A specific super-resolution fluorescent probe for lysine-specific demethylase 1 and its application

Abstract

A specific super-resolution fluorescent probe for lysine-specific demethylase 1 and its application relate to the field of molecular probes. The present invention designs and synthesizes a specific small molecule fluorescent probe LP1 for lysine-specific demethylase 1. The probe is designed based on the active cavity configuration of lysine-specific demethylase 1 and the molecular structure of its specific inhibitor, and has excellent light stability, pH stability, and anti-interference ability against intracellular ions and amino acids. The product released after the probe LP1 reacts with lysine-specific demethylase 1 has an excellent near-infrared fluorescence signal. The probe LP1 successfully and specifically detects the activity of lysine-specific demethylase 1 in different biological samples (pure protein, cell lysate, living cells) and detects the subcellular organelle localization of lysine-specific demethylase 1.

Description

Technical Field

[0001] The present invention relates to the field of molecular probes, and in particular to a specific super-resolution fluorescent probe LP1 of lysine-specific demethylase 1 (LSD1) and applications thereof. Background Art

[0002] Lysine-specific demethylase 1 (LSD1) specifically removes mono- and di-methyl groups from K4 / K9 on histone H3, regulating gene expression. It also directly interacts with related proteins, playing a crucial role in cell cycle regulation, cell differentiation, and stem cell self-renewal. Studies have shown that LSD1 is overexpressed in a variety of malignant tumors (such as neuroblastoma, prostate cancer, lung cancer, and colorectal cancer), promoting tumor proliferation and metastasis. Its selective high expression in tumor tissues makes LSD1 a focus of targeted tumor therapy. Therefore, specific detection of LSD1 activity and subcellular localization are of great significance for studying the physiological and pathological pathways involved in LSD1. However, there is currently a lack of convenient, efficient, and in situ molecular tools for detecting LSD1 in vivo.

[0003] Small molecule fluorescent probe detection technology has the advantages of high sensitivity and good biocompatibility and is widely used in biomarker detection. However, no specific fluorescent probe for LSD1 has been reported. Meanwhile, super-resolution fluorescent probes combined with structured light illumination fluorescence microscopy can break through the diffraction limit and observe more detailed subcellular structures. Therefore, the design and synthesis of specific super-resolution fluorescent probes for LSD1 are crucial for further understanding its biological functions. Summary of the Invention

[0004] The present invention aims to address the aforementioned problems in the prior art by providing a super-resolution fluorescent probe, LP1, specific for lysine-specific demethylase 1 (LSD1), and its applications. The products produced by the reaction between this probe and LSD1 exhibit distinctly different fluorescent properties, making the products easily detectable. Using this probe, LSD1 activity can be detected in a variety of biological samples and the distribution of lysine-specific demethylase 1 in subcellular organelles can be tracked.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A specific super-resolution fluorescent probe for lysine-specific demethylase 1, the structure of which is as follows:

[0007] The compound shown is designated as LP1;

[0008] The synthetic route of LP1 is as follows:

[0009]

[0010] The application of the specific super-resolution fluorescent probe for lysine-specific demethylase 1 is used to detect the activity of human recombinant lysine-specific demethylase 1 protein and lysine-specific demethylase 1 protein in cell lysate.

[0011] The application of the specific super-resolution fluorescent probe for lysine-specific demethylase 1 is used to detect lysine-specific demethylase 1 in cells.

[0012] Specifically, the probe is used as a specific substrate of lysine-specific demethylase 1. After the reaction occurs, the activity of lysine-specific demethylase 1 in different biological systems is measured by quantitatively detecting the fluorescence signal of the product per unit time.

[0013] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0014] 1. High specificity (not affected by amino acids, reactive oxygen species, and various anions and cations): LP1 can react with lysine-specific demethylase 1 with high specificity to generate fluorescent dyes;

[0015] 2. Cheap and easy to obtain: LP1 and its oxidation products can be obtained by chemical synthesis, and the synthesis process is simple and easy;

[0016] 3. High Sensitivity (in vitro detection concentration 0.5 μM): LP1 reacts specifically with lysine-specific demethylase 1 to produce the corresponding C17, which exhibits a well-characterized fluorescence emission spectrum (620-750 nm). While the substrate is non-fluorescent, the product exhibits excellent fluorescence properties, enabling superior differential detection and quantitative determination via standard curve plotting. The product also exhibits high brightness and near-infrared properties, reducing biological background fluorescence and making it suitable for structured light illumination super-resolution imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flow chart for the detection of LP1 activity on lysine-specific demethylase 1;

[0018] Figure 2 is the fluorescence emission spectra of LP1 and C17 (Ex = 580 nm);

[0019] Figure 3 This is the detection spectrum of LP1 against lysine-specific demethylase 1 human recombinant protein;

[0020] Figure 4 is the fluorescence spectrum of LP1 as the concentration of lysine-specific demethylase 1 protein increases;

[0021] Figure 5This is the test result of LP1's anti-interference capability;

[0022] Figure 6 For LP1, the activity of lysine-specific demethylase 1 in cell lysates was detected;

[0023] Figure 7 CCK8 test for LP1 cytotoxicity;

[0024] Figure 8 For live cell imaging experiments, LP1 was co-incubated with U87 cells for 0 to 4 hours;

[0025] Figure 9 This is a super-resolution imaging experiment after LP1 was co-incubated with U87 cells for 3 hours. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0027] Example 1

[0028] Chemical synthesis of LP1

[0029] The reaction formula is as follows:

[0030]

[0031] Step 1, tert-butyl (2-hydroxyethyl)(methyl)carbamate (L2)

[0032] To a round-bottom flask was added N-methyl-2-hydroxyethylamine (1.9 g, 25.3 mmol) and aluminum oxide (3.876 g, 38 mmol). Subsequently, di-tert-butyl dicarbonate (5.28 g, 27.83 mmol) was added. The mixture was stirred at room temperature for 15 minutes. Ethyl acetate (2 × 80 mL) was added to dilute the reaction mixture. Purification by flash column chromatography gave Compound L2 as a colorless oil in an yield of 3.8 g, 87%. 1 H-NMR (500MHz, 298K, DMSO-d6): δ4.63 (s, 1H), 3.47 (t, J = 5.0Hz, 2H), 3.20 (t, J = 5.0Hz, 2H), 2.82 (d, J = 10.0Hz, 3H), 1.38 (s, 9H).

[0033] Step 2, tert-butyl methyl(2-oxoethyl)carbamate (L3)

[0034] Compound L2 (300 mg, 1.7 mmol) was added to a round-bottom flask and dissolved in 8.5 mL of ultra-dry dichloromethane under nitrogen. Stirring was performed in an ice-water bath, and Dess-Martin periodinane (762 mg, 1.8 mmol) was slowly added dropwise. 50 mL of sodium bicarbonate and 50 mL of a saturated sodium thiosulfate solution were then added to the flask and stirred for 30 minutes. The organic phase was collected by extraction and washed with saturated sodium bicarbonate (1 × 20 mL). Purification by flash column chromatography afforded Compound L3 as a colorless oil in a yield of 241 mg (81%). 1 H-NMR (500MHz, 298K, CDCl3): δ9.54 (s, 1H), 3.96 (s, 1H), 3.86 (s, 1H), 2.90 (d, J = 15.0Hz, 3H), 1.41 (t, J = 10.0Hz, 9H).

[0035] Step 3, tert-butyl (2-phenylcyclopropyl)carbamate (1-2)

[0036] Trans-phenylcyclopropylamine hydrochloride (1-1, 4.55g, 25mmol) is dissolved in 200mL of pure water, 25mL of sodium hydroxide (2M) aqueous solution is added, and pH value is adjusted to 8. The organic phase is obtained by extraction with ethyl acetate (3×200mL), washed with salt water, then dried over anhydrous sodium sulfate, filtered, and the organic solvent is evaporated by rotary evaporator to obtain trans-cyclopropylamine. Subsequently, trans-cyclopropylamine is added to a flask, dissolved with 20mL of dichloromethane, and triethylamine (37.5mmol, 5mL) is added dropwise under the condition of stirring in an ice-water bath. T-butyl dicarbonate (8.2g, 37.5mmol) is dissolved in 15mL of ultra-dry dichloromethane and added dropwise to the flask. The reaction is continued at room temperature for 12 hours, and then purified by flash column chromatography to obtain compound 1-2 as a white solid, with an output of 3.1g, 54%. 1 H-NMR (500MHz, 298K, DMSO-d6): δ7.26 (s, 1H), 7.24-7.23 (d, J = 5.0Hz, 2H), 7.15-7.12 (d, J = 5.0Hz, 1H), 7.09- 7.07(d,J=10.0Hz,2H),2.61(s,1H),1.91-1.87(m,1H),1.38(s,9H),1.13-1.09(m,1H),1.05(d,J=5.0Hz,1H).

[0037] Step 4, tert-butyl methyl (2-phenylcyclopropyl) carbamate (1-3)

[0038] Under ice-water bath conditions, compound 1-2 (4.256 g, 18.27 mmol) was added to a round-bottom flask and dissolved with 20 mL of N, N-dimethylformamide. Sodium hydride (692 mg, 95% purity) was then added to the flask. After stirring for 30 minutes, potassium iodide (3.4 mL, 54.81 mmol) was dissolved in 10 mL of ultra-dry N, N-dimethylformamide and added dropwise to the round-bottom flask. The reaction was transferred to room temperature and stirred overnight. Pure water was added to terminate the reaction, and the reaction solution was extracted with dichloromethane (3×100 mL). The organic phase was washed with brine and dried over anhydrous sodium sulfate. Filtered and then purified by flash column chromatography to obtain compound 1-3 as a colorless oil with a yield of 2.43 g, 54%. 1 H-NMR (500MHz, 298K, CDCl3): δ7.29(d,J=5.0Hz,2H),7.2(t,J=5.0Hz,1H),7.13(d,J=5.0Hz,2H),2.9 2(s,3H),2.74-2.71(m,1H),2.61-2.10(m,1H),1.41(s,9H),1.31-1.27(m,1H),1.22(t,J=5.0Hz,1H).

[0039] Step 5, N-methyl-2-phenylcyclopropan-1-amine (1-4)

[0040] Compound 1-3 (2.433 g, 9.815 mmol) was added to a flask and dissolved with 70 mL of dichloromethane. The reaction was placed in an ice-water bath and stirred, and 25 mL of 4 M hydrochloric acid-dioxane solution (100 mmol) was slowly added dropwise. The reaction was warmed to room temperature and stirred for 5 hours. The pH value of the aqueous phase was adjusted to 8 with 2N aqueous sodium hydroxide solution, and then extracted with dichloromethane (3×20 mL). The organic phase was washed with brine and dried over anhydrous sodium sulfate. Purification by flash column chromatography gave compound 1-4 as a brown oil with a yield of 1.36 g, 94%. 1 H-NMR (500MHz, 298K, CDCl3): δ7.29(d,J=5.0Hz,2H),7.2(t,J=5.0Hz,1H),7.13(d,J=5.0Hz,2H),2.9 2(s,3H),2.74-2.71(m,1H),2.61-2.10(m,1H),1.41(s,9H),1.31-1.27(m,1H),1.22(t,J=5.0Hz,1H).

[0041] Step 6, tert-butyl methyl(2-(methyl(2-phenylcyclopropyl)amino)ethyl)carbamate (1-5)

[0042] Compound 1-4 (542 mg, 3.57 mmol), L3 (680 mg, 3.93 mmol) and triethylamine (1.03 mL, 7.44 mmol) were added to a flask in sequence, and 16 mL of ultra-dry chloroform was added to dissolve. Stir at room temperature for 30 minutes. Sodium triacetoxyborohydride (1.13 g, 5.35 mmol) was then added to the flask and stirred at room temperature for 3 hours. The reaction was terminated with 20 mL of sodium bicarbonate, and the organic phase was extracted with dichloromethane (3 × 20 mL), washed with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Purification by flash column chromatography gave compound 1-5 as a light yellow oil with a yield of 720 mg, 64%. 1 H-NMR (500MHz, 298K, CDCl3): δ7.27(t,J=5.0Hz,2H),7.17(d,J=5.0Hz,1H),7.07(d,J=5.0Hz,2H),3.03(s,2H), 2.88(s,3H),2.72(s,2H),2.43(s,3H),1.97(t,J=5.0Hz,2H),1.45(s,9H),1.12-1.08(m,1H),1.01-0.98(m,1H).

[0043] Step 7, N,N-dimethyl-N-(2-phenylcyclopropyl)ethane-1,2-diamine (1-6)

[0044] Compound 1-5 (250 mg, 0.83 mmol) was added to a flask, dissolved in 35 mL of dichloromethane, stirred in an ice-water bath, and slowly added with 4 M hydrochloric acid solution (2.75 mL, 12 mmol). The reaction was warmed to room temperature and stirred for 5 hours. The precipitate was collected by filtration and washed with dichloromethane to obtain the hydrochloride salt of compound 1-6 as a pale white solid. 1 H-NMR (500MHz, 298K, MeOD-d4): δ7.35(t,J=5.0Hz,2H),7.28(t,J=5.0Hz,1H),7.23(d,J=5.0Hz,2H),3 .76(s,2H),3.60(d,J=5.0Hz,2H),3.28(d,J=5.0Hz,1H),2.82(s,4H),1.86(s,1H),1.54-1.50(m,1H). 13 C-NMR (125MHz, 298K, MeOD-d4): δ138.81,129.98,128.36,127.56,53.77,44.66,42.47,34.12,23.70,14.88.

[0045] Step 8, N,N-diallyl-3-bromoaniline (C2)

[0046] 3-Bromoaniline (13.76 g, 80 mmol), 3-bromopropylene (33.87 g, 280 mmol), and potassium carbonate (22 g, 160 mmol) were added to a round-bottom flask in sequence. Then, 80 mL of acetonitrile was added and the mixture was heated to 80°C and refluxed for 14 hours. After cooling, pure water was added to terminate the reaction, and the organic phase was extracted with ethyl acetate. After drying over anhydrous sodium sulfate, the mixture was purified by flash column chromatography to obtain Compound C2 as a yellow oil in a yield of 14 g, 90%. 1 H NMR (400MHz, 298K, CDCl3) δ7.07(t,J=8.1Hz,1H),6.83(m,1H),6.63(dd,J=8.3,2.0Hz,1H),5.86(dd,J=16.8,10.7Hz,1H),5.20(m,3H),3.93(m,3H).

[0047] Step 9, 2-bromo-4-(diallylamino)benzaldehyde (C3)

[0048] Add 17.5mL of N,N-dimethylformamide and 37mL of dichloromethane to the flask, and slowly add phosphorus oxychloride (25.5g, 165mmol) dropwise under stirring in an ice-water bath. Continue stirring for 15 minutes after the addition is complete. Dissolve C2 in 15mL of dichloromethane and add it dropwise to the flask. The reaction is allowed to react at room temperature for 18 hours. Terminate the reaction by adding 1N sodium hydroxide solution in an ice-water bath. The organic phase is extracted with dichloromethane and dried over anhydrous sodium sulfate. Purify by flash column chromatography to obtain a yellow oily compound C3 with a yield of 13.9g, 90%. 1 H NMR (400MHz, 298K, CDCl3) δ10.04 (s, 1H), 7.74 (d, J = 9.5Hz, 1H), 6.79 (s, 2H), 6.61 (m,1H),5.80(dd,J=17.7,10.9Hz,2H),5.17(dd,J=30.5,14.3Hz,4H),3.96(s,4H).

[0049] Step 10, (2-bromo-4-(diallylamino)phenyl)methanol (C4)

[0050] C3 was added to a flask, and 30 mL of methanol and 30 mL of dichloromethane were added to dissolve it. Sodium borohydride (150 mmol) was dissolved in 20 mL of dichloromethane and added dropwise to the flask. The reaction was allowed to react at room temperature for 24 hours. 80 mL of pure water was added to terminate the reaction. The organic phase was extracted with dichloromethane and dried over anhydrous sodium sulfate, and then purified by flash column chromatography to obtain a yellow oily compound C4 in a yield of 11 g, 80%. 1H NMR(400MHz,298K,CDCl3)δ7.20(d,J=8.6Hz,2H),6.86(s,1H),6.60(d,J=11.2Hz,1 H),5.82(dd,J=17.0,10.4Hz,2H),5.16(m,4H),4.57(s,2H),3.89(d,J=4.7Hz,4H).

[0051] Step 11, N,N-diallyl-3-bromo-4-(2-bromo-4-(methylamino)benzyl)aniline (C5)

[0052] C4 (2 g, 7.08 mmol) and 3-bromo-1-N,N-dimethylaniline (1.42 g, 7.08 mmol) were added to a flask and dissolved in 30 mL of ultra-dry dichloromethane. Under nitrogen, 1 mL of boron trifluoride etherate was added. The reaction was allowed to react at room temperature for 24 hours. Saturated sodium bicarbonate solution was added to terminate the reaction, and the product was extracted with dichloromethane. After drying over anhydrous sodium sulfate, the product was purified by flash column chromatography to obtain C5 in a yield of 350 mg (34%). 1 H NMR (400MHz, 298K, CDCl3) δ 6.98 (m, 4H), 6.59 (m, 2H), 5.85 (m, 2H), 5.18 (m, 4H), 4.01 (d, J = 5.1Hz, 2H), 3.89 (d, J = 3.2Hz, 4H), 2.93 (d, 6H).

[0053] Step 12, N3,N3-diallyl-N7,N7,5,5-tetramethyl-5,10-dihydrodibenzo[b,e]silyl-3,7-diamine (C6)

[0054] C5 (1 g, 2.76 mmol) was added to a double-necked flask under nitrogen protection. 8 mL of anhydrous tetrahydrofuran was added with a syringe. Stir at -78 ° C for 20 minutes, slowly add 5.5 mL of sec-butyl lithium and continue stirring for 30 minutes. Then 0.5 mL of dichlorodimethylsilane was added. The reaction was stirred continuously at room temperature for 2 hours. The reaction was quenched with 2N aqueous hydrochloric acid solution. The reaction solution was neutralized with saturated aqueous sodium bicarbonate solution. The organic phase was extracted with dichloromethane. After drying over anhydrous sodium sulfate, C6 was collected by vacuum concentration and directly proceeded to the next step.

[0055] Step 13, 3-(diallylamino)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silanyl-10(5H)-one (C7)

[0056] C6 and potassium permanganate (1.15 g, 7 mmol) were added to a flask, and 10 mL of acetone was added. The reaction was allowed to proceed overnight in an ice-water bath under nitrogen. Filtered through celite, the solid was rinsed with ethyl acetate, and the filtrate was collected. After drying over anhydrous sodium sulfate, the product was purified by flash column chromatography to obtain compound C7 (20%). 1 H NMR(400MHz,298K,CDCl3)δ8.40(dd,J=11.1,9.3Hz,2H),6.82(m,4H),5.89(dd,J =17.6,9.9Hz,2H),5.21(m,4H),4.03(d,J=4.7Hz,4H),3.08(s,6H),0.47(s,6H).

[0057] Step 14, 3-amino-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silan-10(5H)-one (C8).

[0058] C7 (1 g, 2.7 mmol) and 1,3-dimethylbarbituric acid (0.86 g, 5.4 mmol) were added to a flask and dissolved in 35 mL of ultra-dry dichloromethane. The mixture was purged with nitrogen and allowed to react overnight at room temperature. The reaction was terminated by adding 30 mL of saturated aqueous sodium carbonate. The organic phase was extracted with dichloromethane, dried over anhydrous sodium sulfate, and purified by flash column chromatography to yield compound C8. 1 H NMR (400MHz, 298K, CDCl3) δ8.36 (dd, J=22.7, 8.8Hz, 2H), 6.79 (m, 4H), 3.09 (s, 6H), 0.44 (s, 6H).

[0059] Step 15, 3-(dimethylamino)-7-hydroxy-5,5-dimethyldibenzo[b,e]silanyl-10(5H)-one (C9)

[0060] C8 (258 mg, 0.87 mmol) was dissolved in 10 mL of methanol and 10 mL of 6 N sulfuric acid solution. The mixture was placed in an ice-water bath and stirred, and sodium nitrite (600 mg, 8.7 mmol) dissolved in 4 mL of pure water was added dropwise. The reaction mixture was stirred in an ice-water bath under nitrogen for 1 hour, and then 100 mL of 1 N sulfuric acid solution was added dropwise, and stirring continued for 10 minutes. After the reaction reached ambient temperature, the reaction mixture was extracted with dichloromethane. The organic phase was washed with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate, and purified by flash column chromatography to obtain C9 in an output of 81 mg, 31%. 1H NMR (400MHz, 298K, CDCl3) δ8.40(t,J=8.9Hz,2H),7.09(d,J=2.7Hz,1H),7.02(d,J= 2.7Hz,1H),6.83(d,J=2.8Hz,1H),6.78(d,J=2.8Hz,1H),3.11(s,6H),0.44(s,6H).

[0061] Step 16, 3-((tert-Butyldimethylsilyl)oxy)-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silan-10(5H)-one (C15).

[0062] C9 (77 mg, 0.26 mmol), imidazole (176 mg, 2.6 mmol) and tert-butyldimethylsilyl chloride (390 mg, 2.6 mmol) were added to a flask and dissolved in 25 mL of ultra-dry dichloromethane. The mixture was reacted at room temperature for 3 hours under nitrogen protection. Saturated sodium bicarbonate solution was added to terminate the reaction. The organic phase was extracted with dichloromethane. After drying over anhydrous sodium sulfate, the mixture was purified by flash column chromatography to obtain a yellow oily compound C15 in a yield of 53 mg, 50%. 1 H NMR(400MHz,298K,CDCl3)δ8.38(dd,J=8.9,4.6Hz,1H),7.04(d,J=2.6Hz,1H),6.98(d,J=2.6Hz,1H ),6.84(d,J=2.8Hz,1H),6.78(d,J=2.8Hz,1H),3.10(s,6H),1.01(s,9H),0.46(s,6H),0.26(s,6H).

[0063] Step 17, 3-((tert-butyldimethylsilyl)oxy)-7-(dimethylamino)-5,5-dimethyl-3'H,5H-spiro[dibenzo[b,e]silyl-10,1'-isobenzofuran]-3'-one (C16)

[0064] Tert-butyl 2-bromobenzoate (309 mg, 1.5 mmol) was added to a double-necked flask and dissolved in 2 mL of ultra-dry tetrahydrofuran. Stirred at -15 ° C for 10 minutes under nitrogen protection, then iPrMgCl-LiCl (1.3 M dissolved in tetrahydrofuran, 924 μL, 1.2 mmol) was added. Gradually warmed to -5 ° C. C15 (400 mg, 1 mmol) was added to another flask, dissolved in 2 mL of ultra-dry tetrahydrofuran, and protected with nitrogen. The C15 solution was added dropwise to the double-necked flask with a syringe. After stirring for 10 minutes, the reaction was warmed to room temperature and stirred for 30 minutes. Saturated ammonium chloride solution was added to terminate the reaction. The organic phase was extracted with ethyl acetate. After drying over anhydrous sodium sulfate, it was purified by flash column chromatography to obtain oily compound C16 with a yield of 100 mg, 20%. Proceed directly to the next step.

[0065] Step 18, 3-(dimethylamino)-7-hydroxy-5,5-dimethyl-3'H,5H-spiro[dibenzo[b,e]sila-10,1'-isobenzofuran]-3'-one (C17).

[0066] C16 (0.015 mmol, 13 mg) was added to a flask and dissolved in 0.5 mL of tetrahydrofuran (0.5 mL). Tetrabutylammonium fluoride (1.0 M in tetrahydrofuran, 0.045 mmol, 45 μL) was added with stirring at room temperature. After 1 hour, the solvent was removed by rotary evaporation and purified by flash column chromatography to obtain C17 (6.5 mg, 67%). 1 H NMR (400MHz, 298K, CDCl3): δ7.98(d,J=7.6Hz,1H),7.66(m,1H),7.55(t,J=8.0Hz,1H),7.30(d,J=7.7Hz,1H),7.14(d,J=2.8Hz,2H),6.95 (d,J=2.9Hz,2H),6.81(d,J=8.9Hz,2H),6.71(d,J=8.7Hz,2H),6.63(d,J=2.8Hz,1H),6.55(m,2H),2.96(s,9H),0.59(s,3H),0.51(s,3H). 13 C NMR (101MHz, 298K, CDCl3): δ171.57,155.53,154.38,149.48,138.34,136.78,136.18,133.64,130.58,129.50,128.6 7,128.30,126.61,125.94,125.09,120.02,117.25,116.72,113.50,92.11,78.71,77.14,76.82,40.38,0.23,-1.52.

[0067] Step 19, 3-(dimethylamino)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silan-10,1'-isobenzofuran]-7-yl(4-nitrophenyl)carbonate (C18)

[0068] Compound C17 (20 mg, 0.05 mmol) was added to a flask and dissolved in 2 mL of anhydrous dichloromethane. While stirring in an ice-water bath, 0.75 mmol of pyridine and p-nitrophenyl chloroformate (20 mg, 0.1 mmol) were added. The reaction mixture was allowed to warm to room temperature and stirred for 24 hours. The reaction mixture was then diluted with 10 mL of dichloromethane, washed sequentially with saturated sodium bicarbonate and saturated sodium chloride aqueous solutions, and dried over anhydrous sodium sulfate. Purification by flash column chromatography afforded C18 as a white solid in a yield of 11.3 mg (40%). 1 H NMR (500MHz, 298K, CDCl3) δ8.31(d,J=9.2Hz,1H),8.07(s,1H),8.00(d,J=6.7Hz,1H),7.71(s,2H),7.59(d,J=7.0Hz,2H),7.48(d,J=9.2Hz,1H ),7.36(d,J=7.6Hz,1H),7.05(d,J=8.8Hz,1H),6.99(s,1H),6.87(d,J=6.9Hz,1H),6.59(d,J=6.1Hz,1H),2.98(s,6H),0.66(d,J=17.7Hz,6H).

[0069] Step 20, 3-(dimethylamino)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silan-10,1'-isobenzofuran]-7-ylmethyl(2-(methyl(2-phenylcyclopropyl)amino)ethyl)carbamate (LP1)

[0070] Compound 1-6 (0.085 mmol, 24 mg) was dissolved in 3 mL of ultra-dry dichloromethane. Compound C18 (30 mg, 0.053 mmol) and triethylamine (0.24 mmol, 2 mL) dissolved in 2 mL of ultra-dry dichloromethane were added dropwise under stirring in an ice-water bath. The mixture was then left at room temperature and stirred for 12 hours. The organic solvent was removed using a rotary evaporator and the mixture was purified by flash column chromatography to obtain LP1 as a white solid in a yield of 16 mg (30%). 1H NMR(500MHz,298K,DMSO-d6)δ7.99(d,J=7.6Hz,1H),7.82(s,1H),7.69(t,J=7.5Hz, 1H),7.49(s,1H),7.36(t,J=6.7Hz,1H),7.22(t,J=7.5Hz,1H),7.18(t,J=7.4Hz,1H) ,7.06(m,5H),6.91(d,J=8.8Hz,1H),6.68(m,2H),3.51(s,2H),3.03(s,2H),2.92(s ,7H),2.74(s,2H),2.35(s,3H),1.91(s,2H),1.01(s,2H),0.64(s,3H),0.55(s,3H). 13 C NMR(126MHz,298K,DMSO-d6)δ169.16,154.22,151.24,149.43,141.48,137.89,136.24,135.42,130 .67,130.09,128.65,128.20,127.15,126.93,126.19,125.92,125.49,124.88,123.96,123.76,117. 01,114.18,91.17,55.20,54.12,49.78,47.41,42.16,40.49,40.32,40.15,39.99,39.82,39.65,39 .49,35.59,35.23,31.39,29.59,29.29,29.17,27.04,24.85,21.42,17.53,14.42,0.77,-1.06.HRMS of LP1calculate[LP1+Na + ]=654.2764,found:654.2758.

[0071] See also Figure 1 LP1 specifically reacts with lysine-specific demethylase 1 to generate the corresponding C17. In vitro, LP1 is added to a buffer solution containing recombinant human lysine-specific demethylase 1 and reacted for a certain period of time to generate C17, thereby detecting the activity of recombinant human lysine-specific demethylase 1. Cells are cultured in cell culture medium supplemented with LP1. LP1 enters the cells and reacts with lysine-specific demethylase 1 in the cells to generate C17, which can be used to detect the expression and activity of lysine-specific demethylase 1 in the cells. At the same time, the subcellular organelle localization of the C17 fluorescence signal is detected using structured light illumination super-resolution fluorescence microscopy, which can detect the subcellular organelle distribution of lysine-specific demethylase 1.

[0072] Example 2

[0073] Emission spectrum test of LP1 and C17

[0074] Prepare 1 mL of LP1 and C17 solution system, including pH=7.4 PBS buffer, LP1 (0.5 μM), C17 (0.5 μM), and perform fluorescence scanning detection (Ex=580 nm). C17 shows a strong fluorescence signal, while LP1 has no obvious fluorescence signal. Figure 2 .

[0075] Example 3

[0076] Response test of LP1 to lysine-specific demethylase 1

[0077] Prepare 200 μL of LSD1 reaction system, including PBS buffer (pH = 7.4), LSD1 (10 μg / mL), LSD1 inhibitor (PCPA, 200 μM), and LP1 (0.5 μM). Pre-incubate at 37°C with shaking for 1 hour. After 1 hour of reaction, perform fluorescence scanning (Ex = 580 nm). LP1 and LSD1 react to produce enhanced fluorescence. In the control group with the addition of LSD1 inhibitor, there is almost no increase in fluorescence. Figure 3 .

[0078] Example 4

[0079] Response of LP1 to concentration gradient of lysine-specific demethylase 1

[0080] (1) Prepare 200 μL of LSD1 reaction system, including PBS buffer (pH = 7.4), LSD1 (0-11 μg / mL), and LP1 (0.5 μM), and pre-incubate at 37°C with shaking for 1 hour.

[0081] (2) After 1 hour of reaction, fluorescence scanning detection (Ex = 580 nm) was performed. The fluorescence intensity increased with the increase of the concentration of lysine-specific demethylase 1. Figure 4 .

[0082] Example 5

[0083] Selectivity test of LP1 for lysine-specific demethylase 1

[0084] Prepare 200 μL of reaction systems with different interfering factors, including PBS buffer (pH = 7.4) and LP1 (0.5 μM), and pre-incubate at 37°C with shaking for 1 hour. After 1 hour of reaction, measure the fluorescence intensity at 630 nm (Ex = 580 nm). LP1 and LSD1 react to produce fluorescence enhancement, while the reaction system with LP1 and other interfering factors shows almost no fluorescence increase. Figure 5 .

[0085] Example 6

[0086] Detection of Lysine-Specific Demethylase 1 Activity in Cell Lysates

[0087] (1) U87 cells were seeded in a 10-cm culture dish in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin, and 100 IU / mL penicillin. When the cell density reached approximately 90%, the culture dish was completely drained of the medium, and the dish was washed once with PBS buffer (pH = 7.4). 1 mL of PBS buffer (pH = 7.4) was then added, and the cells were broken with a cell scraper. The cell lysate was then collected and centrifuged at 4°C, 12,000 rpm for 10 minutes. The supernatant was collected to obtain a clarified cell lysate.

[0088] (2) Determination of total protein concentration in lysate

[0089] The total protein concentration of the lysate was determined using a BCA protein assay kit.

[0090] (3) Prepare 200 μL of cell lysate reaction system, including PBS buffer (pH = 7.4), cell lysate (total protein concentration 1000 μg / mL), LP1 (10 μM), and pre-incubate at 37°C with shaking for 1 hour.

[0091] (4) After 1 hour of reaction, the fluorescence intensity was detected (Ex = 580 nm, Em = 630 nm). Figure 6 .

[0092] Example 7

[0093] LP1 cytotoxicity test

[0094] U87 cells and SH-SY5Y cells were seeded into 96-well plates in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin, and 100 IU / mL penicillin. When the cell density reached approximately 60%, the medium in the 96-well plate was aspirated and various concentrations of LP1 (0-100 μM) were dissolved in DMEM and added to the 96-well plate. After 24 hours of incubation, cell viability was tested using a CCK8 cell viability kit (see

[15] ). Figure 7 .

[0095] Example 8

[0096] Detection of LP1 activity on lysine-specific demethylase 1 in living cells

[0097] U87 cells were seeded into confocal microplates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin, and 100 IU / mL penicillin. After the cells were observed to have fully adhered, the medium was aspirated and medium containing LP1 (10 μM) was added to the culture dish and incubated for 3 hours. Confocal microscopy imaging (Ex = 561 nm) revealed that the fluorescence intensity of the U87 cells in the control group, which had been incubated for 30 minutes with the addition of the LSD1 inhibitor, was lower than that of the U87 cells without the addition of the LSD1 inhibitor. Figure 8 .

[0098] Example 9

[0099] Super-resolution imaging of LP1 and U87 cells after 3 hours of co-incubation

[0100] U87 cells were seeded into 4 confocal culture dishes. The culture medium was Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin and 100 IU / mL penicillin. After observing that the cells were completely attached, the culture medium was aspirated and the culture medium containing LP1 (10 μM) was added to the culture dish and incubated for 3 hours. Then, mitochondria-targeted commercial dyes, lysosome-targeted commercial dyes, endoplasmic reticulum-targeted commercial dyes and Golgi apparatus-targeted commercial dyes were added according to the instructions of the commercial dyes and incubated for 30 minutes. Structured light illumination fluorescence microscopy was used for imaging (LP1 excitation light 561 nm, commercial dye excitation light 488 nm). The imaging results showed that the fluorescence signal of C17, the product of the reaction between LP1 and LSD1, highly overlapped with the signal of the Golgi apparatus commercial dye, as shown in Figure 2. Figure 9 .

Claims

1. A specific super-resolution fluorescent probe for lysine-specific demethylase 1, characterized in that: The structure is as follows:

2. A specific super-resolution fluorescent probe for lysine-specific demethylase 1 according to claim 1, characterized in that The synthetic route is as follows:

3. Use of a specific super-resolution fluorescent probe for lysine-specific demethylase 1 according to claim 1 or 2, characterized in that: Used to prepare reagents for detecting the activity of human recombinant lysine-specific demethylase 1 protein and lysine-specific demethylase 1 protein in cell lysates.

4. Use of a specific super-resolution fluorescent probe for lysine-specific demethylase 1 according to claim 1 or 2, characterized in that: Used to prepare reagents for detecting intracellular lysine-specific demethylase 1.

Images