Estrogen sulfotransferase specific fluorescent substrate, its preparation method and application

Abstract

The application belongs to the field of biological detection, and particularly relates to a specific fluorescent substrate of estrogen sulfotransferase, and a preparation method and application thereof. The specific fluorescent substrate of estrogen sulfotransferase provided by the application is a 4-(4-hydroxyphenyl)-1,8-naphthalimide derivative, which has simple synthesis process, high economy and efficiency, is used for related detection of SULT1E1 enzyme activity, realizes detection of SULT1E1 enzyme activity in a complex system and high-throughput screening of SULT1E1 related inhibitors, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to an estrogen sulfonate transferase-specific fluorescent substrate, its preparation method, and its application. Background Technology

[0002] Sulfonate transferases (SULTs) are an important class of phase II metabolic enzymes widely distributed in mammals, participating in the biotransformation and detoxification of various endogenous substances (hormones, neurotransmitters, etc.) and exogenous compounds (drugs, food components, and environmental toxins, etc.). According to existing research, human SULTs are divided into 4 families and 13 subtypes. Among them, human estrogen sulfonate transferase (SULT1E1) has attracted much attention due to its crucial role in the balance and homeostasis of estrogen. SULT1E1-mediated sulfonate transfer reactions inactivate endogenous estrogen because sulfonated estrogen cannot bind to the estrogen receptor (ER). Furthermore, SULT1E1 also participates in the metabolic clearance of various estrogen drugs and selective estrogen receptor modulators, such as tamoxifen, pregnenolone, fulvesetron, and ethinylestradiol (EE). Studies have shown that inhibiting SULT1E1 can increase the circulating exposure and oral bioavailability of SULT1E1 drug substrates and bioactive phenolic substances in food (such as flavonoids and other natural phenolic substances), thereby enhancing therapeutic effects. Meanwhile, SULT1E1 inhibitors are also considered potential new targets for various diseases, such as acute kidney injury, type 2 diabetes, and liver ischemia-reperfusion injury.

[0003] Over the past few decades, Western blotting and proteomics techniques have been widely used to detect SULT1E1 expression levels due to their high specificity. However, enzyme expression levels and activity are often inconsistent. Substrate-based enzyme activity assays are the primary means of analyzing target enzyme activity. Current SULT1E1 activity assays mainly rely on its physiological substrates (estrone or estradiol) or drug substrates, using high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS / MS) to detect SULT1E1 activity. However, these methods have limitations such as cumbersome sample pretreatment, reliance on specialized technicians, and expensive equipment. In contrast, enzyme-activated fluorescent probes, due to their ultra-high sensitivity, real-time detection, ideal spatiotemporal resolution, and high-throughput detection, have been widely used to analyze the true activity of target enzymes in complex biological systems and for high-throughput screening of inhibitors. However, to date, there are no reports on small molecules for SULT1E1 activity detection. Therefore, developing a practical and readily available fluorescent tool molecule for real-time quantification of SULT1E1 activity in complex biological samples has become an urgent need in the field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an estrogen sulfonate transferase-specific fluorescent substrate, its preparation method, and its application. The estrogen sulfonate transferase-specific fluorescent substrate provided by this invention solves the technical problems of insufficient accuracy in detecting SULT1E1 enzyme activity in complex systems and the inability to perform high-throughput screening of SULT1E1 inhibitors in existing technologies.

[0005] The technical solution of the present invention is an estrogen sulfonate transferase-specific fluorescent substrate, which is a 4-(4-hydroxyphenyl)-1,8-naphthalimide derivative, with the structural formula shown in formula (1):

[0006]

[0007] Where R is

[0008] This invention provides a method for preparing an estrogen sulfonate transferase-specific fluorescent substrate, comprising the following steps:

[0009] In an alkaline environment, under the action of a catalyst, 4-bromo-1,8-naphthylimide derivatives and 4-hydroxyphenylboronic acid react in an organic solvent to generate 4-(4-hydroxyphenyl)-1,8-naphthylimide derivatives.

[0010] In the above synthesis process, the molar ratio of 4-bromo-1,8-naphthylimide derivative to 4-hydroxyphenylboronic acid is 1:1–2, preferably 1:1–1.5, and more preferably 1:1–1.2; the molar concentration of 4-bromo-1,8-naphthylimide derivative is 50–150 mmol / L, preferably 60–100 mmol / L, and more preferably 75–95 mmol / L.

[0011] In the above synthesis process, the molar ratio of the 4-bromo-1,8-naphthalimide derivative to the catalyst is 1:0.005–0.05, preferably 1:0.01–0.02. The catalyst includes, but is not limited to, any one or any combination of palladium catalysts, nickel catalysts, copper catalysts, ruthenium catalysts, rhodium catalysts, and iridium catalysts. The palladium catalyst includes, but is not limited to, any one or any combination of palladium acetate, palladium chloride, tetra(triphenylphosphine)palladium, tris(dibenzylacetone)palladium, bis(dibenzylacetone)palladium, bis(acetonitrile)palladium dichloride, bis(triphenylphosphine)palladium dichloride, and [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride. The nickel catalyst includes, but is not limited to, any one or any combination of Raney nickel, nickel boride, and zero-valent nickel. The copper catalyst includes, but is not limited to, Cu / SiO2 series, Cu-Zn-Al series, Cu-Cr series, CuO-ZnO series, CuO-Al2O3 series, CuO- The catalysts include any one or any combination of the Cr2O3 series and CuO / SiO2 series; the ruthenium catalysts include, but are not limited to, any one or any combination of ruthenium oxide catalysts, aminoruthenium catalysts, ruthenium chloride catalysts, carbonylruthenium catalysts, and alkylruthenium catalysts; the rhodium catalysts include, but are not limited to, any one or any combination of tris(triphenylphosphine)rhodium chloride (Wilkinson catalyst), 1,4-bis(diphenylphosphine)butanetetrafluoroborate rhodium(I), rhodium(III) chloride hydrate, rhodium(III) acetylacetone, rhodium(I) dicarbonylacetylacetone, rhodium(III) oxide, and hydroxy(cyclooctadiene)rhodium(I) dimer; and the iridium catalysts include, but are not limited to, any one or any combination of homogeneous iridium catalysts and supported iridium catalysts; preferably tetra(triphenylphosphine)palladium.

[0012] In the above synthesis process, the molar concentration of the alkaline substance in the alkaline environment is 0.1–2 mmol / mL, preferably 0.3–1 mmol / mL; the alkaline substance includes inorganic bases and / or organic bases, the inorganic bases including but not limited to any one or any combination of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, and ammonium hydroxide, preferably potassium carbonate; the organic bases including but not limited to any one or any combination of sodium ether, potassium ether, sodium ethoxide, potassium ethoxide, sodium acetate, potassium acetate, ethyl acetate, and sodium acetamide.

[0013] In the above synthesis process, the organic solvent includes, but is not limited to, any one or any combination of methanol, ethanol, n-propanol, isopropanol, ethylene glycol, propylene glycol, glycerol, acetone, tetrahydrofuran, and dimethyl ether, preferably tetrahydrofuran.

[0014] In the above synthesis process, the reaction is carried out at 100–150℃ for 3–4 hours, preferably at 100℃ for 4 hours.

[0015] In the above synthesis process, the reaction solution was filtered, and the product was precipitated by adding acid and water. The obtained product was purified by silica gel column chromatography and recrystallized from ethyl acetate / petroleum ether.

[0016] The structural formula of the 4-(4-hydroxyphenyl)-1,8-naphthalimide derivative is shown in formula (1):

[0017]

[0018] Where R is

[0019] In the above synthesis process, the preparation of the 4-bromo-1,8-naphthalimide derivative includes the following steps: 4-bromo-1,8-naphthalenedicarboxylic anhydride and a primary amine derivative are refluxed in an organic solvent to generate the 4-bromo-1,8-naphthalimide derivative.

[0020] The molar ratio of 4-bromo-1,8-naphthoic anhydride to the primary amine derivative is 1:1–2, preferably 1:1.1–1.5; the molar concentration of 4-bromo-1,8-naphthoic anhydride is 0.1–1 mmol / mL, preferably 0.1–0.5 mmol / mL, and more preferably 0.1–0.3 mmol / mL.

[0021] The organic solvents include, but are not limited to, any one or any combination of alcohols, ketones, esters, acetonitrile, pyridine, and phenol; alcohols include, but are not limited to, any one or any combination of methanol, ethanol, and isopropanol; ketones include, but are not limited to, any one or any combination of acetone, methyl ethyl ketone, and methyl isobutyl ketone; and esters include, but are not limited to, any one or any combination of methyl acetate, ethyl acetate, and propyl acetate; preferably, ethanol.

[0022] The present invention provides an estrogen sulfonyltransferase-specific fluorescent substrate, a 4-(4-hydroxyphenyl)-1,8-naphthylimide derivative (PN derivative), which, under physiological conditions, can be specifically catalyzed by SULT1E1 to generate the corresponding O-sulfonation product. By detecting the product generation rate, accurate quantification of SULT1E1 activity in biological samples can be achieved.

[0023]

[0024] Specifically, the PN derivatives generated under the catalysis of SULT1E1 exhibit significant changes in fluorescence properties: the PN derivatives are “OFF-ON” type SULT1E1 fluorescent substrates with an excitation spectrum of 320–400 nm and an emission spectrum of 450–600 nm.

[0025] The estrogen sulfonate transferase-specific fluorescent substrate provided by the present invention is used to prepare products for SULT1E1 activity detection, enabling qualitative and / or quantitative detection of SULT1E1 activity; it can also be used for SULT1E1 screening and / or evaluation, preparing products for SULT1E1 screening and / or evaluation; and it can also be used to screen and / or evaluate substances related to SULT1E1 activity, including inhibitors and activators of SULT1E1, preparing products for screening and / or evaluating substances related to SULT1E1 activity. The above products can be probes, and / or reagents, and / or kits, and / or detection systems, and / or devices, and / or single-component and / or mixed-component formulations, and can be liquid, and / or semi-solid, and / or solid.

[0026] The present invention provides products containing the estrogen sulfonate transferase-specific fluorescent substrates described above, including but not limited to: products for detecting SULT1E1 activity, and / or products for screening and / or evaluating SULT1E1, and / or products for screening and / or evaluating SULT1E1 activity-related substances.

[0027] The products described above are applicable to samples including, but not limited to, SULT1E1 recombinant single enzymes and biological samples. The biological samples include biological materials obtained from or derived from organisms, including but not limited to remains (including dead embryos, fetuses, etc.), organs, tissues (including embryos, etc.), cells (including fertilized eggs, primary cells, etc.), body fluids (including blood, etc.), secretions, excretions, etc., as well as biological materials derived therefrom, such as DNA, RNA, proteins, microbiota, metabolites, etc.

[0028] The SULT1E1 activity-related substances screening and / or evaluation products of the present invention include SULT1E1 inhibitors and SULT1E1 activators, which can be compounds, and / or plant extracts, and / or biological substances, and / or drugs. They can be single components and / or mixed components, and can be liquid, and / or semi-solid, and / or solid. Biological substances include but are not limited to DNA, and / or RNA, and / or proteins, etc. Drugs include but are not limited to chemical drugs, traditional Chinese medicine, traditional Chinese medicine, biological drugs, etc., such as any one or any combination of tetrabromobisphenol A and ethinylestradiol.

[0029] This invention provides a method for using the estrogen sulfonate transferase-specific fluorescent substrate described above, comprising the following steps: incubating the estrogen sulfonate transferase-specific fluorescent substrate with a sample, adding PAPS to initiate the reaction, detecting the fluorescence intensity of the O-sulfonation product of the estrogen sulfonate transferase-specific fluorescent substrate, and qualitatively and / or quantitatively determining the estrogen sulfonate transferase activity in the sample; or,

[0030] When screening and / or evaluating substances related to estrogen sulfonate transferase activity, the incubation system also contains substances related to estrogen sulfonate transferase activity.

[0031] The samples include, but are not limited to, recombinant single enzymes of estrogen sulfonate transferase and biological samples. The biological samples include biological materials obtained from or derived from organisms, including but not limited to remains (including dead embryos, fetuses, etc.), organs, tissues (including embryos, etc.), cells (including fertilized eggs, primary cells, etc.), body fluids (including blood, etc.), secretions, excretions, etc., as well as biological materials derived therefrom, such as DNA, RNA, proteins, microbiota, metabolites, etc.

[0032] The incubation system includes, but is not limited to, buffer, MgCl2, and DTT, wherein the buffer is Tris-HCl buffer, PBS buffer, or HEPES buffer.

[0033] The estrogen sulfonyltransferase SULT1E1 activity-related substances include inhibitors and activators of SULT1E1. These substances may be compounds, and / or plant extracts, and / or biological substances, and / or drugs. They may be single components and / or mixed components, and may be liquid, and / or semi-solid, and / or solid. Biological substances include, but are not limited to, DNA, and / or RNA, and / or proteins. Drugs include, but are not limited to, chemical drugs, traditional Chinese medicine, traditional Chinese medicine, and biological drugs, such as any one or any combination of tetrabromobisphenol A and ethinylestradiol.

[0034] The fluorescence detection conditions are as follows: the excitation wavelength of the O-sulfonation product of the PN derivative catalyzed by estrogen sulfonyltransferase is 320-400 nm, and the emission wavelength is 450-600 nm; preferably, the excitation wavelength is 360 nm and the emission wavelength is 510 nm.

[0035] Compared with existing technologies, the present invention has the following advantages:

[0036] (1) High specificity: PN derivatives can be catalyzed by SULT1E1 with high specificity, while other SULTs isotypes are basically not involved in its metabolism.

[0037] (2) High affinity: PN derivatives exhibit high affinity with SULT1E1 and conform to classical Michaelis kinetics.

[0038] (3) Sensitive detection: The corresponding O-sulfonated products of PN derivatives all have strong fluorescence emission signals, resulting in high detection sensitivity and easy real-time monitoring.

[0039] (4) Detection at the live cell level: This type of fluorescent substrate can be used to detect the content of SULT1E1 in live cells.

[0040] (5) High detection throughput: The inhibitor screening method established using this fluorescent substrate can achieve efficient, rapid and real-time detection of 96-well and 384-well plates, and can achieve high-throughput detection of 20,000 samples per day, with low cost per test (<0.5 yuan).

[0041] (6) Easy to synthesize: 4-(4-hydroxyphenyl)-1,8-naphthylimide derivatives can all be prepared by simple chemical synthesis, and the synthesis process is economical and efficient. Attached Figure Description

[0042] Figure 1 This is the synthetic route for PN derivatives.

[0043] Figure 2 This provides a specific characterization of PN derivatives.

[0044] Figure 3 Fluorescence spectroscopy analysis of PN derivatives.

[0045] Figure 4 Linear analysis of PN3 enzyme concentration.

[0046] Figure 5 Time linearity analysis for PN3.

[0047] Figure 6 The enzyme activity of SULT1E1 in different cells was measured for PN3.

[0048] Figure 7 The enzyme activity of SULT1E1 in liver tissues of different individuals was measured for PN3.

[0049] Figure 8 To screen for potent inhibitors of SULT1E1 using PN3 as a substrate. Detailed Implementation

[0050] The technical solution of the present invention will be described below with specific embodiments, but these are not intended to limit the scope of protection of the present invention.

[0051] Example 1: Synthesis of 4-(4-hydroxyphenyl)-1,8-naphthylimide derivatives

[0052] Each primary amine derivative (2.0 mmol) was added to a solution of 4-bromo-1,8-naphthalenedicarboxylic anhydride (554 mg, 2.0 mmol) in anhydrous ethanol (15 mL). The mixture was then stirred and refluxed for 6 h. After cooling to room temperature, it was directly filtered to obtain a light yellow powder in high yield. The crude product, 4-bromo-1,8-naphthalimide derivative, did not require further purification and was used directly in the next reaction. 4-bromo-1,8-naphthalimide derivative (1.0 mmol), 4-hydroxyphenylboronic acid (138 mg, 1 mmol), tetrakis(triphenylphosphine)palladium (11.5 mg, 0.01 mmol), K₂CO₃ solution (2 M, 2 mL), and DMF (10 mL) were mixed and stirred at 100 °C for 4 h. After the reaction was complete, an appropriate amount of acidic water was added to the mixture, and the product continued to precipitate. After filtration and drying, the crude product was further purified by silica gel column chromatography and recrystallized from ethyl acetate / petroleum ether. Figure 1 ).

[0053] Primary amine derivatives such as 5-aminomethylthiazole, 2-aminoethylsulfonamide, N-(2-aminoethyl)-4-methylbenzenesulfonamide, and 2,4-difluorobenzylamine, respectively generate 4-(4-hydroxyphenyl)-1,8-naphthalimide derivatives PN1, PN2, PN3, and PN4.

[0054]

[0055] PN1: Yellow powder (180 mg, yield 46.6%). 1 H NMR(600MHz,DMSO-d6)δ9.87(s,1H),9.00(d,J=0.8Hz,1H),8.58–8.53(m,2H),8.35(dd,J=8.5,1.2Hz,1H),7.98(d,J=0 .8Hz,1H),7.85(dd,J=8.5,7.3Hz,1H),7.76(d,J=7.5Hz,1H),7.39(d,J=8.5Hz,2H),6.99(d,J=8.5Hz,2H),5.47(s,2H). 13 C NMR (151MHz, DMSO-d6) δ 163.65, 163.42, 158.51, 155.20, 147.34, 143.71, 133.92, 133.43, 131.70, 131.57, 131.34, 129.91, 129.11, 128.64, 128.28, 127.80, 122.51, 120.64, 116.15, 35.71. HRMS (ESI, m / z) calculated values ​​(C 22 H 14 N₂O₃S, [M+H] +The value was 387.0798, while the actual measured value was 387.0803.

[0056] PN2: Yellow powder (245 mg, yield 64.6%). 1 H NMR(600MHz,DMSO-d6)δ9.86(s,1H),8.53(ddt,J=10.4,6.0,2.0Hz,2H),8.37–8.30(m,1H),7.85(dd,J=8.7,7.0Hz,1H) ,7.76(d,J=7.6Hz,1H),7.40(d,J=8.4Hz,2H),7.07(s,2H),6.99(d,J=8.5Hz,2H),4.50–4.45(m,2H),3.39–3.35(m,2H). 13 C NMR (151MHz, DMSO-d6) δ 163.90, 163.67, 158.46, 147.00, 133.06, 131.67, 131.20, 130.99, 129.87, 129.20, 128.66, 128.18, 127.70, 122.91, 121.07, 116.14, 52.18, 35.27. HRMS (ESI, m / z) calculated values ​​(C 20 H 16 N₂O₅S, [M+H] + The value was 397.0853, while the measured value was 397.0854.

[0057] PN3: Yellow powder (351 mg, yield 72.1%). 1 H NMR(600MHz, DMSO-d6)δ9.86(s,1H),8.47(ddd,J=13.0,6.2,3.3Hz,2H),8.35–8.29(m,1H),7.83(ddt,J=7.0,4.5,1.6Hz,1H),7.79–7.72(m,2H), 7.59(d,J=8.2Hz,2H),7.45–7.38(m,2H),7.22(d,J=7.9Hz,2H),7.00(d, J=8.5Hz,2H),4.13(t,J=6.1Hz,2H),3.11(q,J=6.5Hz,2H),2.25(s,3H). 13C NMR (151MHz, DMSO-d6) δ 164.05, 163.81, 158.43, 146.77, 142.91, 138.09, 132.86, 131.66, 131.07, 130.86, 129.96, 129.79, 129.23, 128.66, 128.12, 127.64, 126.81, 122.95, 121.12, 116.13, 21.35. HRMS (ESI, m / z) calculated values ​​(C 27 H 22 N₂O₅S, [M+H] + The value was 487.1322, while the actual measured value was 487.1308.

[0058] PN4: Yellow powder (250 mg, yield 60.3%) 1 H NMR(400MHz, DMSO-d6)δ9.75(s,1H),8.59–8.50(m,2H),8.36(dd,J=8.6,1.2Hz,1H),7.82(dd,J=8.5,7.3Hz,1H),7.73(d,J=7.6Hz,1H) ,7.39–7.33(m,2H),7.33–7.25(m,1H),7.11(ddd,J=10.5,9.2,2.6Hz,1H),7.02–6.93(m,2H),6.90(td,J=8.6,2.7Hz,1H),5.30(s,2H). 13 C NMR (101MHz, DMSO-d6) δ 163.96, 163.73, 158.49, 147.37, 133.30, 131.47, 131.40, 131.22, 130.05, 129.13, 128.83, 128.06, 127.44, 122.67, 120.81, 116.07, 111.79, 104.31, 104.05, 103.80, 55.04. HRMS (ESI, m / z) calculated values ​​(C 25 H 15 F2N O3, [M+H] + The value was 416.1093, while the measured value was 416.1113.

[0059] Example 2: Specificity of PN derivatives analyzed by metabolic phenotyping

[0060] Prepare a 95 μL reaction system including Tris-HCl buffer (50 mM, pH 7.4), MgCl2 (5 mM), DTT (4 mM), PN derivative (10 μM), and recombinant single enzymes (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C4, SULT1E1, SULT2A1, or SULT2B1). After pre-incubation for 3 min, add 5 μL LPAPS (200 μM) to initiate the reaction. After reacting for 1 h, add 100 μL of ice-cold acetonitrile to terminate the reaction. Centrifuge at 20000g for 30 min, and collect the supernatant for analysis. Detect the fluorescence intensity of the corresponding O-sulfonation products using LC-FD. Figure 2 ).

[0061] Conclusion: PN derivatives can all be specifically catalyzed by SULT1E1 to form the monosulfonated product PN O-sulfate, with a fast reaction rate and good specificity.

[0062] Example 3: Enzymatic reaction kinetics analysis of PN derivatives, specific substrates of SULT1E1

[0063] (1) Prepare a 95 μL reaction system, including Tris-HCl buffer (50 mM, pH 7.4), MgCl2 (5 mM), DTT (4 mM), and a series of PN derivatives, SULT1E1, or human liver S9 at different concentrations. After pre-incubation for 3 min, add 5 μL PAPS (200 μM) to initiate the reaction. After incubation for 1 h, add 100 μL ice-cold acetonitrile and shake vigorously to terminate the reaction. Centrifuge at 20000g for 30 min, and take the supernatant for detection. Detect the amount of PN derivative O-sulfate generated per unit time using LC-FD and fit the enzyme-catalyzed reaction kinetic curve (Table 1).

[0064] (2) Fit the Michaelis constant K according to the Michaelis equation. m With the maximum reaction rate V max .

[0065] V = (V max ×[S]) / (K m +[S])

[0066] Where V is the reaction rate, [S] is the concentration of the PN derivative, and V max It is the maximum reaction rate, K. m It is V max The substrate concentration corresponding to half.

[0067] Conclusion: The O-sulfonation reaction of PN derivatives catalyzed by SULT1E1 follows Michaelis-Menten kinetics, and its enzyme kinetic parameters can be characterized using the Michaelis-Menten equation. This is of great significance for enzyme activity detection and inhibitor screening.

[0068] Table 1. Enzymatic kinetic data for the O-sulfonation of PN derivatives catalyzed by SULT1E1.

[0069]

[0070] Example 4: Study on the fluorescence spectrum changes of PN derivatives before and after incubation with SULT1E1

[0071] Prepare a 95 μL reaction system including Tris-HCl buffer (50 mM, pH 7.4), MgCl2 (5 mM), DTT (4 mM), PN derivative (10 μM), and SULT1E1. The control group does not contain SULT1E1. After a 3-minute pre-incubation, add 5 μL of PAPS (200 μM) to initiate the reaction. After 1 hour, add 100 μL of ice-cold acetonitrile to terminate the reaction. Place the black 96-well microplate in a microplate reader to detect the absorption and fluorescence spectra. Figure 3 ).

[0072] Conclusion: The PN derivatives are specifically catalyzed by SULT1E1 to generate corresponding O-sulfonated products. The maximum emission wavelengths of PN1, PN2, and PN3 are 510 nm, and the maximum emission wavelength of PN4 is 500 nm. The sulfonated products of the PN derivatives exhibit excellent fluorescence properties.

[0073] Example 5: Linear characterization of enzyme concentration and time in fluorescence signals of PN3 metabolized by SULT1E1

[0074] (1) Prepare a 95 μL reaction system, including Tris-HCl buffer (50 mM, pH 7.4), MgCl2 (5 mM), DTT (4 mM), PN3 (1 μM), and different concentrations of SULT1E1. After pre-incubation for 3 min, add 5 μL of PAPS (200 μM) to initiate the reaction. Place the black 96-well microplate in a microplate reader for continuous detection for 1 h. Record the fluorescence intensity of the product and calculate the linear relationship between fluorescence intensity and SULT1E1 enzyme concentration. Figure 4 ).

[0075] (2) Prepare a 95 μL reaction system, including Tris-HCl buffer (50 mM, pH 7.4), MgCl2 (5 mM), DTT (4 mM), PN3 (1 μM), and SULT1E1. After pre-incubation for 3 min, add 5 μL of PAPS (200 μM) to initiate the reaction. Place the black 96-well microplate in a microplate reader and continuously detect for 30 min. Record the fluorescence intensity of the product and calculate the linear relationship between fluorescence intensity and time. Figure 5 ).

[0076] Conclusion: The sulfonation product of PN3 exhibits a good linear response to both SULT1E1 enzyme concentration and reaction time, enabling sensitive and accurate detection of SULT1E1 activity in samples.

[0077] Example 6: PN3 assay of SULT1E1 enzyme activity in different cells

[0078] A certain number of cells were seeded into 96-well plates (HepG2, MCF-7, and A549) and cultured at 37°C and 5% CO2 for 24 h. PN3 (final concentration 20 μM) was added, and the plates were incubated for 2 h. The supernatant was transferred to 1.5 mL EP tubes, and an equal volume of acetonitrile was added. Finally, the samples were centrifuged at 20000 g for 30 min and analyzed by liquid chromatography-fluorescence detection (LC-FD). Figure 6 ).

[0079] Conclusion: The activity of SULT1E1 was highest in HepG2 cells among the three cell types tested. PN3 can be used as a tool molecule to detect SULT1E1 enzyme activity in different living cells.

[0080] Example 7: PN3 assay of SULT1E1 enzyme activity in different human liver tissues

[0081] Prepare a 95 μL reaction system including Tris-HCl buffer (50 mM, pH 7.4), MgCl2 (5 mM), DTT (4 mM), PN3 (1 μM), and different human liver S9 (1 mL). After pre-incubation for 3 min, add 5 μL PAPS (200 μM) to initiate the reaction. After incubation for 1 h, add 100 μL of ice-cold acetonitrile, shake vigorously, and then terminate the reaction. Centrifuge at 20000g for 30 min, collect the supernatant, and detect the fluorescence intensity of the product in each sample using LC-FD. Figure 7 ).

[0082] Conclusion: PN3 can be used as a tool molecule to detect SULT1E1 enzyme activity in human liver tissue preparations.

[0083] Example 8: Construction of a SULT1E1 inhibitor screening method and screening and evaluation of inhibitors

[0084] (1) Prepare a 95 μL reaction system, including Tris-HCl buffer (50 mM, pH 7.4), MgCl2 (5 mM), DTT (4 mM), PN3 (1 μM), and SULT1E1 (1 μg / mL), as well as different inhibitors. After pre-incubation for 3 min, add 5 μL of PAPS (200 μM) to initiate the reaction. Place the black 96-well microplate in a microplate reader for continuous detection for 20 min. Compare the fluorescence intensity values ​​of the inhibitor group with those of the control group (DMSO). Calculate the residual activity and fit the half-maximal inhibitory concentration (IC50) curve. 50 To assess the potential of the compound to inhibit SULT1E1. Figure 8 ).

[0085] Conclusion: PN3 can be used for high-throughput screening of SULT1E1 inhibitors. The screening of potent SULT1E1 inhibitors was also characterized.

[0086] Note: The above examples are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A fluorescent substrate specific to estrogen sulfonate transferase, characterized in that, It is a 4-(4-hydroxyphenyl)-1,8-naphthalimide derivative, with the structural formula shown in formula (1): Where R is 2. The method for preparing an estrogen sulfonate transferase-specific fluorescent substrate according to claim 1, characterized in that, step... include: In an alkaline environment, under the action of a catalyst, 4-bromo-1,8-naphthalimide derivatives react with 4-hydroxyphenylboronic acid in an organic solvent to generate 4-(4-hydroxyphenyl)-1,8-naphthalimide derivatives.

3. The preparation method according to claim 2, characterized in that, The preparation of 4-bromo-1,8-naphthalimide derivatives includes: reflux reaction of 4-bromo-1,8-naphthalenedicarboxylic anhydride with a primary amine derivative in an organic solvent to generate 4-bromo-1,8-naphthalimide derivatives.

4. The estrogen sulfonate transferase-specific fluorescent substrate according to claim 1 is used to prepare at least one of the following products: (1) Products for SULT1E1 activity detection; (2) Products for screening and / or evaluating SULT1E1 inhibitors; (3) Products for screening and / or evaluating SULT1E1 activity-related substances.

5. A product containing an estrogen sulfonate transferase-specific fluorescent substrate as described in claim 1, comprising at least one of the following: (1) Products for SULT1E1 activity detection; (2) Products for screening and / or evaluating SULT1E1 inhibitors; (3) Products for screening and / or evaluating SULT1E1 activity-related substances.

6. The application according to claim 4 or the product according to claim 5, characterized in that, The product is applicable to samples including SULT1E1 recombinant single enzyme, any one or any combination of biological samples.

7. The application according to claim 4 or the product according to claim 5, characterized in that, Substances related to SULT1E1 activity include inhibitors or activators that act on SULT1E1.

8. The application or product according to claim 7, characterized in that, The SULT1E1 activity-related substances include any one or any combination of tetrabromobisphenol A and ethinylestradiol.

9. A method for using the estrogen sulfonate transferase-specific fluorescent substrate as described in claim 1, characterized in that the steps include... include: After incubating the sample with an estrogen sulfonate transferase-specific fluorescent substrate, PAPS is added to initiate the reaction. The fluorescence intensity of the O-sulfonation product of the estrogen sulfonate transferase-specific fluorescent substrate is detected to perform qualitative and / or quantitative determination of estrogen sulfonate transferase activity in the sample; or, When screening and / or evaluating substances related to estrogen sulfonate transferase activity, the incubation system also contains substances related to estrogen sulfonate transferase activity.

10. The operating method according to claim 9, characterized in that, The fluorescence detection conditions were as follows: the excitation wavelength of the O-sulfonation product of the estrogen sulfonate transferase-specific fluorescent substrate was 320–400 nm, and the emission wavelength was 450–600 nm.

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