A lysosome-targeting near-infrared fluorescent probe, a preparation method thereof and application thereof in carboxylesterase detection

Abstract

The application discloses a lysosome-targeting near-infrared fluorescent probe and a preparation method and application thereof in carboxylesterase detection, wherein the structure of the lysosome-targeting near-infrared fluorescent probe is shown in the following formula: The lysosome-targeting near-infrared fluorescent probe of the application shows a rapid response (response time is 40 seconds) to carboxylesterase (CE) in an in-vitro experiment. Cell toxicity test shows that the probe has low biological toxicity, and confocal fluorescence microscopic imaging experiment shows that the fluorescent probe has good light stability in HeLa cells and can effectively target lysosomes (localization coefficient is 0.86), and is suitable for CE near-infrared region fluorescence imaging and detection in lysosomes of living cells.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a lysosome-targeted near-infrared fluorescent probe, its preparation method, and its application in carboxylesterase detection, so as to realize the imaging detection of CE in cellular lysosomes in the near-infrared region, which has the advantages of high selectivity, high sensitivity, rapid response, and low biotoxicity. Background Technology

[0002] Carboxylesterase (CE) is a common serine hydrolase in mammals. Primarily located in the liver, it plays a crucial role in the metabolism of various endogenous esters, ester-containing drugs, and environmental toxins. It catalyzes the cleavage of various ester or amide substrates into their corresponding alcohols or carboxylic acids, and can hydrolyze ester, thioester, amide, and carbamate bonds in various endogenous and exogenous compounds. The important role of CE in human health and the metabolism of exogenous substances, and the potential to influence endogenous metabolism or enhance the efficacy of ester drugs through effective regulation of CE activity levels, have attracted widespread attention from researchers. To date, several methods for CE detection have been reported, including immunological, chromatographic, chemiluminescence, mass spectrometry, and fluorescent probes. Compared to other conventional methods, fluorescent probe bioimaging techniques offer higher selectivity, sensitivity, less invasiveness, and ease of operation, making them increasingly attractive. Although probes for the specific detection of lysosomal CE have been previously reported, most fluorescent probes suffer from drawbacks such as long response times, high detection limits, and short emission wavelengths in the fluorescence channels.

[0003] Lysosomes are organelles that break down biological macromolecules such as proteins, nucleic acids, and polysaccharides, playing a crucial role in a variety of biological functions. Lysosomal enzymes are involved in the intracellular digestion of various carbohydrates, lipids, and proteins, and their malfunction can lead to numerous hereditary lysosomal storage disorders. Therefore, developing readily available enzymes with good water solubility, improved selectivity, and rapid response for tissue imaging and function at the CE level of subcellular lysosomes remains essential.

[0004] Fluorescent probe methods are one of the most effective methods for detecting CE activity. However, most of the reported fluorescent probes for carboxylesterases currently available suffer from problems such as long reaction times, poor sensitivity, and short excitation and emission wavelengths. Summary of the Invention

[0005] This invention aims to provide a lysosome-targeting near-infrared fluorescent probe, its preparation method, and its application in carboxylesterase detection. The technical problem to be solved is to obtain a probe through molecular design that can specifically target lysosomes, rapidly respond to CE (cerebrolysinic esterase), and possess near-infrared fluorescence properties, thereby enabling real-time detection of CE changes in live-cell lysosomes via near-infrared fluorescence imaging. Cell experiments have confirmed that this probe has advantages such as low toxicity, good biocompatibility, fast response, high specificity, ability to locate lysosomal organelles, and near-infrared imaging.

[0006] This invention relates to a lysosomal targeted near-infrared fluorescent probe, abbreviated as CEPB, based on rhodamine, and its structural formula is shown below:

[0007]

[0008] The method for preparing the lysosome-targeted near-infrared fluorescent probe of the present invention includes the following steps:

[0009] Step 1: In an ice bath environment, cyclohexanone (1.98 mL, 20 mmol) was added dropwise to concentrated sulfuric acid (20 mL), and then 4-diethylaminoketo acid (3.13 g, 10 mmol) was slowly added in portions while stirring continuously at 90 °C for 3 h. After the reaction was completed, the mixture was quickly poured into ice water, perchloric acid (2 mL) was added, the precipitate was filtered and washed with cold water to obtain compound 1, which was a red solid and was used directly in the next step of the reaction without purification.

[0010] Step 2: Under N2 environment, p-hydroxybenzaldehyde (508.2 mg, 1.07 mmol) and compound 1 (181.4 mg, 1.49 mmol) were dissolved in acetic acid (15 mL), and the mixture was refluxed at 90 °C overnight. The solvent was evaporated under reduced pressure, diluted with water, and extracted with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, separated by reduced pressure distillation and column chromatography. The developing solvent was dichloromethane / methanol = 30 / 1 (v / v). After purification, CEPB-OH was obtained as a blue-purple solid.

[0011] Step 3: At 0°C, p-nitrophenyl chloroformate (0.54 mg, 2.7 mmol) and a small amount of trimethylamine (600 μL) were added sequentially to acetonitrile containing CEPB-OH (0.43 mg, 0.9 mmol), and then reacted at room temperature for 3 h to obtain compound 2, which can be used directly in the next step without purification.

[0012] Step 4: Compound 2 was dissolved in acetonitrile, and then 4-piperidinylpiperidine (0.34 mg, 2 mmol) and triethylamine (370 μL) were added sequentially. The mixture was stirred for 1 h, and then the solvent was evaporated under reduced pressure. The solvent was then diluted with water and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, then separated by reduced pressure distillation and column chromatography. The developing solvent for column chromatography purification was dichloromethane / methanol = 100:1 to 10 / 1 (v / v). After purification, CEPB was obtained as a blackish-purple solid.

[0013] The reaction route is shown below:

[0014]

[0015] The present invention relates to the application of lysosome-targeted near-infrared fluorescent probes in the preparation of carboxylesterase detection reagents.

[0016] The detection reagent can distinguish between normal cells and cancer cells, and can achieve rapid response detection of CE in near-infrared imaging and lysosomes.

[0017] The specific testing methods are as follows:

[0018] The CEPB of this invention was dissolved in DMSO (5 mL) to prepare a 2 mM stock solution. 15 μL of the CEPB stock solution was added to 3 mL of PBS solvent to obtain a final test solution with a concentration of 10 μM. The spectral properties of CEPB were detected by UV-vis absorption and fluorescence emission spectra obtained in 0.2 M phosphate-buffered saline (PBS, pH = 7.4). After adding CE (the carboxylesterase used in this patent detection) to the reaction system, both the absorption spectrum and fluorescence emission of CEPB showed significant changes. The absorption peak of CEPB increased at 545 nm. The fluorescence at 645 nm increased with increasing CE concentration, making this probe suitable for near-infrared fluorescence imaging. To further investigate the response of CEPB to CE, the time course of the fluorescence response of CEPB to CE was determined by fluorescence spectroscopy. After adding 10 U / mL of CE, the fluorescence intensity reached a stable state within 2 minutes. Based on this, CEPB was added after co-incubating different concentrations of 4-(2-Aminoethyl)benzenesulfonylfluoride hydrochloride (AEBSF, a carboxylesterase inhibitor) with 10 U / mL CE. The fluorescence intensity at 645 nm gradually decreased, further demonstrating the rapid response effect of CEPB to CE.

[0019] The carboxylesterase fluorescent probe provided by this invention can detect CE with high selectivity in the far-infrared to near-infrared region, with an in vitro response time of 40 seconds and a detection limit of 32 mU / mL. Furthermore, given the probe's low toxicity and biocompatibility, it has great application potential in in vivo models. Attached Figure Description

[0020] Figure 1 NMR (A)(B) and HRMS plots (C) of CEPB;

[0021] Figure 2 (A) is the possible mechanism of the probe CEPB responding to CE; (B) HPLC and (C) UV-Vis absorption spectra of CEPB (10 μM) and CE (20 U / mL) before and after the reaction at 37 °C and under PBS (10 mM, pH = 7.4).

[0022] Figure 3 (A) The fluorescence intensity of CEPB (10 μM) with varying CE (0-15 U / mL) concentration in PBS (10 mM, pH = 7.4) at 37℃; (B) The detection limit of CEPB in response to CE; (C) The time dependence of CEPB (10 μM) fluorescence intensity at CE concentrations of 5 U / mL and 10 U / mL; (D) Fluorescence intensity of CEPB (10 μM) against other biological species in PBS (pH = 7.4): 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: TyrE, 21: CE.

[0023] Figure 4 Cell viability of A549 cells after treatment with different concentrations (0 μM, 5 μM, 10 μM, 15 μM and 20 μM) of CEPB for 24 hours.

[0024] Figure 5 (A) Confocal imaging of three cancer cell lines (4T1, HepG2, and HeLa cells) and three normal cell lines (NIH / 3T3, 293T, and MH-S cells) using CEPB (10 μM). Hoechst 33342 is the blue channel, λ ex=360nm, CEPB red channel, λ ex =545nm; (B) is the quantitative representation of the fluorescence intensity of the red channel in (A).

[0025] Figure 6 (A) is a confocal image of HepG2 after additional stimulation with different concentrations (0, 0.2, 0.5, 1.0 mM) of AEBSF for 30 minutes, using 10 μM CEPB; (B) is the fluorescence intensity.

[0026] Figure 7 This study investigated the co-localization of CEPB and Lyso-Green in HeLa cells. Detailed Implementation

[0027] The present invention will be further illustrated by the following examples.

[0028] Example 1: Synthesis of CEPB

[0029] Compound 2 was dissolved in acetonitrile, and then 4-piperidinylpiperidine (336.32 mg, 2 mmol) and triethylamine (370 μL) were added sequentially. The reaction was carried out at room temperature, and the solvent was evaporated under reduced pressure. The solution was then diluted with water and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, purified by distillation under reduced pressure and column chromatography with dichloromethane / methanol = 100:1 to 10 / 1 (v / v). CEPB was then obtained as a blackish-purple solid.

[0030] 1 H NMR (400MHz, DMSO-d6) δ7.93(d,J=7.6Hz,1H),7.78(d,J=7.5Hz,1H),7.68(d,J=7.5Hz,1H),7.45(d,J=8.2Hz,2H ),7.37(s,1H),7.32(d,J=7.6Hz,1H),7.16(d,J=8.1Hz,2H),6.53(s,1H),6.44(d,J=9.2Hz,1H),6.38(d,J=8.9Hz ,1H),3.57-3.45(m,1H),3.38-3.35(m,8H),3.00(s,1H),2.87(d,J=12.1Hz,1H),2.78-2.70(m,1H),2.60(s,1H), 2.53(s,2H),1.99(p,J=7.0,6.5Hz,1H),1.83(dd,J=27.4,12.9Hz,3H),1.61-1.47(m,8H),1.09(t,J=7.0Hz,6H); 13C NMR(101MHz,DMSO-d6)δ169.59,153.21,152.16,150.76,149.53,146.69,135.85,130.80,130.34,128.73,127.21,125.12,1 24.75,124.13,122.27,108.28,104.63,97.35,86.31,61.85,50.19,44.22,27.10,26.62,25.09,23.11,22.46,12.92.ESI-MS m / z:calcd.for C 42 H 48 N3O5 + [CEPB-ClO4 - ] + :674.3588,found:674.3599.

[0031] Example 2: Verification Study of Reaction Mechanism

[0032] like Figure 2 As shown in Figure (A), in the presence of CE, CEPB is reduced to CEPB-OH, thereby restoring the fluorescence of the rhodamine structure. Figure 2 The product formed by the excess reaction of CEPB and CE shown in Figure (B) was confirmed by high performance liquid chromatography (HPLC) to be CEPB-OH. Figure 2 Figure (C) shows the UV-Vis spectrum of a 2 mM stock solution prepared by dissolving CEPB in DMSO (5 mL), adding 15 μL of the CEPB stock solution to 3 mL of PBS solvent (pH = 7.4), and then adding CE (20 U / mL). The UV-Vis absorption spectrum shows that the maximum absorption peak of CEPB is located at approximately 545 nm.

[0033] Example 3: Spectroscopic Study of CEPB

[0034] The CEPB of this invention was dissolved in DMSO (5 mL) to prepare a 2 mM stock solution. 15 μL of the CEPB stock solution was added to 3 mL of PBS solvent (pH = 7.4). Different concentrations (0–15 U / mL) of CE were then added, and the fluorescence spectrum was analyzed. Figure 3 A) It can be seen that the fluorescence intensity at 640 nm increases with increasing CE concentration. For example... Figure 3 As shown in Figure B, the linear correlation coefficient R can be observed. 2 =0.9984, the calculated detection limit is 32 mU / mL ( Figure 3 B). To test the time dependence of CEPB on CE, we used CEPB (10 μM) to test the fluorescence response at CE values ​​of 5 U / mL and 10 U / mL, respectively. Figure 3As shown in C), the fluorescence intensity stabilized after 40 seconds.

[0035] Example 4: Cytotoxicity study of CEPB

[0036] First, we tested the biosafety of the CEPB probe at the cellular level using the CCK8 assay, and the results are as follows: Figure 4 As shown, when the probe concentration is less than 20 μM, the cell survival rate is greater than 90%, proving that the probe has low toxicity at the working concentration.

[0037] Example 5: Imaging study of CE levels in cancer cells using CEPB

[0038] Considering that liver cancer cells express a large amount of carboxylesterase, we pre-incubated cancer cells (including 4T1 cells, HepG2 cells, and HeLa cells) and normal cells (including NIH / 3T3 cells, 293T cells, and MH-S cells) with the probe CEPB for 30 minutes. The results are as follows. Figure 5 As shown, cancer cells exhibited a distinct red fluorescent signal compared to normal cells. When cancer cells were pre-incubated with different concentrations of the inhibitor AEBSF and then incubated with the probe, the results were as follows: Figure 6 As shown, the fluorescence signal in cancer cells gradually weakens, further indicating that the red fluorescence signal is caused by the overexpression of CE.

[0039] Example 6: Co-localization imaging study of CEPB in lysosomes

[0040] The results are as follows Figure 7 As shown, the colocalization coefficient between the probe CEPB and the commercial dye Lyso-Green reaches 0.86.

Claims

1. A lysosome-targeted near-infrared fluorescent probe, characterized in that... Its structural formula is shown below:

2. A method for preparing the lysosome-targeted near-infrared fluorescent probe according to claim 1, characterized in that... Includes the following steps: Step 1: In an ice bath environment, cyclohexanone was added dropwise to concentrated sulfuric acid, and then 4-diethylaminoketo acid was added slowly in batches while stirring continuously. After the reaction was completed, it was quickly poured into ice water, perchloric acid was added, the precipitate was filtered and washed with cold water to obtain compound 1, which is a red solid. It was used directly in the next step of the reaction without purification. Step 2: Under N2 environment, p-hydroxybenzaldehyde and compound 1 were dissolved in acetic acid and refluxed. The solvent was evaporated under reduced pressure, then diluted with water and extracted with dichloromethane. The organic layers were combined and dried with anhydrous sodium sulfate, then purified by vacuum distillation and column chromatography to obtain CEPB-OH, which is a blue-purple solid. Step 3: Under ice bath conditions, p-nitrophenyl chloroformate and a small amount of trimethylamine were added sequentially to acetonitrile containing CEPB-OH, and then the mixture was stirred to obtain compound 2, which can be used directly in the next step without purification. Step 4: Compound 2 was dissolved in acetonitrile, and then 4-piperidinylpiperidine and triethylamine were added sequentially. The mixture was stirred and reacted. The solvent was then evaporated under reduced pressure, diluted with water, and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, then purified by vacuum distillation and column chromatography to obtain CEPB, which was a blackish-purple solid. The reaction route is shown below:

3. The preparation method according to claim 2, characterized in that: In step 2, the developing solvent for column chromatography separation and purification is dichloromethane / methanol = 30 / 1, v / v.

4. The preparation method according to claim 2, characterized in that: In step 4, the developing solvent for column chromatography separation and purification is dichloromethane / methanol = 100:1 to 10 / 1, v / v.

5. The application of the lysosome-targeted near-infrared fluorescent probe of claim 1 in the preparation of carboxylesterase detection reagents.

6. The application according to claim 5, characterized in that: The detection reagent can distinguish between normal cells and cancer cells, and can achieve rapid response detection of CE in near-infrared imaging and lysosomes.

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