CN113234014B - Aggregation-induced emission fluorescent probe for detecting aminopeptidase N and preparation thereof - Google Patents

Aggregation-induced emission fluorescent probe for detecting aminopeptidase N and preparation thereof Download PDF

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CN113234014B
CN113234014B CN202110452636.XA CN202110452636A CN113234014B CN 113234014 B CN113234014 B CN 113234014B CN 202110452636 A CN202110452636 A CN 202110452636A CN 113234014 B CN113234014 B CN 113234014B
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颜梅
卫先哲
张晶
杨小凤
朱彤
李成芳
于京华
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University of Jinan
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D215/14—Radicals substituted by oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
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    • C09K2211/1007—Non-condensed systems
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom

Abstract

The invention discloses a gathering induced emission fluorescent probe for detecting aminopeptidase N and a preparation method thereof, wherein the structure of a probe compound is shown as a formula I. The probe molecule consists of three main components: an aggregation-induced emission fluorophore based on quinoline-malononitrile, a self-cleavable linker and an aminopeptidase n (apn) -specific recognition group. When the N-terminal alanyl site of the probe compound is accurately hydrolyzed into amino by APN, the exposed amino is used as an electron supply group, so that the electron push-pull effect of a conjugated system is promoted, and the fluorescence is enhanced. The probe has the advantages of high response speed, high sensitivity, large Stokes shift and longer emission wavelength, and when the concentration of the solution is higher, the fluorescence emission is stronger, so that the problem of fluorescence quenching induced by aggregation is effectively solved.

Description

Aggregation-induced emission fluorescent probe for detecting aminopeptidase N and preparation thereof
Technical Field
The invention relates to a position and expression level of active enzyme in a cell by a small molecular fluorescent probe in-situ detection, in particular to an accurate detection of endogenous aminopeptidase N content by the fluorescent probe based on aggregation-induced emission, belonging to the technical field of fluorescent probes.
Background
Aminopeptidase N (APN/CD 13), known as membrane-bound zinc ion-dependent type II metalloproteases, degrades neutral or basic amino acids at the N-terminus of the protein polypeptide chain. APN is accordingly involved in many important physiological and pathological processes in the body, such as signal transduction, immunomodulation, hydrolysis of bioactive peptides and invasion of coronaviruses. Many studies have shown that APNs are highly expressed on the surface of most tumor cells compared to normal cells, while APNs play a crucial role in the growth, invasion, metastasis and angiogenesis of malignant tumors. Therefore, APN has the potential to become a tumor cell biomarker, and the development of an effective method for tracking the activity of APN in vivo has great medical significance.
In recent years, the small molecule fluorescence imaging technology has attracted great interest, and the fluorescence imaging has obvious advantages in the aspects of rapid detection, high selectivity, high sensitivity, excellent time and spatial resolution, good biocompatibility and the like. To date, several fluorescent probes have been reported to monitor and visualize APNs in cells. However, most of the conventional fluorescent probes have hydrophobic characteristics of a planar structure. Such probes suffer from aggregation-induced fluorescence quenching problems (ACQ) in concentrated solutions or in aggregated states, which results in their emission being diminished or even quenched. In contrast to common ACQ dyes, Aggregation Induced Emission (AIE) fluorescent dyes have a rotatable structure or a twisted propeller-shaped conformation, which can produce high fluorescence emission due to the restriction of intramolecular movement. In particular, their fluorescence emission is stronger when the solution concentration is higher. Therefore, AIE fluorophores are ideal candidates for detecting APNs in organisms. In addition, probes with longer emission wavelengths (>600 nm) have less light damage and deeper tissue penetration, minimizing the unwanted effects of background fluorescence. However, to our knowledge, few aggregation-induced emission-based fluorescent probes that activate long-emission wavelength detection APN have been reported.
The invention discloses a gathering induced emission fluorescent probe for detecting aminopeptidase N and a preparation method thereof, wherein the structure of a probe compound is shown as a formula I. The probe molecule consists of three main components: an aggregation-induced emission fluorophore based on quinoline-malononitrile, a self-cleavable linker and an aminopeptidase n (apn) -specific recognition group. When the N-terminal alanyl site of the probe compound is accurately hydrolyzed into amino by APN, the exposed amino is used as an electron supply group, so that the electron push-pull effect of a conjugated system is promoted, and the fluorescence is enhanced. The probe has the advantages of high response speed, high sensitivity, large Stokes shift and longer emission wavelength, and when the concentration of the solution is higher, the fluorescence emission is stronger, so that the problem of fluorescence quenching induced by aggregation is effectively solved.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the probe compound has the advantage of carrying out aggregation-induced fluorescence in situ imaging on the endogenous APN of cancer cells and tissues. Secondly, a fluorescence diagnostic reagent is provided to effectively and accurately distinguish the liver cancer cells over expressing APN from normal cells. And thirdly, the fluorescent probe with high sensitivity and good selectivity is provided, and the defects of shallow tissue penetration depth and high background fluorescence of the probe can be overcome.
In order to solve the technical problems, the technical scheme is as follows:
the invention provides a gathering induced emission fluorescent probe for detecting aminopeptidase N, which has the following molecular structural formula:
Figure DEST_PATH_IMAGE002
compounds MQ-N
The invention also provides a preparation method of the aggregation-induced emission fluorescent probe for detecting aminopeptidase N, which comprises the following steps:
(1) (a) in N2Dissolving 2-methylquinoline (1 eq) and methyl iodide (2.5-3 eq) in acetonitrile under the atmosphere, refluxing the reaction solution for 10-15 hours, and concentrating under reduced pressure to obtain a compound 1; (b) dissolving compound 1 (1 eq) and malononitrile (2.5-3 eq) in absolute ethanol, continuously stirring at 0 ℃, adding sodium ethoxide, reacting for 5-8 hours, pouring the mixture into ice water, adjusting the pH (7.5-8.5), and filtering the generated precipitate to obtain compound 2; (c) compound 2 (1 eq) and p-hydroxybenzaldehyde (1.2-1.8 eq) were dissolved in acetonitrile, then piperazine was added to the solution, and the mixture was taken up in N2Refluxing for 8-12 h under atmosphere, and purifying with column (dichloromethane/petroleum ether) to obtain compound 3;
Figure DEST_PATH_IMAGE004
(2) (d) dissolving Boc-L-alanine (1 eq), HATU (1-1.2 eq) and DIPEA (2-3 eq) in an anhydrous dichloromethane solution at 0 ℃, stirring at room temperature for 0.5-1.5 hours, adding p-aminobenzyl alcohol (1-1.2 eq) to the reaction mixture, and further stirring at room temperature for 24-30 hours, column purification (dichloromethane/methanol) to obtain compound 4; (e) dissolving compound 4 (1 eq) in anhydrous tetrahydrofuran andadding PBr at 0 DEG C3(1.2-1.6 eq), stirring the mixture for 2-5 hours, and column purification (petroleum ether/ethyl acetate) to give compound 5;
Figure DEST_PATH_IMAGE006
(3) (f) dissolving Compound 3 (1 eq) and Compound 5 (1.1-1.2 eq) in anhydrous DMF solution, and adding K2CO3And stirred at 120 ℃ for 6-8 hours, column purified (petroleum ether/ethyl acetate) to give the compound MQ-N.
Figure DEST_PATH_IMAGE008
The invention has the advantages that:
the fluorescent probe molecule has the property of aggregation-induced fluorescence emission, can effectively reduce the interference of background fluorescence on detection signals, and improves the tissue penetration depth in vivo imaging.
The fluorescent probe molecule has very high response speed to aminopeptidase N, can completely respond within 20 minutes, and can be applied to quickly detecting the enzyme content in a complex sample.
The fluorescent probe molecule has good sensitivity and selectivity, a fluorescent signal only occurs in the presence of aminopeptidase N, and other common inorganic salts, amino acids, biological enzymes and the like cannot cause the probe solution to generate the change of a fluorescence spectrum.
Therefore, the invention provides a reliable means for non-invasively monitoring the change of the activity of leucine aminopeptidase in vivo. Has wide application prospect in the field of biological analysis and detection.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1
A preparation method of an aggregation-induced emission fluorescent probe for detecting aminopeptidase N comprises the following steps:
1) synthesis of Compound 1:
in N22-methylquinoline (1.79 g, 12.5 mmol) and iodomethane (5.50 g, 35 mmol) were dissolved in acetonitrile (25 mL) under an atmosphere, and the reaction was refluxed for 10 hours and the solvent was evaporated under reduced pressure to give compound 1 in 75% yield.
Synthesis of Compound 2:
compound 1 (1.74 g, 6 mmol) and malononitrile (1.20 g, 18 mmol) were dissolved in absolute ethanol (15 mL) and stirred constantly at 0 ℃, reacted for 6 hours after addition of sodium ethoxide, the mixture was poured into ice water, after adjusting the pH to 8, the resulting precipitate was filtered, washed with water and dried in vacuo to give compound 2 in 85% yield.
Synthesis of Compound 3:
compound 2 (1.40 g, 7.5 mmol) and p-hydroxybenzaldehyde (1.65 g, 13.5 mmol) were dissolved in acetonitrile (30 mL), then piperazine was added to the solution, and the mixture was stirred in N2Reflux under atmosphere for 12 h, evaporation of solvent under reduced pressure, and column purification (dichloromethane/petroleum ether = 2/1) afforded compound 3 in 65% yield.
2) Synthesis of Compound 4:
Boc-L-alanine (0.17 g, 0.9 mmol), HATU (0.42 g, 1.1 mmol) and DIPEA (0.37 mL, 2.3 mmol) were dissolved in anhydrous dichloromethane solution (30 mL) at 0 ℃, stirred at room temperature for 1.2 hours, p-aminobenzyl alcohol (0.14 g, 1.1 mmol) was added to the reaction mixture and further stirred at room temperature for 25 hours, the solvent was evaporated under reduced pressure and column purified (dichloromethane/methanol = 20/1) to give compound 4 in 70% yield.
Synthesis of Compound 5:
(e) compound 4 (0.59 g, 2 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) and PBr was added at 0 deg.C3(0.23 mL, 2.4 mmol), the mixture was stirred for 3 hours, the solvent was evaporated under reduced pressure and column purified (petroleum ether/ethyl acetate = 9/1) to give compound 5 in 40% yield.
3) Synthesis of Compound MQ-N:
compound 3 (0.37 g, 1.1 mmol) and compound 5 (0.43 g, 1.2 mmol) were dissolved in anhydrous DMF solution (5 mL) and K was added2CO3And stirred at 120 ℃ for 7 hours, the solvent was evaporated under reduced pressure and column purified (petroleum ether/ethyl acetate = 10/1) to give compound MQ-N in 65% yield.
Example 2
Measurement of MQ-N absorption and fluorescence spectra of probes
A stock solution (1.0 mM) of the probe MQ-N in DMSO was prepared in a test tube using the MQ-N synthesized in example 1. Preparation of NaCl, KCl, CaCl in distilled water2，ZnCl2，MgCl2，CuCl2，H2O2Stock solutions of NaClO, glucose, GSH, Cys, Hcy, alkaline phosphatase, gamma-glutamyltranspeptidase, nitroreductase, carboxylesterase, and the like. The final volume of each stock was adjusted to 5 mL with phosphate buffer. For spectroscopic experiments, after incubating the MQ-N stock solution with a quantity of analyte in a quartz cuvette for 30 minutes at pH =7.4, 3 mL of the reaction solution was transferred to a 1 cm quartz cuvette with excitation set to 480 nm and emission wavelength 500 and 700 nm. Meanwhile, a control group without aminopeptidase N was prepared and compared under the same conditions. MQ-N shows double absorption peaks near 350 nm and 425 nm, and after APN is added to the micelle probe, the absorption peak centered at 350 nm is weakened, and then the peak at 425 nm is remarkably increased. The characteristics of the probe MQ-N under APN treatment at different concentrations were then evaluated, with increasing APN the fluorescence intensity gradually increasing at 630 nm. Experimental data show that MQ-N has quite high sensitivity to APN and can quantitatively detect APN concentration.
Example 3
Evaluating the influence of different pH values and temperatures on the MQ-N fluorescence of the probe
Taking MQ-N stock solution (10. mu.M) from example 2, the probe was almost non-fluorescent in the pH range of 4.0-8.0 and the temperature range of 23-40 ℃, indicating a high stability of the probe. After reaction with aminopeptidase N, the probe produced a significant fluorescent response throughout the pH range tested, although the maximum fluorescence intensity occurred in the pH range of 6.0-7.4. On the other hand, the strongest fluorescence of the reaction solution occurs at about 37 ℃, which is consistent with the fact that enzymes typically have the greatest activity at 37 ℃. The result shows that the probe MQ-N has good function under normal physiological conditions.
Example 4
Specific detection of APN by probe MQ-N
Taking MQ-N stock solution (10. mu.M) from example 2, the fluorescent response of the probe to APN and some potential interferents, such as some metal ions (Na) that may form complexes with the probe or APN, were tested+，K+，Ca2+，Zn2+，Mg2+And Cu2 +) Possibly oxidizing the active oxygen (NO) of the probe2 -，NO，HNO，1O2，H2O2，ClO-TBHP,. OH and O2 -) Biomolecules (GSH, Hcy and Cys) and APN-related enzymes (alkaline phosphatase, gamma-glutamyltranspeptidase, nitroreductase, carboxylesterase, beta-galactosidase, etc.). When APN was added to the MQ-N solution, the fluorescence intensity at 630 nm increased significantly, but the other substances failed to cause significant fluorescence change. Subsequent further competitive experiments with APN and other interfering substances were performed, and the results indicated that the APN-induced fluorescence response was not affected by other interferences. Therefore, probe MQ-N has superior selectivity for APN in various competitive analytes.
Example 5
Toxicity of MQ-N to cancer cells and determination of endogenous APN
Cell viability was tested by a method of reducing MTT using mitochondrial dehydrogenase. HepG-2 cells at 1X 105cells/mL were seeded in 96-well microplates containing LBS in medium. After 24 hours of cell attachment, the plates were then washed with PBS and the cells were cultured in media containing 0, 1, 2, 5, and 10 μ M probe MQ-N for 24 hours. Meanwhile, cells not cultured with the probe MQ-N were set up as a control. MTT (10. mu.L, 5 mg/mL) was injected into each well and the plates were incubated in 5% CO2Incubate in a humidified incubator for 4 hours. Then removing the culture medium and using the enzymeThe optical density of the solution was measured at 490 nm with a standard instrument to assess cell viability. After 24 hours of treatment of the cells with different concentrations of MQ-N, more than 90% of the cells survived, demonstrating low toxicity of the probe to the cells. Subsequently, MQ-N was evaluated by confocal fluorescence microscopy for the ability to track endogenous APNs in cancer and normal cells. 5% CO at MQ-N (5. mu.M) and 37 ℃2The cells were pretreated for 0.5 hour, and there was almost no fluorescence in normal cells. In contrast, the red fluorescence signal increased dramatically after probe incubation with cancer cells. Therefore, these data indicate that the probe MQ-N exhibits good membrane permeability and can effectively distinguish between cancer cells and normal cells.
The foregoing is only a preferred embodiment of this invention and is not intended to limit the invention in any way, so that any person skilled in the art may, using the teachings disclosed above, modify or adapt for various equivalent embodiments with equivalent modifications. The design concept of the present invention is not limited thereto, and any insubstantial modifications made to the present invention using this concept shall fall within the scope of infringing upon the present invention.

Claims (4)

1. A preparation method of an aggregation-induced emission fluorescent probe for detecting aminopeptidase N is characterized in that the structure of the compound is shown as a formula I:
Figure DEST_PATH_IMAGE001
formula I;
the preparation method comprises the following steps:
(1) (a) in N2Dissolving 2-methylquinoline and methyl iodide in acetonitrile under the atmosphere, refluxing the reaction solution for 10-15 hours, and concentrating under reduced pressure to obtain a compound 1; (b) dissolving the compound 1 and malononitrile in absolute ethyl alcohol, continuously stirring at 0 ℃, adding sodium ethoxide, reacting for 5-8 hours, pouring the mixture into ice water, adjusting the pH, and filtering the generated precipitate to obtain a compound 2; (c) dissolving Compound 2 and p-hydroxybenzaldehyde in acetonitrile, and adding piperazineTo this solution, the mixture is dissolved in N2Refluxing for 8-12 hours under the atmosphere, and purifying by a column to obtain a compound 3;
Figure 590130DEST_PATH_IMAGE002
(2) (d) dissolving Boc-L-alanine, HATU and DIPEA in an anhydrous dichloromethane solution at 0 ℃, stirring at room temperature for 0.5-1.5 hours, adding p-aminobenzyl alcohol to the reaction mixture, further stirring at room temperature for 24-30 hours, and column purifying to obtain compound 4; (e) compound 4 was dissolved in anhydrous tetrahydrofuran and PBr was added at 0 deg.C3Stirring the mixture for 2-5 hours, and purifying the mixture by a column to obtain a compound 5;
Figure DEST_PATH_IMAGE003
(3) (f) dissolving Compound 3 and Compound 5 in anhydrous DMF, and adding K2CO3Stirring at 120 deg.C for 6-8 hr, and purifying with column to obtain compound MQ-N
Figure 507270DEST_PATH_IMAGE004
。
2. The method for preparing the fluorescence probe for detecting aggregation-induced emission of aminopeptidase N according to claim 1, wherein the fluorescence probe comprises: the molar ratio of the 2-methylquinoline to the methyl iodide in the step (1) (a) is in the range of 1: (2.5-3); the molar concentration range of the 2-methylquinoline dissolved in the acetonitrile solution is 0.35-0.55 mol.L-1(ii) a (b) The molar ratio of the compound 1 to the malononitrile is 1: (2.5-3); the molar concentration range of the compound 1 dissolved in the ethanol solution is 0.3-0.45 mol.L-1(ii) a Adjusting the pH range to 7.5-8.5; (c) the mol ratio of the compound 2 to the p-hydroxybenzaldehyde is 1: (1.2-1.8); the molar concentration range of the compound 2 dissolved in the acetonitrile solution is 0.2-0.35 mol.L-1(ii) a Column purityThe volume ratio of dichloromethane to petroleum ether in the reaction is (2-1): 1.
3. the method for preparing the fluorescence probe for detecting aggregation-induced emission of aminopeptidase N according to claim 1, wherein the fluorescence probe comprises: in the step (2) (d), the mole ratio of Boc-L-alanine, p-aminobenzyl alcohol, HATU and DIPEA is in the range of 1: (1-1.2): (1-1.2): (2-3); the molar concentration range of the Boc-L-alanine dissolved in the anhydrous dichloromethane solution is 0.025-0.035 mol.L-1(ii) a The volume ratio of the dichloromethane to the methanol in the column purification is (20-15): 1; (e) compound 4 and PBr3The molar ratio ranges from 1: (1.2-1.6); the molar concentration range of the compound 4 dissolved in the anhydrous tetrahydrofuran solution is 0.2-0.26 mol.L-1(ii) a The volume ratio of petroleum ether to ethyl acetate in column purification is (10-5): 1.
4. the method for preparing the fluorescence probe for detecting aggregation-induced emission of aminopeptidase N according to claim 1, wherein the fluorescence probe comprises: the molar ratio of the compound 3 to the compound 5 in the step (3) (f) is in the range of 1: (1.1-1.2); the molar concentration of the compound 3 dissolved in the anhydrous DMF solution is 0.2-0.24 mol.L-1(ii) a The volume ratio of petroleum ether to ethyl acetate in column purification is (10-6): 1.
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