Polypeptide fluorescent probe capable of specifically detecting enterokinase as well as preparation method and application of polypeptide fluorescent probe

Abstract

The invention relates to the technical field of fluorescent chemical sensors, and particularly discloses a polypeptide fluorescent probe capable of specifically detecting enterokinase (ENTK) as well as a preparation method and application of the polypeptide fluorescent probe. The structural formula of the polypeptide fluorescent probe capable of specifically detecting the ENTK is shown in the specification. At present, molecular fluorescent probes for ENTK detection are not reported, and particularly, a quantitative test technology for ENTK applied to tumor cells or tissues is not developed yet. The polypeptide fluorescent probe disclosed by the invention has high specificity and selective response capability on the ENTK, is high in biological safety and capable of stably imaging for a long time, and can distinguish tumors from normal tissues by detecting the content change of the ENTK and realize in-situ fluorescence imaging of the ENTK. The work provides a new detection means for specifically and rapidly detecting the expression level of the ENTK. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of fluorescent chemical sensors, and in particular relates to a polypeptide fluorescent probe capable of specifically detecting enterokinase, and a preparation method and application thereof. Background Art

[0002] Enterokinase (ENTK), also known as intestinal peptidase, is a serine protease secreted by vertebrate duodenal cells. It can specifically hydrolyze the carboxyl-terminal peptide bond of the polypeptide sequence "DDDDK" and plays an important role in the activation of trypsinogen. ENTK is widely present in glycoproteins in the duodenal and jejunal mucosa. It participates in the synthesis of duodenal trypsin and plays an important role in the pathogenesis of acute pancreatitis. Insufficient enterokinase secretion may lead to clinical symptoms such as diarrhea, developmental disorders, hypoproteinemia and edema. In addition, recent studies have shown that ENTK is overexpressed in tumor cells. For example, in the mitochondria of human cervical cancer cells (HeLa), the expression level of ENTK is much higher than that of other normal cells. ENTK has become a potential tumor biomarker. However, due to the lack of technology for real-time quantitative detection of ENTK content in living cells or tissues, its mechanism of action on tumor cells is still unclear. Therefore, it is urgent to develop a specific method that can specifically and quickly detect the expression level of ENTK.

[0003] At present, the detection method of enterokinase in vitro is still mainly based on enzyme-linked immunosorbent assay (ELISA). This method uses the principle of antigen-antibody reaction. By fixing the antigen or antibody on the test plate, reacting with the antibody or antigen to be detected, and then adding the enzyme-labeled antibody, the content of the antibody or antigen is quantitatively detected by enzyme-catalyzed color development. However, this method has low sensitivity, complex operation steps, easy to form false positive signals, and many environmental interference factors. It is impossible to monitor the content of ENTK in cells or tissues in real time, especially the changes of ENTK in living tumor cells. Fluorescence detection technology has the advantages of simple operation, high specificity and sensitivity, non-invasiveness, and real-time imaging. It is often used to monitor many physiological and pathological processes at the cellular and even molecular levels in real time. The detection principle of common fluorescent probe technology is to analyze the specific response between the substrate and the probe. Under the irradiation of the excitation light source, the probe produces a change in fluorescence signal, and its signal is detected by the instrument, so as to judge the content and position of the target detection according to the signal change. In recent years, fluorescent probes have become an indispensable tool for people to study the level of biological proteases. However, there are currently no reports on molecular fluorescent probes for ENTK detection, and in particular, quantitative testing technology for ENTK in tumor cells or tissues has not yet been developed. Summary of the invention

[0004] In order to solve this problem, the present invention provides a polypeptide fluorescent probe that can specifically detect the expression level of ENTK in tumor cells, as well as a preparation method and application. The probe has high specificity and selective response to ENTK, and the probe has high biological safety, can be imaged stably for a long time, and can distinguish tumors from normal tissues by detecting changes in ENTK content, and realize in situ fluorescence imaging of ENTK.

[0005] To achieve the above object, the present invention discloses the following technical solution:

[0006] In the first aspect, a polypeptide fluorescent probe capable of in situ specific detection of ENTK is provided, which is denoted as ZX-1, and its molecular structure is shown below: The peptide with the sequence DDDDK is used as the probe part of the ZX-1 fluorescent probe, which can specifically recognize and detect ENTK. Under the action of ENTK, the peptide sequence will be cut to form the dye molecule ZX-FL, whose molecular structure is shown below: Produce a strong fluorescent signal, thus achieving the detection effect.

[0007] In the second aspect, a method for preparing a polypeptide fluorescent probe ZX-1 capable of in situ specific detection of ENTK is provided, which specifically comprises the following steps:

[0008] Step 1: preparing 2-(3,5,5-trimethylcyclohex-2-en-1-yl)malononitrile by isophorone (abbreviated as Z1), the required reaction time is 8-12 hours, and the obtained product is finally recorded as Z2;

[0009] Step 2: Prepare ((E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile using Z2 obtained in the first step. The required reaction time is 8-12 hours. Finally, the obtained product is recorded as ZX-FL.

[0010] Step 3: Prepare the fluorescent probe ZX-1 using the ZX-FL prepared in the second step. Specifically, react ZX-FL with the designed polypeptide sequence by solid phase reaction, and finally purify and separate by high performance liquid chromatography to obtain the final product ZX-1.

[0011] In the third aspect, a reaction mechanism of a polypeptide fluorescent probe capable of in situ specific detection of ENTK is provided:

[0012] The present invention uses a polypeptide with a sequence of DDDDK as the response part to ENTK, and uses (E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile as the dye group. In the process of detecting ENTK by the fluorescent probe ZX-1, ENTK can specifically recognize and cut the DDDDK polypeptide chain, thereby releasing the dye ZX-FL with a strong fluorescent signal. Since the solubility of the dye in water after the reaction is poor, a precipitate is formed near the reaction site, and finally a long-term stable imaging effect is achieved. It is worth mentioning that the fluorescent probe ZX-1 not only greatly enhances its water solubility due to the modification of the polypeptide, but also self-assembles to form nanoparticles of a certain particle size due to the difference in hydrophilicity and hydrophobicity between the dye ZX-FL and the polypeptide, thereby enhancing the cell uptake efficiency and tissue aggregation effect.

[0013] In a fourth aspect, a use of a ZX-1 fluorescent probe is provided.

[0014] Specifically, the ZX-1 fluorescent probe can rapidly and specifically detect changes in ENTK content in living cells.

[0015] Specifically, the ZX-1 fluorescent probe can be used to detect the activity of ENTK in tumor sections of tumor-bearing mice.

[0016] The beneficial effects of the present invention are:

[0017] The polypeptide fluorescent probe designed and synthesized by the present invention has the characteristics of strong specificity, good selectivity, strong biosafety, and long-term stable imaging. Compared with the existing detection methods for detecting ENTK, the polypeptide fluorescent probe for in situ specific detection of ENTK designed by the present invention and its preparation method and application have the following advantages:

[0018] 1. Strong specificity: ENTK can specifically recognize the DDDDK sequence peptide of ZX-1, and hydrolyze the peptide during the recognition process to generate the fluorescent dye ZX-FL, forming a strong fluorescent signal and achieving the effect of specific detection.

[0019] 2. Good selectivity: We determined the selectivity of the probe by measuring the fluorescence intensity after the probe was mixed with different anions and cations, amino acids, proteins, and various biological enzymes. The experimental results showed that only when ENTK was added, there would be a significant fluorescence enhancement, showing excellent selectivity.

[0020] 3. High biosafety: We conducted a cytotoxicity experiment on the fluorescent probe molecules, and the results showed that the fluorescent probe molecules have very low biotoxicity to cells and high biosafety.

[0021] 4. Long-term stable in situ imaging capability: The fluorescent probe ZX-1 can not only detect the ENTK content level in different cells by fluorescence, but also exhibits the ability to perform long-term stable in situ imaging of ENTK in tumor tissue sections. We measured the changes in fluorescence intensity of the probe molecule ZX-1 and the commercial dye Rhodamine B for different immersion times in 4T1 tumor tissue sections of tumor-bearing mice, and we found that the in situ imaging performance of the probe is better than that of general water-soluble dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the above and / or other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows:

[0023] Figure 1 The synthetic route of the ENTK-specific probe ZX-1;

[0024] Figure 2 This is the action mechanism diagram of the ENTK-specific probe ZX-1;

[0025] Figure 3 The H-NMR spectrum (i), C-NMR spectrum (ii) and high-resolution mass spectrum (iii) of compound Z2;

[0026] Figure 4 The H-NMR spectrum (i), C-NMR spectrum (ii) and high-resolution mass spectrum (iii) of compound ZX-FL;

[0027] Figure 5 The HPLC flight retention time (i) and high-resolution mass spectrum (ii) of compound ZX-1;

[0028] Figure 6 The transmission electron microscope particle size images of compound ZX-1 at different concentrations;

[0029] Figure 7 This is the response fluorescence spectrum of ENTK-specific fluorescent probe ZX-1 and ENTK;

[0030] Figure 8 To test the kinetic performance of the reaction between ENTK-specific fluorescent probe ZX-1 and ENTK;

[0031] Fig. 9 It is a concentration gradient test of the reaction between ENTK-specific fluorescent probe ZX-1 and ENTK;

[0032] Fig.10 To test the selectivity of ENTK-specific fluorescent probe ZX-1 against 41 substances commonly found in cells;

[0033] Fig.11 To test the cytotoxicity of ENTK probe ZX-1 at different concentrations;

[0034] Fig.12 This is the fluorescence imaging of ENTK probe ZX-1 in different cells;

[0035] Fig.13 The slice images and quantification curves (iii) of 4T1 tumor tissue sections of tumor-bearing mice were incubated with the same concentration of ENTK probe ZX-1 (i) and rhodamine B (ii) and excited at a wavelength of 488nm at different times.

[0036] Fig.14 This is an image of a 4T1 tumor tissue section of a tumor-bearing mouse incubated with the ENTK probe ZX-1 and excited at a wavelength of 488nm. DETAILED DESCRIPTION

[0037] The present invention is described in detail below, specifically in conjunction with the drawings of the present invention specification, to provide a clearer and more complete description of the embodiments of the present invention, without limiting the contents thereof. The embodiments described in the present invention are only a part, not all, of the embodiments, which are all within the protection scope of the present invention.

[0038] Example 1 Reference Figure 1 The synthetic route shown in the figure is to prepare the ZX-1 fluorescent probe

[0039] Step 1: Dissolve Z1 (1.38 g, 10 mmol) and malononitrile (0.8 g, 12 mmol) in 40 mL of ethanol, add glacial acetic acid (5 μL, 0.1 mmol) and piperidine (1 μL, 1 mmol), and heat under reflux for 12 h under nitrogen protection; after the reaction is completed, cool to room temperature and remove ethanol in vacuo, purify by flash column chromatography, and spin dry to obtain 2-(3,5,5-trimethylcyclohex-2-en-1-yl) malononitrile; that is, Z2; the spectrum of Z2 of the present invention is as shown Figure 3 As shown, (i) is the hydrogen nuclear magnetic resonance spectrum, (ii) is the carbon nuclear magnetic resonance spectrum, and (iii) is the high-resolution mass spectrum.

[0040] Step 2: Dissolve Z2 (0.4 g, 2.16 mmol) and 4-aminobenzaldehyde (0.4 g, 2.4 mmol) obtained in the first step in 30 mL of ethanol, add piperidine (1 μL, 1 mmol), and heat under reflux for 12 h under nitrogen protection. After the reaction is completed, cool to room temperature, filter and collect the precipitate, and dry to obtain ((E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile; that is, ZX-FL; the spectrum of ZX-FL of the present invention is as follows Figure 4 As shown, (i) is the hydrogen nuclear magnetic resonance spectrum, (ii) is the carbon nuclear magnetic resonance spectrum, and (iii) is the high-resolution mass spectrum.

[0041] Step 3: Fmoc-Lys(boc)-OH was connected to CTC resin, and then piperidine was deprotected for 30 minutes, then piperidine was removed and washed with dimethylformamide (DMF), and the deprotection color was detected by ninhydrin. Then, the subsequent straight-chain amino acids were connected in sequence until the Boc-Asp(otbu)-OH at the N-terminus was connected to obtain Boc-Asp(otbu)-Tyr((tbu)-Lys(boc)-Asp(otbu)-Asp(otbu)-Asp(otbu)-Asp(otbu)-Lys(boc)-CTC Resin. The above-mentioned protected peptide was cleaved from the resin with trifluoroethanol, washed with ether twice, and dried by rotation. The protected peptide and small molecule ZX-FL were dissolved in DMF respectively, 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) were added after mixing, and the reaction was carried out for 6 hours. After drying by rotation, the peptide protecting group was cut off with trifluoroacetic acid to obtain the crude target product; liquid phase purification was carried out and freeze-dried. The target product ZX-1 was obtained. The spectrum of ZX-1 is shown in FIG. Figure 5 As shown, (i) high performance liquid chromatography flight retention time, (ii) high resolution mass spectrometry.

[0042] Example 2 ZX-1 Characterization and Mechanism

[0043] The intermediate products Z2, ZX-FL and the final structure of the ZX-1 fluorescent probe produced in the process of preparing the ZX-1 fluorescent probe of the present invention can be obtained by Figure 3-5 The results are fully confirmed by the H NMR spectrum, C NMR spectrum, HPLC flight retention time and high resolution mass spectrum. The particle size of ZX-1 at different concentrations can be determined by Figure 6 Transmission electron microscopy characterization was performed as shown.

[0044] like Figure 2 As shown, under the action of ENTK, the probe part of the ZX-1 two-photon fluorescent probe molecule of the present invention is broken to generate corresponding fluorescent groups, thereby achieving the effect of specifically detecting ENTK.

[0045] Example 3 In vitro testing of ZX-1

[0046] The reaction system for in vitro testing is: the total volume of each well is 100μL, including 10μL of fluorescent probe ZX-1 with a stock concentration of 100μM, 1μL of ENTK stock solution with a stock concentration of 100U / mL, and 89μL of 0.1% triton-PBS solution, where the probe stock solution is prepared from PBS, the final test concentration is 10μM, and the test concentration of ENTK is 1U / mL. The above reaction system is incubated at 37°C and 1000rpm on an incubator in a constant temperature and dark environment for 30 minutes. The specific experimental results are as follows:

[0047] (1) Figure 7 As shown in the figure, we tested the response of ZX-1 to ENTK. Under the condition of ENTK concentration of 1U / mL, with 488nm laser as excitation wavelength, ZX-1 showed only weak fluorescence at 650nm, while the fluorescence after the reaction was significantly enhanced (about 30 times). These data show that ZX-1 can well detect changes in ENTK activity.

[0048] (2) Figure 8 As shown in Figure 2, we tested the change of fluorescence over time after the reaction of ZX-1 and ENTK. In the PBS test system with pH = 7.4 (0.1M, 0.1% triton), the concentrations of ZX-1 and ENTK were 10μM and 1U / mL, respectively, and the reaction time kinetic test was performed at 37°C. The kinetic test results showed that the fluorescence of ZX-1 and ENTK gradually increased after the reaction and reached the maximum value (E x =488nm).

[0049] (3) Fig. 9 As shown, in the concentration gradient test of ENTK, specifically, in a PBS test system at pH = 7.4 (0.1M, 0.1% triton), the ZX-1 concentration was 10 μM, different concentrations of ENTK (0-1U / mL) were added, and then reacted at 37°C for 30 minutes to obtain the concentration gradient response spectrum (E x =488nm). The results showed that the fluorescence intensity of ZX-1 after the reaction with ENTK gradually increased with the increase of ENTK concentration.

[0050] (4) The ENTK probe ZX-1 was tested for its selectivity. Specifically, 40 substances commonly found in cells were selected as interfering substances, among which 1-24: F-, Cl-, Br - ,I - , HPO 4 - , HSO 3 -、SO 4 2- , HS - 、SCN - 、Na + , K + , Ca 2+ , Mn 2+ , Fe 2+ , Fe 3 + 、Co 2+ 、Ni + 、Al 3+ 、Ag +, Cu + , Cu 2+ Cr 3+ 、Zn 2+ Mg 2+ (1.0mM); 25-37: D-cysteine, L-aspartic acid, L-serine, L-threonine, L-histidine, L-(-)-proline, methionine, tryptophan, tyrosine, L-arginine, L-cystine, L-alanine, DL-homocysteine ​​(1.0μΜ); 38-39: monoamine oxidase A, monoamine oxidase B (10μg / mL), 40-41: glutamine transferase, ENTK (1U / mL). Then, in a PBS test system (0.1M, 0.1% triton) at pH = 7.4, under an environment with a ZX-1 concentration of 10μM, various interfering substance solutions were added in turn, and the fluorescence emission spectra (E) after 30min of response were tested respectively. x =488nm).

[0051] The results are as follows Fig.10 As shown, there is a significant fluorescence enhancement only when ENTK is added, and other interfering substances have no effect, which proves that ZX-1 has a high specificity for detecting ENTK.

[0052] Example 4 ZX-1 intracellular test

[0053] (1) Cytotoxicity of ENTK-specific probe ZX-1 was detected. Specifically, standard MTT analysis was used for analysis. HeLa and 4T1 cells were digested with 0.25% trypsin, respectively, and then cell suspensions were prepared with cell culture medium and inoculated into 96-well plates. The plates were cultured at 37°C and 5% carbon dioxide for 24 hours. Probes of different concentrations (0 μM, 40 μM, 80 μM, 120 μM, 160 μM, 200 μM, 240 μM, and 280 μM) were added to the plates and cultured for 24 hours. MTT was added and cultured for four hours. Finally, a triple dissolution solution was added for 2 hours. The absorbance value of each well was tested with an ELISA reader. A control well and a zeroing well were set at the same time.

[0054] The results are as follows Fig.11 As shown, different concentrations of the fluorescent probe material ZX-1 were incubated in HeLa and 4T1 cells respectively. Even at a high concentration of 280 μM ZX-1, the survival rate of the cells can still reach more than 80%, indicating that the toxicity of the probe ZX-1 is very low.

[0055] (2) In order to verify the cell imaging effect of the probe, a preliminary cell imaging test was performed on ZX-1. In previous experiments, we have determined that HeLa cells can specifically express ENTK, while breast cancer cells MCF -7 had low ENTK expression levels. Therefore, we tried to express ENTK in HeLa cells and MCF - 7 cells were incubated with probe ZX-1 and the imaging was observed. Fig.12 As shown, ZX-1 can be specifically imaged in HeLa cells.

[0056] Example 5 ZX-1 In-tissue testing

[0057] At the same time, in order to prove the in situ imaging ability of the probe ZX-1 for tumor tissue, we immersed the probe material ZX-1 and the commercial dye Rhodamine B in 4T1 tumor tissue of tumor-bearing mice at 37°C for imaging, and observed the changes in fluorescence intensity at different times. Fig.13 As shown, the fluorescence intensity loss of the probe material ZX-1 within 8 hours is less than that of rhodamine B, which proves that the probe material ZX-1 has good long-term stable imaging capability.

[0058] In order to verify the tissue penetration effect of the probe, ZX-1 was tested for imaging of 4T1 tumor tissue slices of tumor-bearing mice. We imaged 4T1 tumor tissue and normal tissue slices of tumor-bearing mice under a 488nm laser as two groups of comparative experiments. The 4T1 tumor tissue of tumor-bearing mice with a slice thickness of 300μm was placed in a 1.5mL centrifuge tube, and 200μL of ZX-1 with a concentration of 100μM was added. The tissue slices were incubated for 50 minutes, and then the tissue slices were placed on a slide, covered with a coverslip, and the tissue slices were imaged under a confocal fluorescence microscope.

[0059] The results are as follows Fig.14 As shown, the fluorescence intensity of 4T1 tumor tissue in tumor-bearing mice was much higher than that of normal tissue sections, and the penetration depth was 157.46 μm. This result proves that ZX-1 has a superior penetration effect on tissue sections.

[0060] The materials, methods and embodiments described herein are exemplary only and not restrictive. A person skilled in the art may refer to the contents of this article and appropriately replace and / or modify the process parameters. However, it should be particularly noted that all similar replacements and / or modifications are obvious to a person skilled in the art and do not deviate from the spirit of the present invention or exceed the scope defined by the appended claims. Therefore, they are all considered to be included in the present invention.

Claims

1. A polypeptide fluorescent probe capable of specifically detecting enterokinase, characterized in that: Its structural formula is as follows:

2. A method for preparing a polypeptide fluorescent probe capable of in situ specific detection of enterokinase as claimed in claim 1, characterized in that: The preparation scheme is as follows: Step 1: preparing 2-(3,5,5-trimethylcyclohex-2-en-1-yl)malononitrile by isophorone (abbreviated as: Z1), the required reaction time is 8-12 hours, and the obtained product is finally recorded as Z2; Step 2: Prepare ((E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile using Z2 obtained in the first step. The required reaction time is 8-12 hours. Finally, the obtained product is recorded as ZX-FL. Step 3: Connect Fmoc-Lys(boc)-OH to CTC resin, then remove piperidine after 30 minutes of piperidine deprotection reaction, wash with dimethylformamide (DMF), and detect the deprotection color with ninhydrin. Then connect the subsequent straight-chain amino acids in sequence until the N-terminal Boc-Asp(otbu)-OH is connected, and condense with the dye molecule ZX-FL to obtain the target ZX-1.

3. The method for preparing enterokinase capable of in situ specific detection according to claim 2, characterized in that: The preparation scheme is as follows: Step 1: Dissolve Z1 (1.38 g, 10 mmol) and malononitrile (0.8 g, 12 mmol) in 40 mL of ethanol, add glacial acetic acid (5 μL, 0.1 mmol) and piperidine (1 μL, 1 mmol), and heat under reflux for 12 h under nitrogen protection; after the reaction is completed, cool to room temperature and remove ethanol in vacuo, purify by flash column chromatography, and spin dry to obtain 2-(3,5,5-trimethylcyclohex-2-en-1-yl) malononitrile; that is, Z2. Step 2: Dissolve Z2 (0.4 g, 2.16 mmol) and 4-aminobenzaldehyde (0.4 g, 2.4 mmol) prepared in the first step in 30 mL of ethanol, add piperidine (1 μL, 1 mmol), and heat under reflux for 12 h under nitrogen protection. After the reaction is completed, cool to room temperature, filter and collect the precipitate, and dry to obtain ((E)-2-(3-(4-aminophenylvinyl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile; that is, ZX-FL. Step 3: Fmoc-Lys(boc)-OH was connected to CTC resin, and then piperidine was deprotected for 30 minutes, then piperidine was removed and washed with dimethylformamide (DMF), and the deprotection color was detected by ninhydrin. Then, the subsequent straight-chain amino acids were connected in sequence until the Boc-Asp(otbu)-OH at the N-terminus was connected to obtain Boc-Asp(otbu)-Tyr((tbu)-Lys(boc)-Asp(otbu)-Asp(otbu)-Asp(otbu)-Asp(otbu)-Lys(boc)-CTC Resin. The above-mentioned protected polypeptide was cleaved from the resin with trifluoroethanol, washed with ether twice, and dried by rotation. The protected polypeptide and the small molecule ZX-FL were dissolved in DMF respectively, 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylcarbodiimide (DIC) were added after mixing, and the reaction was carried out for 6 hours. After drying by rotation, the polypeptide protecting group was cut off with trifluoroacetic acid to obtain the crude target product; liquid phase purification and freeze-drying were performed to obtain the target product ZX-1.

4. The polypeptide fluorescent probe for specific detection of enterokinase according to claim 1, wherein the polypeptide fluorescent probe prepared by the preparation method according to any one of claims 2 to 3 is used in the detection of enterokinase.

5. Use of the polypeptide fluorescent probe for specifically detecting enterokinase according to claim 1 or the polypeptide fluorescent probe prepared by the preparation method according to any one of claims 2 to 3 in detecting enterokinase in a tumor-bearing mouse model.

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