorescent probe prepared by the above-described preparation method in myrosinase detection.

[0015] Preferably, the applications are in vitro myrosinase detection, myrosinase detection in plants and microorganisms, screening of myrosinase inhibitors, identification of myrosinase heterologous expression in microorganisms, and high-throughput rapid screening.

[0016] The beneficial effects of this invention are as follows: When the fluorescent probe provided by this invention is used to detect myrosinase, it undergoes an enzymatic reaction after entering the active cavity of myrosinase, generating a ring-closed product. This suppresses the photoinduced electron transfer (PET) effect between the aryl group and the fluorophore, thereby restoring the fluorescence signal and achieving a "lighting up" detection effect from nothing to something. The fluorescent probe is not only suitable for the quantitative detection and analysis of myrosinase in vitro, but also for the in-situ, non-destructive detection and fluorescence imaging of endogenous myrosinase in live plant and microbial samples. This method has advantages such as speed, visualization, high sensitivity, and high-throughput screening in myrosinase detection, effectively overcoming the technical limitations of traditional detection methods, such as complex and destructive sample pretreatment and difficulty in real-time and rapid observation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The fluorescence spectral response changes of probes BDP-GSL1 and BDP-GSL5 in the reaction with myrosinase; Figure 2 The time-dependent fluorescence growth of probes BDP-GSL1 and BDP-GSL5 after reaction with myrosinase; Figure 3 To test the selective response of probes BDP-GSL1 and BDP-GSL5 to myrosinase and other interfering agents; Figure 4 The fluorescence response spectrum and linear relationship of the probe BDP-GSL5 to myrosinase concentration-dependent fluorescence response; Figure 5 The time-dependent response of probe BDP-GSL5 to myrosinase at different temperatures; Figure 6 The fluorescence response of probe BDP-GSL5 after incubation with different vegetable lysates was measured. Figure 7 Time-dependent fluorescence confocal imaging of probe BDP-GSL1 after co-incubation with living Arabidopsis root tissue; Figure 8 The probe BDP-GSL5 was used for fluorescence confocal imaging screening of human intestinal bacteria that can metabolize glucosinolates. Figure 9 A schematic diagram illustrating the specific mechanism of the PET response of the myrosinase detection probe; Figure 10 A schematic diagram illustrating the specific enzymatic hydrolysis mechanism of the reaction between BDP-GSL1 / BDP-GSL5 and myrosinase. Detailed Implementation

[0019] This invention provides a BODIPY-type fluorescent probe for rapid detection of myrosinase, having the structure shown in Formula I: Formula I; In Equation I, n = 2 or 3; R1, R3, R4 and R6 are independently hydrogen, halogroup, cyanogroup, mercaptogroup, or C1-C8 straight-chain or branched saturated alkane group; the halogroup in R1, R3, R4 and R6 can independently be chlorine, bromine, or iodine; R2 and R5 are independently hydrogen, ethyl, propyl, isopropyl, halogen, cyano, nitro, carboxyl, sulfonic acid, methoxy, acetyl, phenyl, benzyl, ester, or pyrrole rings with different substitutions; The halogen groups in R2 and R5 can be fluorine, chlorine, bromine or iodine; the ester groups in R2 and R5 can be COOMe or COOEt.

[0020] R7, R8, R9 and R 10 It can be independently hydrogen, cyano, nitro, aldehyde, carboxyl, sulfonic acid, methoxy, acetyl, or ester group; R7, R8, R9 and R 10 The alkyl group mentioned is methyl, ethyl, n-propyl, isopropyl, n-butyl, or tert-butyl; R7, R8, R9, and R 10 The ester group can be COOMe or COOEt.

[0021] As one embodiment of the present invention, the BODIPY-type fluorescent probe has a structure shown in any one of formulas BDP-GSL1 to BDP-GSL8: .

[0022] That is, when n = 2, R1, R3, R4, and R6 are all methyl groups, and R7, R8, R9, and R... 10 When all are hydrogen, and R2 and R5 are hydrogen, chlorine, ethyl, and ester (COOEt) respectively, they correspond to BDP-GSL1~BDP-GSL4; when n = 3, R1, R3, R4, and R6 are all methyl, and R7, R8, R9, and R... 10 When all are hydrogen, and R2 and R5 are hydrogen, bromine, isopropyl, and methoxy respectively, they correspond to BDP-GSL5~BDP-GSL8 respectively.

[0023] This invention also provides a method for preparing the fluorescent probe described in the above technical solution, comprising the following steps: (1) The iodo-BODIPY fluorescent precursor with the structure shown in Formula II, the first catalyst, potassium carbonate, and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal are dissolved in the first solvent, and then the first nucleophilic substitution reaction is carried out to obtain compound A-1 or compound A-2, wherein R1~R in the compound shown in Formula (II) 10 The definition is the same as that in Equation I; Formula II; (2) Mix the solution of compound A-1 or compound A-2 with the solution of fluorenyl chloroformate, and carry out the second nucleophilic substitution reaction and acid-catalyzed hydrolysis reaction in sequence to obtain compound B-1 or compound B-2; The structural formulas of compounds B-1 and B-2 are as follows: A-1; A-2; B-1 (n=2) / B-2 (n=3); (3) The compound B-1 or compound B-2, hydroxylamine hydrochloride, the second catalyst and the third solvent are mixed and oxime reaction is carried out to obtain compound C-1 or compound C-2; C-1(n=2) / C-2(n=3); (4) The compound C-1 or C-2, the fourth solvent, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate and triethylamine are mixed and subjected to a third nucleophilic substitution reaction to obtain compound D-1 or compound D-2; D-1(n=2) / D-2(n=3); (5) Mix the compound D-1 or compound D-2, pyridine sulfur trioxide and acetonitrile, and carry out an electrophilic substitution reaction to obtain compound E-1 or compound E-2; E-1 (n=2) / E-2 (n=3); (6) Mix the compound E-1 or compound E-2, potassium methoxide and the fifth solvent, and carry out an alkaline-catalyzed hydrolysis reaction to obtain the BODIPY fluorescent probe.

[0024] In this invention, the first catalyst comprises one or more of cuprous iodide, L-proline, diethylamine, triethylamine, pyridine, pyrrole, and N,N-diisopropylethylamine; the first solvent comprises one or more of dimethyl sulfoxide, N,N-dimethylformamide, and N-methylpyrrolidone; when the first catalyst is cuprous iodide and L-proline, the molar ratio of compound 1, cuprous iodide, L-proline, potassium carbonate, and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal can be 1.0:(0.05~0.4):(0.2~0.6):(1.0~3.0). The temperature of the first nucleophilic substitution reaction can be 50~100℃, and the reaction time can be 1.5~3 hours.

[0025] In this invention, the solution of compound A-1 or compound A-2 is obtained by dissolving compound A-1 or compound A-2 in a mixed solvent; the mixed solvent is obtained by mixing a second solvent and an aqueous sodium bicarbonate solution; the concentration of the aqueous sodium bicarbonate solution can be 5~15 wt%, preferably 10 wt%; the molar ratio of compound 2-1 or compound 2-2 and chloroformate-9-fluorenyl methyl ester can be 1:(1.0~2.0); in this invention, the temperature of the second nucleophilic substitution reaction can be 0~25°C, and the reaction time can be 30~60 minutes; the temperature of the acid-catalyzed hydrolysis reaction can be room temperature, and the reaction time can be 6~9 hours.

[0026] In this invention, the second catalyst comprises one or more of diethylamine, trimethylamine, triethylamine, morpholine, and N,N-diisopropylethylamine; the third solvent may be one or more of dichloromethane, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; when the second catalyst is triethylamine, the molar ratio of compound B-1 or compound B-2, hydroxylamine hydrochloride, and triethylamine may be 1:(1.5~3.5):(3.0~6.0); in this invention, the oxime reaction may be carried out at room temperature for 3~5 hours.

[0027] In this invention, the fourth solvent may be one or more of dichloromethane, 1,2-dichloroethane, and N,N-dimethylformamide; the third catalyst includes one or more of pyridine, diethylamine, triethylamine, and N,N-diisopropylethylamine; when the third catalyst is pyridine and triethylamine, the molar ratio of compound C-1 or compound C-2, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate, and triethylamine may be 1.0:(2.0~5.0):(1.0~2.5):(2.0~4.5):(3.0~6.0); the third nucleophilic substitution reaction is carried out at room temperature for 4~8 hours.

[0028] In this invention, the molar ratio of compound D-1 and compound D-2 to pyridine sulfur trioxide can be 1.0:(3.5~6.5); in this invention, the electrophilic substitution reaction can be carried out at a temperature of 60~90°C for 5~8 hours.

[0029] In this invention, the fifth solvent can be one or more of methanol, ethanol, acetonitrile, and isopropanol; the molar ratio of compound E-1 or compound E-2 to potassium methoxide can be 1.0:(5.0~12.0); the alkaline-catalyzed hydrolysis reaction is carried out at room temperature for 2~4 hours.

[0030] This invention also provides a method for preparing BDP-GSL1 or BDP-GSL5, comprising the following steps: (1) Dissolve compound 1, cuprous iodide, L-proline, potassium carbonate and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal in the first solvent and carry out the first nucleophilic substitution reaction to obtain compound 2-1 or compound 2-2. ; (2) Mix the solution of compound 2-1 or compound 2-2 with the solution of chloroformate-9-fluorenyl methyl ester, and carry out the second nucleophilic substitution reaction and acid-catalyzed hydrolysis reaction in sequence to obtain compound 3-1 or compound 3-2; ; ; ; (3) The compound 3-1 or compound 3-2, hydroxylamine hydrochloride, triethylamine and a third solvent are mixed and oxime reaction is carried out to obtain compound 4-1 or compound 4-2; (4) Mix the compound 4-1 or compound 4-2, the fourth solvent, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate and triethylamine, and carry out the third nucleophilic substitution reaction to obtain compound 5-1 or compound 5-2; ; ; (5) Mix the compound 5-1 or compound 5-2, pyridine sulfur trioxide and acetonitrile, and carry out an electrophilic substitution reaction to obtain compound 6-1 or compound 6-2; ; (6) Mix the compound 6-1 or compound 6-2, potassium methoxide and the fifth solvent, and carry out an alkaline-catalyzed hydrolysis reaction to obtain compound BDP-GSL1 or BDP-GSL5.

[0031] In this invention, compound 1, cuprous iodide, L-proline, potassium carbonate, and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal are dissolved in a first solvent, and then a first nucleophilic substitution reaction is carried out to obtain compound 2-1 or compound 2-2.

[0032] In this invention, the first solvent can be dimethyl sulfoxide and / or N,N-dimethylformamide, preferably dimethyl sulfoxide. In this invention, the molar ratio of compound 1, cuprous iodide, L-proline, potassium carbonate, and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal can be 1.0:(0.05~0.4):(0.2~0.6):(1.0~3.0), preferably 1.0:0.2:0.3:2.0. In this invention, the temperature of the first nucleophilic substitution reaction can be 50~100℃, and the reaction time can be 1.5~3 hours; preferably, the temperature of the first nucleophilic substitution reaction can be 85℃, and the reaction time can be 2 hours.

[0033] After obtaining compounds 2-1 and 2-2, the present invention mixes a solution of compound 2-1 or compound 2-2 with a solution of 9-fluorenyl chloroformate, and sequentially performs a second nucleophilic substitution reaction and an acid-catalyzed hydrolysis reaction to obtain compound 3-1 or compound 3-2.

[0034] In this invention, the solution of compound 2-1 or compound 2-2 is obtained by dissolving compound 2-1 or compound 2-2 in a mixed solvent; the mixed solvent is obtained by mixing a second solvent and an aqueous sodium bicarbonate solution; the concentration of the aqueous sodium bicarbonate solution can be 5 to 15 wt%, preferably 10 wt%.

[0035] In this invention, the second solvent can be 1,4-dioxane and / or tetrahydrofuran; the molar ratio of compound 2-1 or compound 2-2 to fluorenyl chloroformate can be 1:(1.0~2.0). In this invention, the temperature of the second nucleophilic substitution reaction can be 0~25℃, and the reaction time can be 30~60 minutes.

[0036] In this invention, the specific steps of the acid-catalyzed hydrolysis reaction are as follows: adding an aqueous hydrochloric acid solution and a second solvent to the second nucleophilic substitution reaction solution to carry out the acid-catalyzed hydrolysis reaction. The concentration of the aqueous hydrochloric acid solution can be 3.0~5.0M, preferably 4.0M; in this invention, the temperature of the acid-catalyzed hydrolysis reaction can be room temperature, and the time can be 6~9 hours.

[0037] After obtaining compound 3-1 or compound 3-2, the present invention mixes compound 3-1 or compound 3-2, hydroxylamine hydrochloride, triethylamine and a third solvent to carry out an oxime reaction to obtain compound 4-1 or compound 4-2.

[0038] In this invention, the third solvent can be dichloromethane and / or 1,2-dichloroethane, preferably dichloromethane; the molar ratio of compound 3-1 or compound 3-2, hydroxylamine hydrochloride, and triethylamine can be 1:(1.5~3.5):(3.0~6.0), preferably 1.0:2.2:4.0. In this invention, the oxime reaction can be carried out at room temperature for 3~5 hours.

[0039] After obtaining compound 4-1 or compound 4-2, the present invention mixes compound 4-1 or compound 4-2, a fourth solvent, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate and triethylamine, and carries out a third nucleophilic substitution reaction to obtain compound 5-1 or compound 5-2.

[0040] In this invention, the fourth solvent can be dichloromethane and / or 1,2-dichloroethane, preferably dichloromethane; the molar ratio of compound 4-1 or compound 4-2, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate, and triethylamine can be 1.0:(2.0~5.0):(1.0~2.5):(2.0~4.5):(3.0~6.0), preferably 1.0:3.0:1.5:3.0:4.5. In this invention, the third nucleophilic substitution reaction is carried out at room temperature for 4~8 hours.

[0041] After obtaining compound 5-1 or compound 5-2, the present invention mixes compound 5-1 or compound 5-2, pyridine sulfur trioxide and acetonitrile, and carries out an electrophilic substitution reaction to obtain compound 6-1 or compound 6-2.

[0042] In this invention, the molar ratio of compound 5-1 and compound 5-2 to pyridine sulfur trioxide can be 1.0:(3.5~6.5), preferably 1.0:5.0. In this invention, the electrophilic substitution reaction can be carried out at a temperature of 60~90℃, preferably 80℃, for a time of 5~8 hours, preferably 6 hours.

[0043] In this invention, compound 6-1 or compound 6-2, potassium methoxide and a fifth solvent are mixed and subjected to an alkaline-catalyzed hydrolysis reaction to obtain compound BDP-GSL1 or BDP-GSL5.

[0044] In this invention, the fifth solvent can be methanol and / or ethanol, preferably methanol; the molar ratio of compound 6-1 or compound 6-2 to potassium methoxide can be 1.0:(5.0~12.0), specifically 1.0:8.0. In this invention, the temperature of the alkaline-catalyzed hydrolysis reaction can be room temperature, and the time is 2~4 hours, preferably 3 hours; the alkaline-catalyzed hydrolysis reaction can be carried out under stirring conditions.

[0045] The preparation method provided by this invention has the advantages of simple synthesis steps, mild reaction conditions, and controllable yield.

[0046] This invention also provides the application of the fluorescent probe described in the above technical solution in the detection of myrosinase.

[0047] In this invention, the specific applications can be in vitro myrosinase detection, myrosinase detection in plants and microorganisms, screening of myrosinase inhibitors, identification of myrosinase heterologous expression in microorganisms, and high-throughput rapid screening.

[0048] In this invention, the in vitro myrosinase detection, or the detection of myrosinase in plants and microorganisms, includes qualitative and / or quantitative detection of myrosinase; the in vitro myrosinase detection, or the detection of myrosinase in plants and microorganisms, includes the detection of commercially available myrosinase and / or endogenous myrosinase in plants and microorganisms. In this invention, the screening of myrosinase inhibitors is performed using the fluorescent probe as a substrate. In this invention, the experimental identification and high-throughput rapid screening of heterologous myrosinase expression in microorganisms is performed using the fluorescent probe as a substrate for the identification and high-throughput rapid screening of heterologously expressed myrosinase.

[0049] In this invention, such as Figure 9As shown, the PET response mechanism of the fluorescent probe provided by this invention is as follows: Due to photoinduced electron transfer (PET) between the recognition group (aryl moiety) of the fluorescent probe and the fluorophore, the parent fluorescence is completely quenched, resulting in extremely low background fluorescence. During incubation, after entering the myrosinase cavity, the sulfonic acid group and glucose moiety of the probe molecule can interact with specific amino acids of myrosinase (such as Arg259, Gln187, etc.) via hydrogen bonds and catalyze the cleavage of the thioglucoside bond, releasing a molecule of glucose and an unstable glycoside ligand intermediate. This intermediate spontaneously undergoes a Rosen rearrangement reaction, rapidly generating a highly electrophilic isothiocyanate group (RN=C=S), which then attacks the exposed secondary amine site on the aryl side chain of the fluorescent parent, leading to an intramolecular ring-closure reaction and generating a thiourea ring with an electron-withdrawing effect. The enzymatic hydrolysis of the ring-closure product blocks the PET process between the aryl group and the fluorophore, causing the fluorescence signal to recover in situ, achieving a "lighting up" process from zero to positive fluorescence, thereby enabling the detection of myrosinase. The driving force behind PET is the orbital energy difference between the donor (fluorophore) and the acceptor (recognition group). If the highest occupied molecular orbital (HOMO) of the recognition group lies between the HOMO and lowest unoccupied molecular orbital (LUMO) of the fluorophore, electrons will transfer from the HOMO orbital of the recognition group to the HOMO orbital of the fluorophore, resulting in the PET process and quenching of the fluorescence of the parent fluorophore. When the analyte binds to the recognition group to form a product, changing its orbital energy, the highest occupied molecular orbital (HOMO) of the product acceptor fragment is lower than that of the HOMO orbital of the fluorophore. Electrons cannot transfer from the HOMO orbital of the product acceptor fragment to the HOMO orbital of the fluorophore, the PET process is suppressed, the fluorescence signal of the parent fluorophore is restored, and thus sensitive detection of the target analyte is achieved. Taking BDP-GSL1 and BDP-GSL5 as examples, the specific enzymatic hydrolysis mechanism of BDP-GSL1 and BDP-GSL5 with myrosinase is described in [link to documentation]. Figure 10 .

[0050] The fluorescent probes provided by this invention have the following significant advantages for detecting changes in myrosinase activity in vitro or monitoring the expression of this enzyme in plants and microorganisms in vivo: (1) Good water solubility: The strong hydrophilic sulfonic acid group of glucosinolate and the four hydroxyl groups carried by the glucose unit in this type of probe molecule endow it with good water solubility, effectively avoiding the problem that most dyes are prone to aggregation and difficult to disperse uniformly in water due to excessive hydrophobicity. Thanks to its outstanding hydrophilic properties, the probe can be fully dispersed in the aqueous phase used as the incubation medium, which is conducive to entering the cavity of myrosinase protein and forming hydrogen bond interactions with adjacent amino acid residues, thereby promoting the catalysis of the enzymatic reaction.

[0051] (2) High stability: The rigid structure in its molecular skeleton and the extended aromatic system effectively reduce the rotation of high-energy bonds and molecular vibrations, thereby improving the overall chemical stability. In addition, the introduction of hydrophilic groups also reduces the probability of the probe coming into contact with unstable factors such as reactive oxygen species in the environment, enabling it to remain stable for a long time in complex biological incubation media.

[0052] (3) Strong specific response: The glucosinolate in the probe molecule is specifically recognized by myrosinase and undergoes an enzymatic hydrolysis reaction. The final enzymatic hydrolysis ring-closing product blocks the PET process between the acceptor fragment and the fluorophore. The fluorescence signal is not significantly enhanced, but the probe enhances the fluorescence signal for a variety of interfering substances other than myrosinase (such as various ionic compounds, proteins, enzymes, biomacromolecules, amino acids, and active substances). (4) Fast detection speed: The enzymatic reaction between this series of probes and myrosinase can achieve complete transformation within 10 minutes. This rapid kinetic characteristic, combined with the high stability and specific response of the probes themselves, makes them very suitable for real-time analysis of myrosinase activity for rapid, accurate, and high-throughput screening.

[0053] (5) High sensitivity: The probe itself undergoes photoinduced electron transfer (PET) between the recognition group (aryl part) and the fluorophore, which completely quenches the parent fluorescence and results in extremely low background fluorescence. After contact with myrosinase and enzymatic hydrolysis, the newly generated enzymatic hydrolysis ring-closing product blocks the PET process between the aryl group and the fluorophore, causing the fluorescence signal to recover in situ. By monitoring the fluorescence signal of the compound, high-sensitivity detection of myrosinase can be achieved.

[0054] (6) Wide range of applications: This type of probe has a wide range of applications, combining in vitro detection and in vivo analysis capabilities. It can not only be used for the quantitative analysis of myrosinase in solution, but more importantly, it can perform non-invasive and non-destructive detection of endogenous myrosinase in living plants and microbial systems, effectively overcoming the destructive drawbacks of traditional methods that require sample pulverization. By monitoring the enhancement of fluorescence signals, the enzymatic reaction process can be tracked intuitively and in real time, and further, fluorescence confocal imaging of live samples can be achieved. In addition, this probe is also suitable for the identification and high-throughput screening of enzyme molecules expressed heterologously in microorganisms.

[0055] In summary, the fluorescent probe for rapid detection of myrosinase prepared by this invention exhibits good water solubility, high stability, strong specificity, fast detection speed, and high sensitivity. When the probe is not bound to myrosinase, photoinduced electron transfer (PET) occurs between the recognition group and the fluorophore, resulting in complete quenching of the parent fluorescence ("Off" state) and extremely low background fluorescence. Once the probe reacts with myrosinase, the PET process is blocked, and fluorescence is restored ("On" state), achieving a "lighting up" process from zero fluorescence, thus enabling rapid and sensitive detection of myrosinase. The fluorescent probe for rapid detection of myrosinase of this invention can be used not only for in vitro myrosinase detection and screening of myrosinase inhibitors, but also for the detection and fluorescence imaging of endogenous myrosinase in plants or microorganisms. It is also suitable for the experimental identification, rapid detection, and high-throughput rapid screening of heterologous expression of myrosinase in microorganisms.

[0056] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1 4-Iodobenzoyl chloride (4.26 g, 16.0 mmol) and 2,4-dimethylpyrrole (3.3 mL, 32.0 mmol) were added to a reaction vessel and dissolved in anhydrous dichloromethane (350 mL). The reaction mixture was stirred under reflux at 35 °C for 3 hours under an argon atmosphere. The reaction mixture was then cooled to 0 °C, and triethylamine (8.90 mL, 64.0 mmol) and boron trifluoride diethyl ether (11.84 mL, 96.0 mmol) were added. The reaction mixture was stirred at room temperature for another 2 hours. After the reaction was complete, the solvent was removed by evaporation under reduced pressure. The residue was extracted with chloroform, and the organic layer was washed three times with water. The organic layers were combined and dried over anhydrous sodium sulfate. The organic phase was transferred to a flask, and the solvent was removed by evaporation under reduced pressure. The crude product was purified by column chromatography (0–10% ethyl acetate / petroleum ether, linear gradient) to give compound 1 as a brown solid (yield: 5.04 g, 11.2 mmol, 70%). TLC (V / V, PE / EtOAc = 10 / 1): R f = 0.67.

[0058] The synthesis reaction formula for compound 1 is as follows: Identification of Compound 1 by NMR and mass spectrometry: 1H NMR (500 MHz, Chloroform-d) δ 7.86 (d, J = 8.3Hz, 2H), 7.06 (d, J = 8.3 Hz, 2H), 6.01 (s, 2H), 2.57 (s, 6H), 1.44 (s, 6H). 13 C NMR (126 MHz, Chloroform-d) δ 155.90, 142.93, 140.08, 138.37, 134.56,121.46, 94.77, 14.67, 14.62. HRMS-ESI (m / z): [M+Na] + calcd for C 19 H 18 BF2IN2Na + :473.0473, found: 473.0483. Example 2 (1) The general synthetic method for compounds 2-1 and 2-2, and the synthetic reaction formulas are as follows: Compound 1 (1.80 g, 4.0 mmol), cuprous iodide (0.15 g, 0.8 mmol), L-proline (0.14 g, 1.2 mmol), potassium carbonate (1.10 g, 8.0 mmol), and 1-amino-3,3-diethoxypropane (for the synthesis of compound 2-1, 1.3 mL, 8.0 mmol) or 4-aminobutyraldehyde dimethyl acetal (for the synthesis of compound 2-2, 1.1 mL, 8.0 mmol) were added to a dry flask. Anhydrous dimethyl sulfoxide (80.0 mL) was then added to the reaction vessel under an argon atmosphere to dissolve the reactants. The resulting mixture was stirred at 85 °C for 2 hours. After the reaction was complete, water was added to quench the reaction, followed by extraction three times with ethyl acetate and saturated sodium chloride brine. The organic layers were combined and dried over anhydrous sodium sulfate. The organic phase was transferred to a flask and the solvent was removed by evaporation under reduced pressure. The crude product was purified by column chromatography to obtain compounds 2-1 and 2-2.

[0059] Purification and NMR / MS identification of compound 2-1: Compound 2-1 was purified from the crude product by column chromatography (0–20% ethyl acetate / petroleum ether, linear gradient) as a brownish-red solid (yield: 1.69 g, 3.60 mmol, 90%). TLC (V / V, PE / EtOAc = 4 / 1): R f = 0.55. 1H NMR (500 MHz, Chloroform-d) δ 7.00 (d,J = 8.4 Hz, 2H), 6.68 (d, J = 8.3 Hz, 2H), 5.96 (s, 2H), 4.66 (t, J = 5.2 Hz,1H), 3.74-3.68 (m, 2H), 3.57-3.51 (m, 2H), 3.27 (t, J = 6.3 Hz, 2H), 2.54 (s,6H), 1.99 (q, J = 6.2 Hz, 2H), 1.50 (s, 6H), 1.24 (t, J = 7.0 Hz, 6H). 13 C NMR(126 MHz, Chloroform-d) δ 154.79, 148.75, 143.23, 143.05, 128.91, 120.86,113.08, 102.31, 61.83, 39.96, 32.92, 15.39, 14.71, 14.55. HRMS-ESI (m / z): [M+H] + calcd for C 26 H 35 BF2N3O2 + : 470.2790, found: 470.2792. Purification and NMR / MS identification of compound 2-2: Compound 2-2 was obtained by column chromatography (0–25% ethyl acetate / petroleum ether, linear gradient) as a brownish-red solid (yield: 1.58 g, 3.48 mmol, 87%). TLC (V / V, PE / EtOAc = 3 / 1): R f = 0.57. 1 H NMR (600 MHz, Chloroform-d) δ 7.08 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.1 Hz, 2H), 5.97 (s, 2H), 4.42 (d, J = 5.0 Hz, 1H), 3.34(s, 6H), 3.23 (t, J = 4.8 Hz, 2H), 2.54 (s, 6H), 1.75-1.74 (m, 4H), 1.47 (s, 6H). 13C NMR (151 MHz, Chloroform-d) δ 155.10, 143.14, 142.31, 131.98, 129.19,121.05, 115.05, 104.26, 53.11, 45.32, 30.10, 23.64, 14.69, 14.59. [M+Na] + calcd for C 25 H 32 BF2N3NaO2 + : 478.2452, found: 478.2463. (2) General synthetic method for compounds 3-1 and 3-2: 9-fluorenyl chloroformate (1.06 g, 4.08 mmol) was dissolved in 1,4-dioxane (30.0 mL), and then slowly added to a mixed solution of 10 wt% sodium bicarbonate aqueous solution (40.0 mL) and 1,4-dioxane (20.0 mL) containing either compound 2-1 (1.60 g, 3.4 mmol) or 2-2 (1.55 g, 3.4 mmol). The reaction was stirred at 0 °C for 40 minutes. The mixture was then allowed to warm naturally to room temperature and stirred overnight. After the reaction was complete, the reaction mixture was extracted with ethyl acetate and washed with saturated sodium bicarbonate solution and sodium chloride brine. The organic phases were combined and dried over anhydrous sodium sulfate. After evaporating the organic solvent under reduced pressure, 4.0 M hydrochloric acid aqueous solution (5.0 mL) and tetrahydrofuran (20.0 mL) were added to the residue, and the reaction mixture was stirred at room temperature for 8 hours. After the reaction was complete, the mixture was extracted with ethyl acetate and washed with saturated sodium chloride brine, then dried over anhydrous sodium sulfate. After evaporating the organic solvent under reduced pressure, the crude product was purified by column chromatography to give compounds 3-1 and 3-2.

[0060] Purification and NMR / MS identification of compound 3-1: Compound 3-1 was obtained by column chromatography (0–35% ethyl acetate / petroleum ether, linear gradient) as a red solid (yield: 1.58 g, 3.48 mmol, 87%). TLC (V / V, PE / EtOAc = 2 / 1): R f = 0.52. 1H NMR (500 MHz, Chloroform-d) δ 9.62 (s, 1H), 7.74 (d,J = 7.6 Hz, 2H), 7.47 (d, J = 6.8 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.29 (d,J = 7.5 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 5.97 (s,2H), 4.62 (s, 2H), 4.12 (s, 1H), 3.94 (s, 2H), 2.56 (s, 8H), 1.32 (s, 6H). 13 CNMR (126 MHz, Chloroform-d) δ 199.89, 155.74, 154.91, 143.67, 142.95, 141.40,131.33, 128.88, 127.83, 127.13, 124.71, 121.37, 120.01, 67.24, 47.28, 42.67,14.57 (d, J = 9.9 Hz). HRMS-ESI (m / z): [M+Na] + calcd for C 37 H 34 BF2N3NaO3 + :640.2560, found: 640.2548. Purification and NMR / MS identification of compound 3-2: Compound 3-2 was purified from the crude product by column chromatography (0–40% ethyl acetate / petroleum ether, linear gradient) as a red solid (yield: 2.15 g, 3.40 mmol, 85%). TLC (V / V, PE / EtOAc = 2 / 1): R f = 0.37. 1H NMR (600 MHz, Chloroform-d) δ 9.66 (s, 1H),7.75 (d, J = 7.6 Hz, 2H), 7.49 (d, J = 7.1 Hz, 2H), 7.38 (t, J = 7.5 Hz, 2H),7.28 (t, J = 7.4 Hz, 2H), 7.17 (d, J = 7.8 Hz, 2H), 7.10 (s, 2H), 5.96 (s,2H), 4.65 (d, J = 5.5 Hz, 2H), 4.12 (q, J = 7.2 Hz, 1H), 3.64 (s, 2H), 2.55(s, 6H), 2.29 (s, 2H), 1.68 (s, 2H), 1.31 (s, 6H). 13 C NMR (151 MHz, Chloroform-d) δ 201.03, 155.72, 155.12, 143.77, 142.99, 141.46, 131.38,128.77, 127.82, 127.14, 124.78, 121.37, 119.99, 66.88, 49.37, 47.46, 40.66,20.47, 14.58 (d, J = 17.0 Hz). HRMS-ESI (m / z): [M+Na] + calcd for C 38 H 36 BF2N3NaO3 + : 654.2717, found: 654.2716. (3) General synthetic method for compounds 4-1 and 4-2: Compound 3-1 (1.85 g, 3.0 mmol) or compound 3-2 (1.89 g, 3.0 mmol) and hydroxylamine hydrochloride (0.44 g, 6.4 mmol) were added to a round-bottom flask, followed by anhydrous dichloromethane (80.0 mL) and triethylamine (1.67 mL, 12.0 mmol). The reaction was stirred at room temperature for 4 hours. After the reaction was completed, the solvent and triethylamine were removed by evaporation under reduced pressure. The residual mixture was extracted with dichloromethane and saturated sodium chloride brine, and the organic phases were combined and dried over anhydrous sodium sulfate. After evaporation of the organic solvent under reduced pressure, the crude product was purified by column chromatography to obtain compounds 4-1 and 4-2.

[0061] Purification and NMR / MS identification of compound 4-1: Compound 4-1 was purified from the crude product by column chromatography (0–40% ethyl acetate / petroleum ether, linear gradient) as a red solid (yield: 1.33 g, 2.1 mmol, 70%). TLC (V / V, PE / EtOAc = 2 / 1): R f = 0.35. 1 H NMR (600 MHz, Chloroform-d) δ 7.74 (t, J =6.5 Hz, 2H), 7.45 (s, 2H), 7.38 (q, J = 7.0 Hz, 2H), 7.28 (d, J = 7.7 Hz, 2H), 7.22-7.20 (m, 2H), 7.15 (s, 2H), 5.96 (d, J = 3.8 Hz, 2H), 4.59 (s, 2H), 4.10 (s, 2H), 3.86 (s, 3H), 2.55 (s, 6H), 1.31 (d, J = 9.2 Hz, 6H). 13 C NMR(151 MHz, Chloroform-d) δ 155.79, 155.09, 143.70, 143.61, 142.96, 142.91,141.36, 131.31, 129.01, 128.89, 127.85, 127.81, 127.11, 124.75, 124.73,121.39, 120.01, 67.43, 47.24, 47.20, 28.63, 14.60, 14.53, 14.50. HRMS-ESI (m / z): [M+Na] + calcd for C 37 H 35 BF2N4NaO3 + : 655.2669; found: 655.2675. Purification and NMR / MS identification of compound 4-2: Compound 4-2 was purified from the crude product by column chromatography (0–45% ethyl acetate / petroleum ether, linear gradient) as a brownish-red solid (yield: 1.23 g, 1.95 mmol, 65%). TLC (V / V, PE / EtOAc = 1 / 1): R f = 0.62. 1H NMR (600 MHz, Chloroform-d) δ 7.75 (d,J = 7.6 Hz, 2H), 7.53 – 7.43 (m, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.27 (s, 2H),7.18 (d, J = 7.9 Hz, 2H), 7.15-7.05 (m, 2H), 5.97 (s, 2H), 4.66 (d, J = 10.7Hz, 2H), 4.13 (s, 1H), 3.67 (s, 2H), 2.55 (s, 6H), 2.08 (s, 2H), 1.60 (s,2H), 1.31 (s, 6H). 13 C NMR (151 MHz, Chloroform-d) δ 155.77, 155.21, 151.17,143.73, 142.94, 141.44, 140.69, 131.36, 128.84, 128.82, 127.85, 127.83,127.14, 124.77, 124.75, 121.39, 120.01, 67.09, 49.88, 49.51, 47.41, 26.53,24.79, 14.64, 14.51, 14.50. HRMS-ESI (m / z): [M+H] + calcd for C 38 H 38 BF2N4O3 + :647.3001; found: 647.3006 (4) General synthetic method for compounds 5-1 and 5-2: A mixed solution of N-chlorosuccinimide (0.20 g, 1.5 mmol) dissolved in dichloromethane (8.0 mL) was added dropwise under an argon atmosphere to anhydrous dichloromethane (15.0 mL) containing pyridine (0.16 mL, 3.0 mmol) and either compound 4-1 (0.63 g, 1.0 mmol) or compound 4-2 (0.65 g, 1.0 mmol). After stirring at room temperature for 4 hours, a mixed solution of 1-thio-β-d-glucose-tetraacetate (1.09 g, 3.0 mmol) dissolved in dichloromethane (15.0 mL) was slowly added, followed by dropwise addition of triethylamine (0.63 mL, 4.5 mmol). The reaction was continued to be stirred at room temperature for 4 hours, and then the solvent and triethylamine were removed by vacuum evaporation. The residue was extracted with dichloromethane and washed with 2.0 M hydrochloric acid aqueous solution and saturated sodium chloride brine. The combined organic phases were dried over anhydrous sodium sulfate. After evaporation of the organic solvent under reduced pressure, the crude product was further purified by column chromatography to give compounds 5-1 and 5-2.

[0062] Purification and NMR / MS identification of compound 5-1: The crude product was purified by column chromatography (0–50% ethyl acetate / petroleum ether, linear gradient) to give compound 4-1 as a red solid (yield: 0.32 g, 0.32 mmol, 32%). TLC (V / V, PE / EtOAc = 1 / 1): R f = 0.42. 1H NMR (600 MHz, Chloroform-d) δ 7.74 (d, J =7.6 Hz, 2H), 7.47 (d, J = 7.5 Hz, 2H), 7.40-7.37(m, 2H), 7.29-7.26 (m, 2H),7.18 (d, J = 8.0 Hz, 2H), 7.11 (s, 2H), 5.97 (s, 2H), 5.35 (t, J = 9.3 Hz,1H), 5.09 (dt, J = 34.7, 9.7 Hz, 2H), 4.65-4.56 (m, 2H), 4.19 (dd, J = 12.4,4.8 Hz, 1H), 4.17-4.09 (m, 2H), 1.98 (s, 3H), 1.30 (s, 6H). 13 C NMR (151 MHz, Chloroform-d) δ 170.57, 170.23,169.46, 169.22, 155.80, 155.04, 143.68, 143.59, 142.92, 142.40, 141.38,140.53, 133.41, 131.32, 128.87, 127.88, 127.15, 127.10, 124.82, 124.69,121.39, 120.08, 120.07, 79.51, 75.42, 73.83, 70.21, 68.03, 67.65, 62.07, 47.18, 31.36, 20.65, 20.61, 14.63, 14.61. Purification protocol and NMR mass spectrometry identification of compound 5-2: The crude product was purified by column chromatography (0 ~ 60% ethyl acetate / petroleum ether, linear gradient) to obtain compound 5-1 as a red solid (yield: 0.30 g, 0.30 mmol, 30%). 1HNMR (600 MHz, Chloroform-d) δ 7.75-7.74 (m, 2H), 7.47 (s, 2H), 7.40-7.36 (m,2H), 7.27 (s, 2H), 7.21 (d, J = 7.9 Hz, 2H), 7.15 (s, 2H), 5.97 (s, 2H),5.31 (t, J = 8.8 Hz, 1H), 5.12 (t, J = 9.8 Hz, 1H), 5.07 (d, J = 8.3 Hz, 2H),4.60 (dd, J = 10.7, 6.1 Hz, 2H), 4.21 (dd, J = 12.5, 4.9 Hz, 1H), 4.17-4.12(m, 2H), 3.80 (s, 1H), 3.72 (s, 2H), 2.55 (s, 6H), 2.03 (dd, J = 12.8, 5.5Hz, 12H), 1.85 (s, 2H), 1.32 (s, 6H). 13 C NMR (151 MHz, Chloroform-d) δ 170.56,170.22, 169.38, 169.19, 155.78, 155.27, 143.74, 143.71, 142.94, 141.42,141.37, 140.66, 131.36, 128.86, 127.85, 127.10, 127.10, 124.79, 121.39,120.05, 79.96, 75.93, 73.77, 70.16, 67.41, 61.99, 47.26, 29.95, 25.35, 20.73,20.66, 20.61, 20.59, 14.64, 14.58, 0.03. HRMS-ESI (m / z): [M+Na] + calcd forC 52 H 55 BF2N4NaO 12 S + : 1031.3499; found: 1031.3498. (5) General synthetic method for compounds 6-1 and 6-2: Pyridine sulfur trioxide (0.16 g, 1.0 mmol) was added to anhydrous acetonitrile (15.0 mL) in which compound 5-1 (0.20 g, 0.20 mmol) or compound 5-2 (0.30 g, 0.20 mmol) was dissolved. The reaction was stirred at 80 °C for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate and washed with saturated sodium chloride brine. The organic phases were combined and dried over anhydrous sodium sulfate. After the organic solvent was evaporated under reduced pressure, the crude product was further purified by column chromatography to obtain compounds 6-1 and 6-2.

[0063] Purification and mass spectrometry identification of compound 6-1: Compound 6-1 was purified from the crude product by column chromatography (0–10% methanol / dichloromethane, linear gradient) as a red solid (85.9 mg, 0.08 mmol, 40%). TLC (V / V, DCM / CH3OH = 10 / 1): R f = 0.58. TLC (V / V, DCM / CH3OH = 8 / 1): R f = 0.35. HRMS-ESI(m / z): [M] - calcd for C 51 H 52 BF2N4O 15 S2 - : 1073.2937; found: 1073.2905. Purification and mass spectrometry identification of compound 6-2: The crude product was purified by column chromatography (0–15% methanol / dichloromethane, linear gradient) to obtain compound 6-1 as a red solid (65.2 mg, 0.06 mmol, 30%). TLC (V / V, DCM / CH3OH = 8 / 1): R f = 0.35. HRMS-ESI (m / z): [M] - calcd for C 52 H 54 BF2N4O 15 S2 - :1087.3094; found: 1087.3098. (6) General synthetic method for compounds BDP-GSL1 and BDP-GSL5: Potassium methoxide (35.1 mg, 0.50 mmol) was slowly added to anhydrous methanol (2.0 mL) in which compound 6-1 (85.9 mg, 0.08 mmol) or compound 6-2 (65.2 mg, 0.06 mmol) was dissolved. The reaction was stirred at room temperature for 3 hours. After the organic solvent was evaporated under reduced pressure, the crude product was purified by reversed-phase high-performance liquid chromatography to obtain compounds BDP-GSL1 and BDP-GSL5.

[0064] Purification and NMR / MS identification of compound BDP-GSL1: Probe BDP-GSL1 was purified by reversed-phase high-performance liquid chromatography (30–100% acetonitrile / water, linear gradient 0–65 min, addition of 0.1% V / V trifluoroacetic acid, t = 15.2 min) as a red solid (yield: 5.5 mg, 0.008 mmol, 10%). TLC (V / V, DCM / CH3OH = 3 / 1): R f =0.46. 1 H NMR (600 MHz, DMSO-d6) δ 7.03 (d, J = 8.3 Hz, 2H), 6.77 (d, J = 8.3Hz, 2H), 6.15 (s, 2H), 4.79 (d, J = 9.8 Hz, 1H), 3.24-3.21 (m, 3H), 3.14 (t,J = 9.2 Hz, 2H), 3.07 (t,J = 9.2 Hz, 2H), 2.88-2.85 (m, 2H), 2.44 (s, 6H),1.50 (s, 6H). HRMS-ESI (m / z): [M] - calcd for C 28 H 34 BF2N4O9S2 - : 683.1842; found: 683.1839. Purification and NMR / MS identification of compound BDP-GSL5: Probe BDP-GSL7 was purified by reversed-phase high-performance liquid chromatography (30–100% acetonitrile / water, linear gradient 0–65 min, addition of 0.1% V / V trifluoroacetic acid, t = 11.2 min) as a red solid (yield: 4.2 mg, 0.006 mmol, 10%). TLC (V / V, DCM / CH3OH = 3 / 1): R f = 0.46. 1H NMR (500 MHz, DMSO-d6) δ 6.98-6.96 (m, 2H), 6.74 (d, J = 8.6 Hz,2H), 6.15 (s, 2H), 4.75 (d, J = 9.7 Hz, 1H), 3.69 (dd, J = 12.1, 2.0 Hz, 1H),3.22 (dd, J = 10.1, 7.3 Hz, 3H), 3.14-3.05 (m, 4H), 2.81-2.68 (m, 2H), 2.55(s, 1H), 2.44 (s, 6H), 1.97-1.87 (m, J = 6.7 Hz, 2H), 1.51 (s, 6H). HRMS-ESI(m / z): [M] - calcd for C 29 H 36 BF2N4O9S2 - : 697.1990; found: 697.1982. Example 3: Fluorescence spectral response changes of probes BDP-GSL1 and BDP-GSL5 in the reaction with myrosinase Incubation procedure and conditions: Take 1.0 μL of the 1.0 mM probe BDP-GSL1 or BDP-GSL5 prepared in Example 2 and 4.0 μL of commercial myrosinase (purchased from Sigama-Aldrich, derived from white mustard) at a concentration of 4000 U / L into PBS buffer (20 mM, pH=7.4) to make a total volume of 400 μL. Keep the concentration of probe BDP-GSL1 or BDP-GSL5 prepared in Example 2 at 2.5 μM and the concentration of myrosinase at 40 mU / mL. Mix the above mixture thoroughly and transfer it to a PE tube. Place the PE tube in a water bath at 37 ℃ and incubate for 10 minutes. Then, take 300 μL of the reaction solution from the PE tube into a micro-volume quartz dish for fluorescence testing at an excitation wavelength of 485 nm.

[0065] Figure 1 (a) shows the fluorescence spectra of the probe BDP-GSL1 prepared in Example 2 before and after reaction with myrosinase; Figure 1(b) Fluorescence spectra of probe BDP-GSL5 prepared in Example 2 before and after reaction with myrosinase. The parent fluorescence of probes BDP-GSL1 / BDP-GSL5 is completely quenched due to photoinduced electron transfer (PET) between the recognition group (aryl moiety) and the fluorophore, resulting in extremely low background fluorescence. After 10 minutes of reaction with myrosinase, a significant increase in fluorescence signal at 510 nm was observed, and green fluorescence could be observed with the naked eye under 365 nm UV light. Specifically, the fluorescence intensity of BDP-GSL1 increased 51-fold at 510 nm after reaction with myrosinase, while that of BDP-GSL5 increased 110-fold. Compared to BDP-GSL1, the enzymatic hydrolysis cyclization product generated after the reaction of BDP-GSL5 with myrosinase exhibits a higher fluorescence quantum yield in water.

[0066] Example 4: Time-dependent fluorescence growth of probes BDP-GSL1 and BDP-GSL5 after reaction with myrosinase Incubation Procedure and Conditions: For each time point, 1.0 μL of the 1.0 mM probe BDP-GSL1 / BDP-GSL5 prepared in Example 2 and 4.0 μL of a commercially available myrosinase (purchased from Sigama-Aldrich, derived from white mustard) at a concentration of 4000 U / L were added to PBS buffer (20 mM, pH = 7.4) to make a total volume of 400 μL. The concentrations of the probe BDP-GSL1 / BDP-GSL5 prepared in Example 2 were maintained at 2.5 μM and the myrosinase concentration at 40 mU / mL. The mixtures of samples from each time point were thoroughly mixed and transferred to PE tubes. The PE tubes were incubated in a 37 ℃ water bath for different times (0–20 min). Samples were taken every 2 min, and 300 μL of the reaction solution from the PE tubes was transferred to a microplate for fluorescence testing. The excitation wavelength was 485 nm, and the fluorescence intensity at the optimal emission wavelength of 510 nm was measured.

[0067] Figure 2 The graph shows the increase in fluorescence intensity at 510 nm over time after the probes BDP-GSL1 / BDP-GSL5 prepared in Example 2 react with myrosinase. The fluorescence intensity at 510 nm increases synchronously with the reaction time, reaching equilibrium around 10 minutes, reflecting the complete conversion of the probes into enzymatic products by myrosinase. Furthermore, compared to BDP-GSL1, BDP-GSL5 exhibits a higher fluorescence intensity at 510 nm after the reaction with myrosinase reaches equilibrium, indicating a better signal-to-noise ratio.

[0068] Example 5: Selective response test of probes BDP-GSL1 and BDP-GSL5 to myrosinase and other interfering agents Incubation Procedure and Conditions: Take 1.0 μL of the 1.0 mM probe BDP-GSL1 / BDP-GSL5 prepared in Example 2 and 4.0 μL of a commercially available myrosinase (purchased from Sigama-Aldrich, derived from white mustard) at a concentration of 4000 U / L into PBS buffer (20 mM, pH = 7.4), making a total volume of 400 μL. Maintain the probe concentration at 2.5 μM and the myrosinase concentration at 40 mU / mL. Mix the above mixture thoroughly and transfer it to a PE tube. Incubate the PE tube in a water bath at 37 ℃ for 10 minutes. After incubation, perform fluorescence testing on the reaction solution in the PE tube. The excitation wavelength is 485 nm, and the fluorescence intensity at the optimal emission wavelength of 510 nm is measured.

[0069] In response tests involving interfering agents other than myrosinase (various enzymes, proteins, amino acids, biomacromolecules, and ionic compounds), each interfering agent (50-fold equivalent concentration) was incubated with 2.5 μM probe in PBS buffer in PE tubes at 37 °C for 10 min. After incubation, 300 μL of the reaction solution from the PE tube was aspirated into a micro-quartz dish for fluorescence testing. The excitation wavelength was 485 nm, and the fluorescence intensity at the optimal emission wavelength of 510 nm was measured. At the same time, the background fluorescence intensity was measured using PBS solutions of the same concentration of probe without myrosinase and various interfering agents. Figure 3 In Figures (a) and (b), the selective response tests of BDP-GSL1 and BDP-GSL5 to myrosinase and other interfering agents are shown, respectively. It can be seen that the fluorescence intensity of the experimental group incubated with BDP-GSL1 / BDP-GSL5 and incubated with myrosinase increased by nearly 51 times and 110 times, respectively, compared with the control group without any interfering agents. In contrast, the experimental groups incubated with various interfering agents showed almost no fluorescence increase compared with the control group. This proves that the probes BDP-GSL1 / BDP-GSL5 prepared in Example 2 have high selectivity for myrosinase and achieve a specific response to myrosinase.

[0070] Example 6: Fluorescence response spectrum and linear relationship of probe BDP-GSL5 to myrosinase concentration-dependent fluorescence spectrum In the same incubation system as in Example 3, the concentration of myrosinase was varied (0 mU / mL to 70 mU / mL). After incubation, the fluorescence of the reaction solution was analyzed. A standard curve was plotted between the fluorescence intensity of the enzymatic digest at 510 nm and the protein concentration. The limit of detection (LOD) of probe BDP-GSL5 for myrosinase was calculated using the formula DL = 3σ / k, where σ represents the standard deviation of the blank sample, and k represents the linear coefficient (slope) between the fluorescence intensity at 510 nm and the enzyme concentration after the reaction of probe BDP-GSL5 prepared in Example 2 with myrosinase. Figure 4 As shown, Figure 4 (a) shows the fluorescence intensity curves of the reaction solution after incubation with different concentrations of myrosinase. Figure 4 (b) shows the linear relationship between myrosinase protein concentration and fluorescence intensity, and the calculated limit of detection for myrosinase; the excitation wavelength is 485 nm. Figure 4 As shown in (b), within the concentration range of myrosinase protein (0–40 mU / mL), the fluorescence peak intensity at 510 nm gradually increases after the enzymatic digestion reaction, exhibiting a good linear relationship with the myrosinase concentration. Incubation with myrosinase at concentrations above 40 mU / mL showed fluorescence approaching saturation. Based on the linear relationship, the detection limit for myrosinase was determined to be as low as 0.08 mU / mL. These results demonstrate that the probe BDP-GSL5 prepared in Example 2 can achieve sensitive detection of myrosinase. The excitation wavelength was 485 nm, and the fluorescence intensity at the optimal emission wavelength of 510 nm was measured.

[0071] Example 7: Time-dependent response of probe BDP-GSL5 to myrosinase at different temperatures In the same incubation system as in Example 4 of this invention, the incubation temperature was changed and the incubation time was varied. After the incubation was completed, the fluorescence of the incubation reaction solution was tested. Figure 5 As shown, the fluorescence intensity of the enzymatic hydrolysate at 510 nm varies significantly with time under different incubation temperatures: the time required for fluorescence to reach saturation is longer at lower temperatures, while it only takes about 90 seconds at a higher temperature of 50 °C. This indicates that temperature has a significant impact on enzyme activity; appropriately increasing the incubation temperature can greatly increase enzyme activity and significantly reduce the time required for the reaction to approach saturation. Considering that subsequent practical applications involve live plant samples, a relatively mild condition of 37 °C was ultimately chosen as the incubation temperature for this system. Testing showed that the time required for the fluorescence of the enzymatic hydrolysate to approach saturation was 10 minutes. The excitation wavelength was 485 nm, and the fluorescence intensity at the optimal emission wavelength of 510 nm was measured.

[0072] Example 8: Fluorescence response determination of probe BDP-GSL5 after incubation with different vegetable lysates To verify whether there are differences in the enzymatic hydrolytic activity of myrosinase expressed by different cruciferous vegetable varieties on the probe BDP-GSL5 prepared in Example 2, different cruciferous vegetables (white radish, Chinese cabbage, kale, cauliflower, and broccoli) were selected as the experimental group, while two non-cruciferous vegetables (lettuce and cucumber) were selected as the control group. In this invention, equal masses of different vegetables were weighed and thoroughly pulverized and lysed. The lysates were diluted with the same volume of PBS to maintain a final concentration of 2.5 mg / mL. The lysates of different vegetables were then centrifuged for 10 minutes (6200 rpm) to remove bottom vegetable residue, retaining the supernatant. 2.0 μL of the 1.0 mM probe BDP-GSL5 prepared in Example 2 and the supernatant of the different vegetable lysates were taken to bring the total volume to 400 μL, maintaining the concentration of the probe BDP-GSL5 prepared in Example 2 at 5.0 μM. The above mixture was thoroughly mixed and transferred to a PE tube. The PE tube was then incubated in a water bath at 37 ℃ for 5 minutes. After incubation, the reaction solution in the PE tube was subjected to fluorescence testing. The excitation wavelength was 485 nm, and the fluorescence intensity at the optimal emission wavelength of 510 nm was measured.

[0073] like Figure 6 As shown, the myrosinases expressed in different cruciferous vegetables exhibit significant differences in their enzymatic hydrolysis rate of the probe BDP-GSL5 prepared in Example 2, resulting in varying fluorescence intensities measured at 510 nm. Overall, the myrosinases expressed in radish, cabbage, and Chinese cabbage showed high enzymatic hydrolysis activity against the probe BDP-GSL5 prepared in Example 2, exhibiting a high fold increase in background fluorescence at 510 nm compared to the probe itself. In contrast, the myrosinases in broccoli and cauliflower showed slightly lower enzymatic hydrolysis activity against the probe BDP-GSL5, with a lower fluorescence fold increase. Lettuce and cucumber, serving as the control group, are from the Asteraceae and Cucurbitaceae families respectively, and are not cruciferous, thus they cannot express myrosinase and cannot enzymatically hydrolyze the probe BDP-GSL5. Consequently, the fluorescence intensity measured at 510 nm showed almost no fold increase compared to the background fluorescence of GSL5 itself at 510 nm.

[0074] Example 9: Time-dependent fluorescence confocal imaging after co-incubation of probe BDP-GSL1 with living Arabidopsis root tissue Since the model plant Arabidopsis thaliana belongs to the Brassicaceae family and can express myrosinase, seeds of the Colombian wild-type (Col-0) Arabidopsis thaliana were sterilized and sown on 1 / 2 MS solid medium. They were vernalized at 4 ℃ in the dark for 48 hours, and then transferred to an intelligent artificial climate chamber for cultivation (conditions: 22 ℃, 65% humidity, 16-hour 6000 lx light / 8-hour dark cycle). Seedlings that had grown for 5 days were harvested, their above-ground parts removed, and their roots incubated with the probe BDP-GSL1 (5.0 μM) synthesized in Example 2 at 37 ℃ for 0 to 5 hours (experimental groups were set up at 1-hour intervals). After each incubation, the roots were thoroughly washed three times with water, placed on a glass slide, and imaged using a confocal fluorescence microscope (excitation wavelength 470 nm, collection signal in the 520-520 nm band). The results are as follows: Figure 7 As shown, the fluorescence intensity in the root tip region significantly increased with prolonged incubation time, exhibiting a clear time-dependent accumulation trend. This phenomenon indicates that the probe BDP-GSL1 needs a certain amount of time to be transported into the root tip cells and undergo a specific enzymatic reaction with endogenous myrosinase to generate a fluorescence signal; therefore, its fluorescence accumulation process has a significant time dependence.

[0075] Example 10: Screening of human intestinal bacteria capable of metabolizing glucosinolates by fluorescence confocal imaging using probe BDP-GSL5 This invention purchased five types of human intestinal bacteria and corresponding culture media from Beina Biotechnology Co., Ltd., including two aerobic bacteria (Enterococcus faecalis and Proteus mirabilis); two anaerobic bacteria (Bacteroides polymorpha and Bifidobacterium bifidum); and one facultative anaerobic bacterium (Escherichia coli). All intestinal bacteria were cultured in their respective culture media at 37 °C until OD (Organic Depth). 600 The concentration of each bacterial suspension was set to 0.6–0.75. Then, 600 μL of each suspension was centrifuged (6000 rpm, 20 min), and the supernatant was discarded. Each enteric bacterium was washed twice with PBS and then resuspended in PBS. BDP-GSL5 was added to the resuspended enteric bacterium suspension, maintaining a BDP-GSL1 concentration of 5.0 µM and a total resuspended volume of 400 µL. All resuspended bacterial suspensions with the probe were incubated at 37 °C for 4 hours. After centrifugation, the supernatant was discarded, and each enteric bacterium was washed twice again with PBS and then resuspended in PBS. 5 µL of the resuspended bacterial suspension was plated onto a glass slide, covered with a coverslip, and used for fluorescence confocal imaging (excitation wavelength 470 nm, signal collection in the 520–520 nm band). Figure 8As shown, under the same excitation light intensity and other parameter settings, *Bacteroides polymorpha* exhibited significant fluorescence release, proving that the probe was effectively converted into enzymatic cyclization products upon entering its cells, resulting in fluorescence "lighting up." In contrast, the other four intestinal bacteria did not generate significant fluorescence signals, indicating that they lack the ability to metabolize GSLs. In summary, the probe prepared in this invention can effectively screen human intestinal bacteria capable of metabolizing GSLs.

[0076] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A rapid detection of myrosinase using a BODIPY-type fluorescent probe, characterized in that, It has the structure shown in Equation I: Equation I; In Equation I, n = 2 or 3; R1, R3, R4 and R6 are independently hydrogen, halogroup, cyanogroup, mercaptogroup, or C1-C8 straight-chain or branched saturated alkane group; R2 and R5 are independently hydrogen, ethyl, propyl, isopropyl, halogen, cyano, nitro, carboxyl, sulfonic acid, methoxy, acetyl, phenyl, benzyl, ester, or pyrrole rings with different substitutions; R7, R8, R9 and R 10 It can be hydrogen, cyano, nitro, aldehyde, carboxyl, sulfonic acid, methoxy, acetyl, or ester group independently.

2. The BODIPY-type fluorescent probe as described in claim 1, characterized in that, It has any of the structures shown in formulas BDP-GSL1 to BDP-GSL8: 。 3. The method for preparing the fluorescent probe of formula BDP-GSL1 or BDP-GSL5 according to claim 2, characterized in that, Includes the following steps: (1) Dissolve compound 1, cuprous iodide, L-proline, potassium carbonate and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal in the first solvent, and then carry out the first nucleophilic substitution reaction to obtain compound 2-1 or compound 2-2. ； (2) Mix the solution of compound 2-1 or compound 2-2 with the solution of fluorenyl chloroformate, and carry out the second nucleophilic substitution reaction and acid-catalyzed hydrolysis reaction in sequence to obtain compound 3-1 or compound 3-2; The structural formulas of compounds 2-1, 2-2, 3-1, and 3-2 are as follows: ； ； ； (3) The compound 3-1 or compound 3-2, hydroxylamine hydrochloride, triethylamine and a third solvent are mixed and oxime reaction is carried out to obtain compound 4-1 or compound 4-2; (4) Mix the compound 4-1 or compound 4-2, the fourth solvent, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate and triethylamine, and carry out the third nucleophilic substitution reaction to obtain compound 5-1 or compound 5-2; The structural formulas of compounds 4-1, 4-2, 5-1, or 5-2 are as follows: ； ； (5) The compound 5-1 or 5-2, pyridine sulfur trioxide, and acetonitrile are mixed and subjected to an electrophilic substitution reaction to obtain compound 6-1 or 6-2; the structural formula of compound 6-1 or 6-2 is as follows: ； (6) Mix the compound 6-1 or compound 6-2, potassium methoxide and the fifth solvent, and carry out an alkaline-catalyzed hydrolysis reaction to obtain compound BDP-GSL1 or BDP-GSL5.

4. The preparation method according to claim 3, characterized in that, The first solvent is dimethyl sulfoxide and / or N,N-dimethylformamide; The molar ratio of compound 1, cuprous iodide, L-proline, potassium carbonate, and 1-amino-3,3-diethoxypropane or 4-aminobutyraldehyde dimethyl acetal is 1.0:(0.05-0.4):(0.2-0.6):(1.0-3.0). The temperature of the first nucleophilic substitution reaction is 50~100℃, and the reaction time is 1.5~3 hours.

5. The preparation method according to claim 3, characterized in that, The solution of compound 2-1 or compound 2-2 is obtained by dissolving compound 2-1 or compound 2-2 in a mixed solvent; the mixed solvent is obtained by mixing a second solvent and an aqueous solution of sodium bicarbonate; the second solvent is 1,4-dioxane and / or tetrahydrofuran; the molar ratio of compound 2-1 or compound 2-2 to fluorenyl chloroformate is 1:(1.0~2.0); The temperature for the second nucleophilic substitution reaction is 0~25℃, and the reaction time is 30~60 minutes; The acid-catalyzed hydrolysis reaction is carried out by adding hydrochloric acid aqueous solution and a second solvent to the second nucleophilic substitution reaction solution; the acid-catalyzed hydrolysis reaction is carried out at room temperature for 6 to 9 hours.

6. The preparation method according to claim 3, characterized in that, The third solvent is dichloromethane and / or 1,2-dichloroethane; the molar ratio of compound 3-1 or compound 3-2, hydroxylamine hydrochloride and triethylamine is 1:(1.5~3.5):(3.0~6.0); the oxime reaction is carried out at room temperature for 3~5 hours.

7. The preparation method according to claim 3, characterized in that, The fourth solvent is dichloromethane and / or 1,2-dichloroethane; the molar ratio of compound 4-1 or compound 4-2, pyridine, N-chlorosuccinimide, 1-thio-β-d-glucose tetraacetate, and triethylamine is 1.0:(2.0~5.0):(1.0~2.5):(2.0~4.5):(3.0~6.0); the third nucleophilic substitution reaction is carried out at room temperature for 4~8 hours.

8. The preparation method according to claim 3, characterized in that, The molar ratio of compound 5-1 and compound 5-2 to pyridine sulfur trioxide is 1.0:(3.5~6.5); the electrophilic substitution reaction is carried out at a temperature of 60~90℃ for 5~8 hours. The fifth solvent is methanol and / or ethanol; the molar ratio of compound 6-1 or compound 6-2 to potassium methoxide is 1.0:(5.0~12.0); the alkaline-catalyzed hydrolysis reaction is carried out at room temperature for 2~4 hours.

9. The application of the fluorescent probe according to claim 1 or 2 or the fluorescent probe prepared by the preparation method according to any one of claims 3 to 8 in the detection of myrosinase.

10. The application as described in claim 9, characterized in that, The applications include in vitro myrosinase detection, myrosinase detection in plants and microorganisms, screening of myrosinase inhibitors, identification of myrosinase heterologous expression in microorganisms, and high-throughput rapid screening.

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