Two-photon fluorescent probe and application thereof in sulfatase detection

Abstract

The invention discloses a two-photon fluorescent probe and application thereof in sulfatase detection. The structure of the endoplasmic reticulum targeting two-photon fluorescent probe is shown as follows: # imgabs0 #. The endoplasmic reticulum targeting two-photon fluorescent probe responds to sulfatase with different concentrations, the detection limit is as low as 0.12 U / L, and the two-photon absorption cross section is 81 GM. A cytotoxicity test shows that the probe is relatively low in biotoxicity. Confocal fluorescence microscopic imaging shows that the probe can effectively target the endoplasmic reticulum (the positioning coefficient is 0.86) in HeLa cells, and is suitable for fluorescence imaging detection of sulfatase in the endoplasmic reticulum of living cells.

Description

Technical Field

[0001] The present invention relates to a two-photon fluorescent probe and its application in sulfatase detection, so as to realize the characteristics of fluorescence imaging of sulfatase in solution and cells and endoplasmic reticulum fluorescence tracing, and has the advantages of selectivity, high detection efficiency and good biocompatibility. Background Art

[0002] The endoplasmic reticulum (ER) is a vesicular, vesicular and tubular structure formed by a layer of unit membrane, forming a continuous reticular system. It is the main site for the synthesis of intracellular proteins, lipids and carbohydrates. It regulates various physiological processes in human cells, including gluconeogenesis metabolism, protein processing, and lipid biosynthesis. ER stress can induce abnormal accumulation of unfolded proteins, leading to various diseases, including endocrine disorders, diabetes, neurodegeneration, stroke and even cancer. Therefore, in-depth research on the biological characteristics and functions of the endoplasmic reticulum is of great significance for understanding and treating these diseases.

[0003] Sulfatase is a general term for enzymes that free inorganic sulfuric acid from organic sulfate. It is mainly found in lysosomes, endoplasmic reticulum and Golgi apparatus, and participates in physiological processes such as cell signal transduction and hormone regulation. It can catalyze the esterification reaction between sulfate groups and other molecules and transfer sulfate groups to other molecules. Sulfatase is mainly involved in the metabolism of chemical substances. It can accelerate the dissociation reaction of sulfate esters and hydrolyze sulfate esters into sulfuric acid and corresponding alcohols or phenols. This reaction is very important because it can make many organic molecules more hydrophilic and easier to be absorbed by cells, so as to better participate in the metabolic process. In medicine, sulfatase is used to treat metabolic diseases, such as metabolic disorders and genetic diseases. Overexpression of sulfatase can lead to increased estrogen levels in organisms, accelerate cell growth, and induce hormone-dependent cancers. Therefore, the accurate detection of sulfatase is of great significance for exploring the physiological functions of sulfatase and diagnosing related diseases. So far, several detection methods for sulfatase have been reported, including enzyme-linked immunosorbent assay, colorimetry, flow cytometry, and fluorescent probe technology. Compared with other conventional methods, bioimaging techniques based on fluorescent probes are becoming increasingly attractive due to their higher selectivity, sensitivity, less invasiveness, and ease of operation.

[0004] In recent years, fluorescence imaging methods have been widely used to monitor biological processes, but there are still very few specific imaging of sulfatases targeting the endoplasmic reticulum, especially fluorescent probes with two-photon performance. Therefore, the development of two-photon fluorescent probes that can respond to sulfatases will provide a powerful tool for real-time imaging studies of intracellular sulfatases fluctuations. Summary of the invention

[0005] The present invention aims at the deficiencies of the above-mentioned prior art and provides a two-photon fluorescent probe for endoplasmic reticulum targeted detection of sulfatase and its preparation method and application. The technical problem to be solved is to obtain a molecular structure of two-photon response to sulfatase and endoplasmic reticulum fluorescence tracing through molecular design, requiring that the molecular structure not only has the ability of endoplasmic reticulum tracing, but also has a strong fluorescence signal after responding to sulfatase.

[0006] In the present invention, a two-photon fluorescent probe SE-SA for endoplasmic reticulum-targeted detection of sulfatase is designed and synthesized with naphthalene as a two-photon fluorophore, p-toluenesulfonamide as an endoplasmic reticulum targeting group, and sulfate as a response group. Hydroxyl sulfation blocks the intramolecular charge transfer (ICT) effect of SE-OH. SE-SA is initially almost non-fluorescent. After reacting with sulfatase, the sulfate part of SE-SA is specifically cleaved and removed, resulting in the release of SE-OH and the recovery of the ICT effect, thereby activating the fluorescence signal. The tests of optical properties and cytotoxicity show that the SE-SA has sulfatase specificity and good biocompatibility. This study evaluated the feasibility of SE-SA for living cell sulfatase-specific fluorescence imaging and endoplasmic reticulum imaging through the analysis of cell confocal fluorescence imaging, aiming to provide a convenient chemical tool for the visualization detection of sulfatase and the study of related diseases.

[0007] The two-photon fluorescent probe of the present invention is abbreviated as SE-SA, and its structure is as follows:

[0008] .

[0009] The preparation method of the two-photon fluorescent probe of the present invention comprises the following steps:

[0010] Step 1: Under ice bath conditions, 3.01 g of 4-toluenesulfonyl chloride and 4.00 g of 3-bromopropylamine hydrobromide were dissolved in 14 mL of anhydrous dichloromethane, and then 4.02 g of triethylamine was added dropwise and stirred for 30 min. After the reaction was completed, the reaction solution was diluted with 150 mL of dichloromethane, and then the pH of the diluent was adjusted to neutral with 2×60 mL of 2M hydrochloric acid. The resulting solution was extracted with dichloromethane, and the organic layers were combined and dried over anhydrous sodium sulfate, distilled under reduced pressure, and separated and purified by column chromatography (eluent: petroleum ether: ethyl acetate = 30:1) to obtain compound 1 as a white oil.

[0011] Step 2: Under nitrogen protection, 0.88 g of compound 1, 0.48 g of p-hydroxyacetophenone and 0.21 g of potassium carbonate were dissolved in 6 mL of N,N-dimethylformamide, and the mixture was heated at 45°C for 12 h. After the reaction was completed, it was cooled to room temperature and 50 mL of 5% cold HCl solution (0-5°C) was added dropwise to produce a white precipitate, which was filtered and rinsed with pure water and air-dried to obtain a crude white powder of compound 2, which was slurried with dichloromethane (10 mL) and ethyl acetate (30 mL) to obtain compound 2;

[0012] Step 3: Under nitrogen protection, 0.35 g of compound 2, 0.07 g of malononitrile and 0.16 g of hexamethyldisilazane were dissolved in 2.5 mL of acetic acid and refluxed with stirring for 8 h. After the reaction was completed, the mixture was cooled to room temperature, 50 mL of water was added to dilute the reaction solution, and then the pH of the dilution solution was adjusted to neutral with sodium carbonate. The resulting solution was extracted with dichloromethane, and the organic layers were combined and dried over anhydrous sodium sulfate, distilled under reduced pressure, and separated and purified by column chromatography (dichloromethane: methanol = 200: 1) to obtain compound 3 as a white solid;

[0013] Step 4: At 20-22°C, 0.26 g of 6-hydroxy-2-naphthaldehyde was dissolved in 3 mL of tetrahydrofuran, and 0.15 g of sodium tert-butoxide was slowly added, stirred for 15 min, and then 0.28 g of solid trimethylammonium trisulfur oxide copolymer was added, and stirring was continued for 1 h. After the reaction was completed, vacuum distillation and column chromatography (eluent: dichloromethane: methanol = 30:1) were used to separate and purify compound 4, which was a light yellow oil;

[0014] Step 5: In N 2 Under the environment, 0.2 g of compound 3 and 0.14 g of compound 4 were dissolved in 4 mL of ethanol, and one drop of piperidine was added. The mixture was stirred at 25 °C for 4 h, and then vacuum distilled and purified by column chromatography (dichloromethane: methanol = 30:1) to obtain compound SE-SA as a yellow solid.

[0015] The synthetic route is as follows:

[0016]

[0017] The invention discloses an application of a two-photon fluorescent probe in sulfatase detection.

[0018] Specifically, the two-photon fluorescent probe is used to prepare a sulfatase detection reagent, thereby realizing the detection of sulfatase.

[0019] The detection reagent can distinguish normal cells from cancer cells and achieve a rapid response of sulfatase in the endoplasmic reticulum of cells.

[0020] The two-photon fluorescent probe for endoplasmic reticulum targeted detection of sulfatase of the present invention blocks the intramolecular charge transfer (ICT) effect of SE-OH by hydroxyl sulfation, and SE-SA initially has weak fluorescence. After reacting with sulfatase, the sulfate part of SE-SA is specifically cleaved and removed, resulting in the generation of SE-OH and the recovery of the ICT effect, thereby activating a strong fluorescent signal. SE-SA has good cell compatibility and has the prospect of being used in cell confocal imaging.

[0021] The detection method is as follows:

[0022] The SE-SA of the present invention was dissolved in DMSO to prepare a 2 mM mother solution, and 15 μL of the mother solution was taken in 3 mL of a mixed solvent of PBS and DMSO containing different concentrations of sulfatase (PBS: DMSO = 9: 1), and UV-vis absorption spectra and fluorescence emission spectra were obtained to detect the spectral properties of SE-SA. After adding sulfatase (the sulfatase with CAS number: 9016-17-5 was used in the detection of this patent) to the reaction system, the absorbance of SE-SA shifted from 390 nm to 440 nm as the concentration of sulfatase increased. The fluorescence red-shifted from 520 nm to 580 nm, and the fluorescence intensity gradually increased. In order to further study the response of SE-SA to sulfatase, we measured the time course of the fluorescence response of SE-SA to sulfatase by fluorescence spectroscopy. After adding 150 U / L of sulfatase, the fluorescence intensity reached a stable state at 40 min.

[0023] The sulfatase fluorescent probe provided by the present invention can detect sulfatase with high selectivity, an in vitro response time of 40 minutes, and a detection limit of 0.12 U / L. Cytotoxicity tests showed that SE-SA had good biocompatibility. Confocal fluorescence microscopy experiments showed that SE-SA had good photostability for HeLa cells and had the ability to monitor intracellular concentration changes. On this basis, different concentrations of 6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c][1]benzopyran-3-yl sulfamate (STX-64, a steroid sulfatase inhibitor) were co-incubated with 150 U / L of sulfatase and then added to SE-SA. The fluorescence intensity at 580 nm gradually weakened, further proving the rapid response of SE-SA to sulfatase. SE-SA also had the ability to image the endoplasmic reticulum (Pr=0.86), and was suitable for confocal fluorescence imaging of endoplasmic reticulum tracing in cells. In addition, given the low toxicity and biocompatibility of the probe, it has great application potential in living models. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 (A) Possible mechanism of probe SE-SA responding to sulfatase; Figure 1 (B) UV-visible absorption spectra of SE-SA (10 μM) before and after the reaction with sulfatase (150 U / L) at 37 °C in PBS:DMSO=9:1. Figure 1 (C) HPLC of SE-SA (100 μM) before and after reaction with sulfatase (150 U / L) at 37°C in PBS:DMSO=9:1.

[0025] Figure 2 (A) Fluorescence intensity of SE-SA (10 μM) changes with the concentration of sulfatase (0-150 U / L) at 37°C and PBS:DMSO=9:1; (B) Detection limit of SE-SA in response to sulfatase; (C) Time dependence of fluorescence intensity of SE-SA (10 μM) at 150 U / L sulfatase; (D) Fluorescence intensity of SE-SA (10 μM) in PBS:DMSO=9:1 to other biological species 1: blank, 2: ClO - , 3:CO 3 2- , 4:S 2- , 5:SO 3 2- , 6:H 2 O 2 , 7:Mg 2+ , 8:NH 4 + , 9:Fe 3 + , 10:Cu 2+ , 11:Hcy, 12:Cys, 13:Glu, 14:GSH, 15:Gly, 16:Tyr, 17:Ser, 18:AchE, 19:GGT, 20:CE, 21:Sulfatase.

[0026] Figure 3 Two-photon absorption cross sections of SE-SA and Sulfatase at different wavelengths.

[0027] Figure 4 Cell viability of HeLa cells treated with different concentrations (0 μM, 5 μM, 10 μM, 15 μM, 20 μM) of SE-SA for 24 h.

[0028] Figure 5(A) Confocal imaging of two tumor cells (A549 cells and HeLa cells) and two normal cells (NTH / 3T3 cells and MH-S cells) by SE-SA (10 μM); the red channel of SE-SA, λ ex =440 nm. Figure 5 (B) Yes Figure 5 (A) Quantification of red channel fluorescence intensity.

[0029] Figure 6 (A) Confocal images of HeLa cells stimulated with 10 μM SE-SA after additional stimulation with different concentrations of sulfatase inhibitor (STX-64) for 40 min; Figure 6 (B) Yes Figure 6 (A) Quantification of red channel fluorescence intensity.

[0030] Figure 7 Co-localization imaging experiment of SE-SA and ER-Green in HeLa cells. DETAILED DESCRIPTION

[0031] The present invention will be further described below by way of examples.

[0032] Example 1: Synthesis of SE-SA

[0033] In N 2 Under the environment, 0.2 g of compound 3 and 0.14 g of compound 4 were dissolved in 4 mL of ethanol, and a drop of piperidine was added, and stirred at room temperature for 4 h, and then the solvent was evaporated under reduced pressure, and then column chromatography (eluent: dichloromethane: methanol = 20:1) was used for separation and purification to obtain compound SE-SA as a yellow solid. (Yield 40%).

[0034] 1 H NMR (400 MHz, DMSO-D 6) δ 8.03 (s, 1H), 7.80 (d, J = 8.9 Hz, 1H), 7.75 (s, 2H), 7.71 (s, 1H), 7.69 (s, 1H), 7.64 (t, J = 5.8 Hz, 1H), 7.54 (d,J = 15.4 Hz, 1H), 7.49 (s, 1H), 7.47 (s, 1H), 7.40 (s, 1H), 7.38 (s, 1H), 7.16 (d, J = 2.3 Hz, 1H), 7.12 (dd, J = 8.8, 2.4 Hz, 1H), 7.10 – 7.04 (m,3H), 4.05 (t, J = 6.1 Hz, 2H), 2.94 (q, J = 6.6 Hz, 2H), 2.37 (s, 3H), 1.87(t, J = 6.5 Hz, 2H). 13 C NMR (151 MHz, DMSO-D 6 ) δ 158.27, 149.91, 143.15,132.70, 131.79, 131.43, 130.19, 127.75, 127.08, 124.34, 123.41, 120.10,115.27, 115.15, 109.74, 79.11, 65.50, 40.07, 29.32, 21.50.

[0035] Example 2: Reaction mechanism verification study

[0036] like Figure 1 As shown in the middle (A) figure, in the presence of sulfatase, SE-SA reacts to SE-OH, and the fluorescence turns on. Figure 1 Figure (B) shows a 2 mM stock solution of SE-SA dissolved in DMSO (5 mL), 15 μL of SE-SA stock solution was added to 3 mL of PBS:DMSO = 9:1, and (150 U / L) sulfatase was added to obtain the UV spectrum. From the UV-visible absorption spectrum, it can be seen that the maximum absorption peak of SE-SA is located at around 390 nm, and after the sulfatase response, the maximum absorption shifts to 440 nm, which is consistent with the UV absorption position of SE-OH. Figure 1 The product generated by the overreaction of SE-SA with sulfatase shown in Figure (C) was confirmed to be SE-OH by high performance liquid chromatography (HPLC).

[0037] Example 3: Spectral study of SE-SA

[0038] The SE-SA of the present invention was dissolved in DMSO (5 mL) to prepare a 2 mM stock solution. 15 μL of the SE-SA stock solution was added to 3 mL of PBS:DMSO = 9:1. Sulfatase of different concentrations (0-150 U / L) was added. The fluorescence spectrum was used to analyze the SE-SA. Figure 2 (A) It can be seen that the fluorescence intensity at 580 nm increases with the increase of sulfatase concentration. Figure 2 As shown in (B), we can see the linear correlation coefficient R 2 =0.9984, and the calculated detection limit was 0.12 U / L. To test the time dependence of SE-SA on sulfatase, we used SE-SA (10 μM) to test the fluorescence response of sulfatase at 150 U / L. Figure 2 As shown in (C), the fluorescence intensity reached a stable level within 40 min.

[0039] Example 4: Two-photon absorption cross sections of SE-SA and Sulfatase at different wavelengths

[0040] Sulfatase (150 U / L) was added to the test solution containing SE-SA (10 mM) and incubated at 37 °C for 40 min. The effective two-photon absorption cross section was measured on a two-photon exciter, and the two-photon absorption cross section (δ) was calculated by measuring the fluorescence quantum yield. It can be found that the maximum two-photon absorption cross section appears at a wavelength of 860 nm, with a value of 81 GM, such as Figure 3 It is shown that SE-SA has good two-photon performance after responding to Sulfatase.

[0041] Example 5: Cytotoxicity studies

[0042] First, we used CCK8 assay to test the biosafety of SE-SA probe at the cellular level. Figure 4 As shown, when the concentration of the probe was less than 20 μM, the cell survival rate was greater than 90%, proving that the probe had low toxicity at the working concentration.

[0043] Example 6: SE-SA imaging of sulfatase levels in cancer cells

[0044] Considering that liver cancer cells express a large amount of sulfatase, we pre-incubated cancer cells (including A549 cells, HeLa cells) and normal cells (including NTH / 3T3 cells, MH-S cells) with the probe SE-SA for 30 minutes. Figure 5 As shown in Figure 2, cancer cells showed obvious red fluorescence signals compared with normal cells. When cancer cells were pre-incubated with different concentrations of the sulfatase inhibitor STX-64 and then incubated with the probe, the results were as follows: Figure 6As shown, the fluorescence signal in cancer cells gradually weakened, further indicating that the fluorescence signal was caused by overexpressed sulfatase.

[0045] Example 7: Co-localization imaging study of SE-SA in the endoplasmic reticulum

[0046] In order to explore the ability of SE-SA to image the endoplasmic reticulum in cells, we used the endoplasmic reticulum dye (ER-TrackerGreen) and SE-SA to perform co-localization studies in HeLa cells. Figure 7 As shown, the fluorescence images of the red channel of SE-SA and the green channel of ER-TrackerGreen overlap well, and the Pearson colocalization coefficient of SE-SA and ER-Tracker Green is calculated to be 0.86. The results show that SE-SA has the ability to trace the endoplasmic reticulum.

Claims

1. A two-photon fluorescent probe, referred to as SE-SA, characterized in that Its structure is as follows: 。 2. The method for preparing the two-photon fluorescent probe according to claim 1, characterized in that The steps include: Step 1: Under ice bath conditions, 4-toluenesulfonyl chloride and 3-bromopropylamine hydrobromide are dissolved in anhydrous dichloromethane, and then triethylamine is added dropwise and stirred for reaction; after the reaction is completed, the reaction solution is diluted with dichloromethane, and then the pH of the diluent is adjusted with hydrochloric acid, the resulting solution is extracted with dichloromethane, the organic layers are combined and dried over anhydrous sodium sulfate, distilled under reduced pressure, and separated and purified by column chromatography to obtain compound 1 as a white oil; Step 2: Under nitrogen protection, compound 1, p-hydroxyacetophenone and potassium carbonate were dissolved in N,N-dimethylformamide, and the mixture was heated at 45°C for 12 h. After the reaction, the mixture was cooled to room temperature, and a 5% HCl cold solution was added dropwise to produce a white precipitate, which was filtered, rinsed with pure water and air-dried to obtain a crude white powder of compound 2, which was slurried with dichloromethane and ethyl acetate to obtain compound 2; Step 3: Under nitrogen protection, compound 2, malononitrile and hexamethyldisilazane are dissolved in acetic acid, refluxed and stirred for reaction, cooled to room temperature after the reaction is completed, diluted with water, and then the pH of the dilution solution is adjusted to neutral with sodium carbonate. The resulting solution is extracted with dichloromethane, the organic layers are combined and dried over anhydrous sodium sulfate, distilled under reduced pressure, and separated and purified by column chromatography to obtain compound 3 as a white solid; Step 4: at 20-22°C, 6-hydroxy-2-naphthaldehyde was dissolved in tetrahydrofuran, sodium tert-butoxide was slowly added, and the mixture was stirred for 15 min, followed by the addition of trimethylammonium trisulfur oxide copolymer, and the mixture was stirred for 1 h. After the reaction, the compound 4 was obtained by vacuum distillation and column chromatography separation and purification as a light yellow oil. Step 5: Under N2 environment, compound 3 and compound 4 were dissolved in ethanol, piperidine was added dropwise, and the mixture was stirred at 25°C for 4 h, followed by vacuum distillation and column chromatography separation and purification to obtain compound SE-SA as a yellow solid; The synthetic route is as follows: 。 3. Use of the two-photon fluorescent probe according to claim 1 in sulfatase detection.

4. The use according to claim 3, characterized in that: The two-photon fluorescent probe is used to prepare a sulfatase detection reagent, thereby realizing the detection of sulfatase.

5. The use according to claim 4, characterized in that: The detection reagent can target the endoplasmic reticulum of the cell.

6. The use according to claim 5, characterized in that: The detection reagent can distinguish normal cells from cancer cells and achieve a rapid response of sulfatase in the endoplasmic reticulum of cells.

7. The use according to claim 4, characterized in that: The detection limit of the detection reagent for sulfatase is 0.12 U / L.

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