Specific super-resolution fluorescent probe of lysine specific demethylase 1 and application of specific super-resolution fluorescent probe

Abstract

The invention discloses a specific super-resolution fluorescent probe of lysine specific demethylase 1 and application of the specific super-resolution fluorescent probe, and relates to the field of molecular probes. According to the designed and synthesized specific micromolecular fluorescent probe LP1 of the lysine specific demethylase 1, the probe is designed on the basis of an active cavity configuration of the lysine specific demethylase 1 and a molecular structure of a specific inhibitor of the lysine specific demethylase 1, and the probe has excellent light stability, pH stability and anti-interference capability on ions and amino acids in cells. A product released after the reaction of the probe LP1 and the lysine specific demethylase 1 has an excellent near-infrared fluorescence signal. The probe LP1 successfully and specifically detects the activity of the lysine specific demethylase 1 in different biological samples (pure protein, cell lysis buffer and living cells), and detects the subcellular organelle positioning of the 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) can specifically remove the mono- and di-methyl groups of K4 / K9 on histone H3, regulate gene expression, and directly interact with related proteins, playing an important 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. The selective high expression in tumor tissues makes LSD1 the focus of tumor targeted therapy. Therefore, the specific detection of LSD1 activity and subcellular organelle localization are of great significance for the study of 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 the detection of biomarkers. However, there is no report on specific fluorescent probes for LSD1. At the same time, super-resolution fluorescent probes combined with structured light illumination fluorescence microscopy can break through the diffraction limit and observe more detailed subcellular structures. Therefore, designing and synthesizing specific super-resolution fluorescent probes for LSD1 is very important for further analyzing its biological functions. Summary of the invention

[0004] The purpose of the present invention is to solve the above-mentioned problems in the prior art, and to provide a specific super-resolution fluorescent probe LP1 of lysine-specific demethylase 1 (LSD1) and its application. The fluorescent properties of the product after the reaction between the probe and LSD1 are significantly different, and the product is easy to detect. Using the probe reaction, the activity of LSD1 in a variety of biological samples can be detected and the distribution of lysine-specific demethylase 1 in subcellular organelles can be traced.

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

[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 of 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 determined 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 0.5μM): LP1 reacts specifically with lysine-specific demethylase 1 to generate the corresponding C17, which has good fluorescence emission spectrum characteristics (620-750nm). The substrate is non-fluorescent, and the product has good fluorescence properties, which can be well distinguished and detected, and can be quantitatively determined by drawing a standard curve. The product also has high brightness and near-infrared properties, which can reduce biological background fluorescence and is suitable for structured light illumination super-resolution imaging technology. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 to 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 The activity of lysine-specific demethylase 1 in cell lysates was detected for LP1;

[0023] Figure 7 CCK8 test for LP1 cytotoxicity;

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

[0025] Fig. 9 This is a super-resolution imaging experiment after LP1 and U87 cells were co-incubated 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 in conjunction with 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] N-methyl-2-hydroxyethylamine (1.9 g, 25.3 mmol) and aluminum oxide (3.876 g, 38 mmol) were added to a round-bottom flask. Di-tert-butyl dicarbonate (5.28 g, 27.83 mmol) was subsequently added. Stir 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 with a 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-bottomed flask and dissolved with 8.5 mL of ultra-dry dichloromethane and protected with nitrogen. Stirred in an ice-water bath, and Dess-Martin periodinane (762 mg, 1.8 mmol) was slowly added dropwise. Then 50 mL of sodium bicarbonate and 50 mL of a saturated solution of sodium thiosulfate were 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), and purified by flash column chromatography to obtain a colorless oily compound L3 with a yield of 241 mg, 81%. 1 H-NMR (500MHz, 298K, CDCl 3 ): 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 brine, 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. Tert-butyl dicarbonate (8.2g, 37.5mmol) is dissolved in 15mL of ultra-dry dichloromethane and added dropwise to a flask. The reaction is continued at room temperature for 12 hours, and then purified by flash column chromatography to obtain white solid compound 1-2, 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. Then sodium hydride (692 mg, purity 95%) was 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 colorless oily compound 1-3 with a yield of 2.43 g, 54%. 1 H-NMR (500MHz, 298K, CDCl 3 ): δ7.29(d,J=5.0Hz,2H),7.2(t,J=5.0Hz,1H),7.13(d,J=5.0Hz,2H),2.92(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 sodium hydroxide aqueous solution, and then extracted with dichloromethane (3×20 mL). The organic phase was washed with brine and dried over anhydrous sodium sulfate. Compound 1-4 was purified by flash column chromatography to obtain a brown oily compound with a yield of 1.36 g, 94%. 1 H-NMR (500MHz, 298K, CDCl 3 ): δ7.29(d,J=5.0Hz,2H),7.2(t,J=5.0Hz,1H),7.13(d,J=5.0Hz,2H),2.92(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. Then sodium triacetoxyborohydride (1.13 g, 5.35 mmol) was added to the flask and continued to stir at room temperature for 3 hours. The reaction was terminated with 20 mL of sodium bicarbonate, the organic phase was extracted with dichloromethane (3 × 20 mL), the organic phase was washed with a saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Purification by flash column chromatography gave a light yellow oily compound 1-5 with a yield of 720 mg, 64%. 1 H-NMR (500MHz, 298K, CDCl 3 ): 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 a pale white solid compound 1-6 hydrochloride. 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, and then 80 mL of acetonitrile was added and 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, it was purified by flash column chromatography to obtain a yellow oily compound C2 with a yield of 14 g, 90%. 1 H NMR (400 MHz, 298 K, CDCl 3 )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) 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. Add 1N sodium hydroxide solution to the ice-water bath to terminate the reaction. 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 (400 MHz, 298 K, CDCl 3 )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, 30 mL of methanol and 30 mL of dichloromethane were added to dissolve, and sodium borohydride (150 mmol) was dissolved in 20 mL of dichloromethane and added dropwise to the flask. The reaction was allowed to react for 24 hours at room temperature. 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 with a yield of 11 g, 80%. 1 H NMR (400 MHz, 298 K, CDCl 3 )δ7.20(d,J=8.6Hz,2H),6.86(s,1H),6.60(d,J=11.2Hz,1H),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, dissolved in 30 mL of ultra-dry dichloromethane, and 1 mL of boron trifluoride ether was added under nitrogen protection. The reaction was allowed to react at room temperature for 24 hours. Saturated sodium bicarbonate solution was added to terminate the reaction, and the mixture was extracted with dichloromethane. After drying over anhydrous sodium sulfate, the mixture was purified by flash column chromatography to obtain C5 with a yield of 350 mg, 34%. 1 H NMR (400 MHz, 298 K, CDCl 3 )δ6.98(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. The reaction solution was neutralized with saturated aqueous sodium bicarbonate. 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 protection. The solid was washed with ethyl acetate and the filtrate was collected. After drying over anhydrous sodium sulfate, the compound was purified by flash column chromatography to obtain compound C7 (20%). 1 H NMR (400 MHz, 298 K, CDCl 3 )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 with 35 mL of ultra-dry dichloromethane. After nitrogen protection, the mixture was reacted at room temperature overnight. 30 mL of saturated sodium carbonate aqueous solution was added to terminate the reaction. The organic phase was extracted with dichloromethane, dried over anhydrous sodium sulfate, and purified by flash column chromatography to obtain compound C8. 1 H NMR (400 MHz, 298 K, CDCl 3 )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 (258mg, 0.87mmol) was dissolved in 10mL of methanol and 10mL of 6N sulfuric acid solution. Placed in an ice-water bath and stirred, and sodium nitrite (600mg, 8.7mmol) dissolved in 4mL of pure water was added dropwise. The reaction mixture was stirred in an ice-water bath for 1 hour under nitrogen protection, and then 100mL of 1N sulfuric acid solution was added dropwise, and stirring continued for 10 minutes. After reacting to ambient temperature, the reaction mixture was extracted with dichloromethane. The organic phase was washed with saturated sodium chloride aqueous solution and then dried over anhydrous sodium sulfate, and purified by flash column chromatography to obtain C9, with a yield of 81mg, 31%. 1 H NMR (400 MHz, 298 K, CDCl 3 )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 25 mL of ultra-dry dichloromethane was added to dissolve. The reaction was carried out 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, it was purified by flash column chromatography to obtain a yellow oily compound C15 with a yield of 53 mg, 50%. 1 H NMR (400 MHz, 298 K, CDCl 3 )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]silanyl-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, and 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 the flask and dissolved with 0.5 mL of tetrahydrofuran (0.5 mL). Tetrabutylammonium fluoride (1.0 M in tetrahydrofuran, 0.045 mmol, 45 μL) was added under stirring at room temperature. After 1 hour, the solvent was removed by rotary evaporator and purified by flash column chromatography to obtain C17 (6.5 mg, 67%). 1 H NMR (400 MHz, 298 K, CDCl 3 ): 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). 13C 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 with 2 mL of anhydrous dichloromethane. Under stirring in an ice-water bath, 0.75 mmol of pyridine and p-nitrophenyl chloroformate (20 mg, 0.1 mmol) were added. The reaction was warmed to room temperature and stirred for 24 hours. The reaction mixture was then diluted with 10 mL of dichloromethane, washed with saturated sodium bicarbonate and saturated sodium chloride aqueous solution in turn, and dried over anhydrous sodium sulfate. Purification by flash column chromatography gave C18 as a white solid with 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 (0.085mmol, 24mg) was dissolved in 3mL of ultra-dry dichloromethane, and compound C18 (30mg, 0.053mmol) and triethylamine (0.24mmol, 2mL) dissolved in 2mL of ultra-dry dichloromethane were added dropwise under stirring in an ice-water bath. The mixture was then placed at room temperature and stirred for 12 hours. After removing the organic solvent with a rotary evaporator, it was purified by flash column chromatography to obtain a white solid LP1 with a yield of 16mg, 30%. 1 H 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 1LP1 reacts specifically 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 a 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 detect the expression and activity of lysine-specific demethylase 1 in the cells. At the same time, the subcellular organelle localization of the fluorescence signal of C17 is detected by structured light illumination super-resolution fluorescence microscopy, and the subcellular organelle distribution of lysine-specific demethylase 1 can be detected.

[0072] Example 2

[0073] Emission spectrum test of LP1 and C17

[0074] 1 mL of LP1 and C17 solution systems were prepared, including pH = 7.4 PBS buffer, LP1 (0.5 μM), and C17 (0.5 μM), and fluorescence scanning detection (Ex = 580 nm) was performed. C17 showed a strong fluorescence signal, while LP1 had no obvious fluorescence signal. Figure 2 .

[0075] Example 3

[0076] Testing the responsiveness of LP1 to lysine-specific demethylase 1

[0077] Prepare 200 μL of LSD1 reaction system, including PBS buffer at pH = 7.4, LSD1 (10 μg / mL), LSD1 inhibitor (PCPA, 200 μM), LP1 (0.5 μM), and pre-incubate at 37°C for 1 hour with shaking. After 1 hour of reaction, perform fluorescence scanning detection (Ex = 580 nm). LP1 and LSD1 respond to produce fluorescence enhancement, while the control group with LSD1 inhibitor has almost no fluorescence increase. 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 at pH = 7.4, LP1 (0.5 μM), and pre-incubate at 37°C for 1 hour with shaking. After 1 hour of reaction, measure the fluorescence intensity at 630 nm (Ex = 580 nm). LP1 and LSD1 respond to produce fluorescence enhancement, while the reaction system with LP1 and other interfering factors has 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 inoculated in a 10 cm culture dish in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin and 100 IU / mL penicillin. When the cell density was about 90%, the culture dish was emptied of the culture medium, and the dish was washed once with PBS buffer (pH=7.4), and then 1 mL of PBS buffer (pH=7.4) was added, and the cells were broken with a cell scraper, and then the cell lysate was collected and centrifuged at 4°C and 12000 rpm for 10 minutes, and the supernatant was taken 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 the BCA protein concentration 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 were inoculated into 96-well plates in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin and 100 IU / mL penicillin. When the cell density was about 60%, the medium in the 96-well plate was aspirated, and different concentrations of LP1 (0-100 μM) were dissolved in DMEM and added to the 96-well plate. After incubation for 24 hours, the cell viability was tested using the CCK8 cell viability kit, see Figure 7 .

[0095] Example 8

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

[0097] U87 cells were inoculated into confocal culture dishes in 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 to the wall, the culture medium was aspirated and the culture medium containing LP1 (10 μM) was added to the culture dish and incubated for 3 hours. Using confocal microscopy imaging (Ex = 561 nm), the imaging results showed that the fluorescence intensity of the control group U87 cells incubated for 30 minutes with the addition of LSD1 inhibitor in advance was lower than the fluorescence produced by the U87 cells without the addition of LSD1 inhibitor, as shown in Figure 2. 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 to the wall, 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. Imaging was performed using a structured light illumination fluorescence microscope (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. Fig. 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 detect the activity of human recombinant lysine-specific demethylase 1 protein and lysine-specific demethylase 1 protein in cell lysate.

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 detect intracellular lysine-specific demethylase 1.

5. The use of a specific super-resolution fluorescent probe for lysine-specific demethylase 1 as claimed in claim 4, characterized in that: 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 determined by quantitatively detecting the fluorescence signal of the product per unit time.

Images