A two-photon fluorescent probe and its application in sulfatase detection

Abstract

The present invention discloses a two-photon fluorescent probe and its application in sulfatase detection, wherein the structure of the endoplasmic reticulum-targeted two-photon fluorescent probe is shown below: #imgabs0#. The endoplasmic reticulum-targeted two-photon fluorescent probe of the present invention responds to different concentrations of sulfatase, with a detection limit as low as 0.12 U / L and a two-photon absorption cross section of 81 GM. Cytotoxicity tests demonstrate that the probe has low biological toxicity. Confocal fluorescence microscopy imaging demonstrates that the probe can effectively target the endoplasmic reticulum in HeLa cells (localization coefficient of 0.86), making it suitable for fluorescent 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, which can realize the characteristics of fluorescence imaging of sulfatase in solution and cells and fluorescence tracing of endoplasmic reticulum, and has the advantages of selectivity, high detection efficiency and good biocompatibility. Background Art

[0002] The endoplasmic reticulum (ER) is a continuous membrane system composed of vesicular, vesicular, and tubular structures formed by a single unit membrane. It is the primary site for the synthesis of proteins, lipids, and carbohydrates within cells. It regulates various physiological processes within human cells, including gluconeogenesis, protein processing, and lipid biosynthesis. ER stress can induce the 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 ER is crucial for understanding and treating these diseases.

[0003] Sulfatase is a general term for enzymes that liberate inorganic sulfate from organic sulfate esters. They are primarily found in lysosomes, the endoplasmic reticulum, and the Golgi apparatus, and participate in physiological processes such as cell signaling and hormone regulation. They catalyze esterification reactions between sulfate groups and other molecules, transferring the sulfate groups to other molecules. Sulfatase is primarily involved in chemical metabolism, accelerating the dissociation of sulfate esters, hydrolyzing them into sulfuric acid and the corresponding alcohol or phenol. This reaction is crucial because it renders many organic molecules more hydrophilic, making them more readily absorbed by cells and thus better able to participate in metabolic processes. In medicine, sulfatase is used to treat metabolic diseases, such as metabolic disorders and genetic diseases. Overexpression of sulfatase can lead to elevated estrogen levels in the body, accelerate cell growth, and cause hormone-dependent cancers. Therefore, accurate detection of sulfatase is crucial for understanding the physiological functions of sulfatase and diagnosing related diseases. To date, several methods for sulfatase detection have been reported, including enzyme-linked immunosorbent assays, colorimetric methods, 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 specific imaging of sulfatases targeting the endoplasmic reticulum is still very rare, 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 of intracellular sulfatases fluctuations. Summary of the Invention

[0005] The present invention addresses the shortcomings of the aforementioned prior art and provides a two-photon fluorescent probe for endoplasmic reticulum-targeted detection of sulfatase, as well as its preparation method and application. The technical problem to be solved is to design a molecular structure that is both responsive to sulfatase and capable of endoplasmic reticulum fluorescence tracing. This molecular structure is required to not only possess endoplasmic reticulum tracing capabilities but also produce a strong fluorescence signal after responding to sulfatase.

[0006] This study designed and synthesized a two-photon fluorescent probe, SE-SA, for the ER-targeted detection of sulfatases, using naphthalene as a two-photon fluorophore, p-toluenesulfonamide as an ER-targeting group, and sulfate as a response group. The hydroxyl group sulfation blocks the intramolecular charge transfer (ICT) effect of SE-OH, rendering SE-SA initially nearly non-fluorescent. Upon reaction with sulfatases, the sulfate moiety of SE-SA is specifically cleaved and removed, releasing SE-OH and restoring the ICT effect, thereby activating the fluorescent signal. Optical properties and cytotoxicity tests demonstrated that SE-SA exhibits sulfatase specificity and good biocompatibility. This study evaluated the feasibility of SE-SA for live-cell sulfatase-specific fluorescence imaging and ER imaging through confocal fluorescence imaging of cells, aiming to provide a convenient chemical tool for the visualization of sulfatases and related disease studies.

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

[0008] .

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

[0010] Step 1: Dissolve 3.01 g of 4-toluenesulfonyl chloride and 4.00 g of 3-bromopropylamine hydrobromide in 14 mL of anhydrous dichloromethane in an ice bath. Then, add 4.02 g of triethylamine dropwise and stir for 30 minutes. After the reaction, dilute the reaction solution with 150 mL of dichloromethane, adjust the pH to neutral with 2 × 60 mL of 2M hydrochloric acid, and extract the resulting solution with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, evaporated under reduced pressure, 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, 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 heated at 45°C for 12 h. After the reaction, the mixture was cooled to room temperature and 50 mL of a cold 5% HCl solution (0-5°C) was added dropwise to produce a white precipitate. This was filtered, rinsed with pure water, and air-dried to obtain a crude white powder of compound 2. This was then 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 and diluted with 50 mL of water. The pH of the dilution was then 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: Dissolve 0.26 g of 6-hydroxy-2-naphthaldehyde in 3 mL of tetrahydrofuran at 20-22°C. Slowly add 0.15 g of sodium tert-butoxide and stir for 15 minutes. Then, add 0.28 g of solid trimethylammonium sulfur trioxide copolymer and continue stirring for 1 hour. After the reaction, vacuum distillation and column chromatography (eluent: dichloromethane:methanol = 30:1) yield compound 4 as a pale yellow oil.

[0014] Step 5: Under N2 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 dropwise. The mixture was stirred at 25°C for 4 h. Then, the mixture was distilled under reduced pressure 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 relates to 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] This two-photon fluorescent probe targets the endoplasmic reticulum (ER) for sulfatase detection. The sulfation of the hydroxyl group blocks the intramolecular charge transfer (ICT) effect of SE-OH, resulting in a weak initial fluorescence. Upon reaction with sulfatase, the sulfate moiety of SE-SA is specifically cleaved and removed, leading to the generation of SE-OH and restoration of the ICT effect, thereby activating a strong fluorescence signal. SE-SA exhibits excellent cytocompatibility and has promising applications in confocal cell imaging.

[0021] The detection method is as follows:

[0022] SE-SA of the present invention was dissolved in DMSO to prepare a 2 mM stock solution. 15 μL of each stock solution was then added to 3 mL of a PBS / DMSO mixture (PBS:DMSO = 9:1) containing varying concentrations of sulfatase. UV-vis absorption and fluorescence emission spectra were obtained to examine the spectral properties of SE-SA. Upon addition of sulfatase (CAS No. 9016-17-5 was used in this assay), the absorbance of SE-SA shifted from 390 nm to 440 nm with increasing sulfatase concentration. The fluorescence intensity also red-shifted from 520 nm to 580 nm, gradually increasing. To further investigate the response of SE-SA to sulfatase, the time course of the fluorescence response of SE-SA to sulfatase was measured using fluorescence spectroscopy. After adding 150 U / L of sulfatase, the fluorescence intensity reached a steady state 40 minutes after the addition of 150 U / L of sulfatase.

[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 in 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 decreased, further proving the rapid response of SE-SA to sulfatase. SE-SA also had the ability to image the endoplasmic reticulum (Pr=0.86), making it 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 SE-SA probe's response to sulfatase. Figure 1 (B) UV-visible absorption spectra of SE-SA (10 μM) before and after reaction with sulfatase (150 U / L) at 37°C in PBS:DMSO = 9:1. Figure 1 (C) HPLC chromatograms 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 in PBS:DMSO = 9:1; (B) Detection limit of SE-SA in response to sulfatase; (C) Time dependence of the 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 in response to other biological species (1: blank, 2: ClO - ,3:CO3 2- , 4:S 2- , 5:SO3 2- ,6:H2O2,7:Mg 2+ ,8:NH4 + , 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) treated with 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] Under a nitrogen atmosphere, 0.2 g of compound 3 and 0.14 g of compound 4 were dissolved in 4 mL of ethanol. One drop of piperidine was added dropwise, and the mixture was stirred at room temperature for 4 h. The solvent was then evaporated under reduced pressure. Compound SE-SA was then isolated and purified by column chromatography (eluent: dichloromethane:methanol = 20:1) to obtain compound SE-SA as a yellow solid (yield 40%).

[0034] 1 H NMR (400 MHz, DMSO-D6) 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). 13C NMR (151 MHz, DMSO-D6) 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 fluorescence turns on. Figure 1 Figure (B) shows the UV spectrum of a 2 mM stock solution of SE-SA prepared by dissolving it in DMSO (5 mL). 15 μL of the SE-SA stock solution was added to 3 mL of PBS:DMSO = 9:1, and 150 U / L of sulfatase was added. The UV-visible absorption spectrum shows that the maximum absorption peak of SE-SA is located at around 390 nm. After the sulfatase reaction, 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 and the fluorescence spectrum was used to analyze the effect of the sulfatase on 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 state 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 minutes. The effective two-photon absorption cross section was measured on a two-photon excitation device, and the two-photon absorption cross section (δ) was calculated by measuring the fluorescence quantum yield. It was found that the maximum two-photon absorption cross section appeared at a wavelength of 860 nm, with a value of 81 GM, as shown in Figure 2. Figure 3 It is shown that SE-SA has good two-photon performance after responding to Sulfatase.

[0041] Example 5: Cytotoxicity Study

[0042] First, we tested the biosafety of the SE-SA probe using CCK8 assay at the cellular level. Figure 4 As shown in the figure, when the concentration of the probe was less than 20 μM, the cell viability was greater than 90%, demonstrating 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 sulfatase inhibitor STX-64 and then incubated with the probe, the results were as follows: Figure 6 As shown, the fluorescence signal in cancer cells gradually weakened, further indicating that the fluorescence signal was caused by overexpressed sulfatase.

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

[0046] To explore the ability of SE-SA to image the endoplasmic reticulum in cells, we used 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-TrackerGreen is calculated to be 0.86. These results indicate that SE-SA has the ability to track the endoplasmic reticulum.

Claims

1. A two-photon fluorescent probe, abbreviated 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: 4-Toluenesulfonyl chloride and 3-bromopropylamine hydrobromide were dissolved in anhydrous dichloromethane under ice-bath conditions, and triethylamine was then added dropwise and stirred for reaction. After the reaction, the reaction solution was diluted with dichloromethane, and the pH of the dilution solution was adjusted with 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 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 solution was added dropwise to produce a white precipitate. The precipitate was filtered, rinsed with pure water, and air-dried to obtain a crude white powder of compound 2, which was then slurried with dichloromethane and ethyl acetate to obtain compound 2. Step 3: Under nitrogen protection, compound 2, malononitrile and hexamethyldisilazane were dissolved in acetic acid, refluxed and stirred for reaction, and cooled to room temperature after the reaction was completed. Water was added to the reaction solution, and 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 to obtain compound 3 as a white solid; Step 4: Dissolve 6-hydroxy-2-naphthaldehyde in tetrahydrofuran at 20-22°C, slowly add sodium tert-butoxide, stir for 15 minutes, then add trimethylammonium sulfur trioxide copolymer, and continue stirring for 1 hour. After the reaction, vacuum distillation and column chromatography separation and purification give compound 4 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. Then, the mixture was distilled under reduced pressure and purified by column chromatography 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 the preparation of a sulfatase detection reagent.

4. The use according to claim 3, characterized in that: The detection reagent can target the endoplasmic reticulum of the cell.

5. The use according to claim 4, 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.

6. The use according to claim 3, characterized in that: The detection limit of the detection reagent for sulfatase is 0.12 U / L.

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