FRET hypoxia enzyme fluorescent probe based on perylene imide, preparation method and application thereof

Abstract

This case involves a FRET hypoxia enzyme fluorescent probe based on perylene imide, its preparation method and application. The material uses perylene imide as the main luminescent unit, azo derivatives as the modifying group, and BODIPY as the intramolecular energy donor to construct an intramolecular FRET molecule with the following structural formula: #imgabs0# wherein R1 is H or -N((CH2) m CH3)2, m is an integer of 2 to 10; R2 is a long-chain alkyl group. The hypoxia enzyme material prepared by the present invention modifies the azo group onto the perylene imide derivative for the first time, introduces it into the perylene imide through simple steps, and prepares a series of hypoxia enzyme perylene imide fluorescent materials with good light stability, obvious selective recognition of hypoxia enzyme, and strong anti-interference ability. It has strong absorption and fluorescence response to azoreductase in cells, and can realize dual signal detection of hypoxia enzyme in tumor cells.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probes, and in particular to a FRET hypoxic enzyme fluorescent probe based on perylene diimide, and a preparation method and application thereof. Background Art

[0002] Fluorescence imaging technology is widely used in cell and living biological imaging analysis due to its advantages of visualization, in situ and non-destructiveness. Among the commonly used fluorescent probes, small molecule fluorescent probes have the advantages of easy synthesis and modification, easy spectrum adjustment, high fluorescence quantum yield, good biocompatibility, etc. Through various principles (such as PET mechanism, ICT mechanism and FRET mechanism), fluorescent probes with high sensitivity, good selectivity and fast response can be constructed, thereby realizing responsive recognition of specific target substances.

[0003] Hypoxia is a common phenomenon in most solid tumors. Hypoxia can induce accelerated biological reduction reactions and lead to the expression of intracellular reductases, such as quinone reductase, azo reductase (NZR) and nitroreductase. Among them, azoreductase is the most representative. This enzyme catalyzes the reduction of azo to amino groups and has been proven to be an effective positioning and hypoxia imaging principle. At present, there are reports on recognition studies based on this NZR mechanism. However, most of the related studies are based on the single-molecule fluorescence response of rhodamine and cyanine dyes, while studies based on intramolecular FRET and azo recognition mechanisms are relatively lacking. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a fluorescent probe with intramolecular FRET and azo recognition, which can be used for near-infrared imaging of tumor hypoxia.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A FRET hypoxic enzyme fluorescent probe based on perylene imide, the material uses perylene imide as the main luminescent unit, an azo derivative as the modifying group, and BODIPY as the intramolecular energy donor to construct an intramolecular FRET molecule, and has the following structural formula:

[0007]

[0008] Wherein R1 is H or -N((CH2) m CH3)2, m is an integer from 2 to 10; R2 is a long-chain alkyl group.

[0009] The second object of the present invention is to provide a method for preparing the above-mentioned FRET hypoxia enzyme fluorescent probe based on perylene diimide, comprising the following steps:

[0010] S1: Add 1-bromoperylenetetramethylenedianhydride and an amino compound in a molar ratio of 1:1 into a reaction bottle, use ethanol as a solvent, heat to 85°C in an oil bath, and react for 48 hours. After the reaction is completed, separate and purify to obtain intermediate 1;

[0011] S2: Add intermediate 1 and 4-aminoazobenzene derivative to a reaction bottle, add glacial acetic acid / imidazole, raise the temperature to 125-135°C and react for 8 hours. After the reaction is completed, separate and purify to obtain intermediate 2;

[0012] S3: Under nitrogen protection, the intermediate 2, 5-hydroxypentyne and potassium carbonate were dissolved in ethylene glycol monomethyl ether and heated under reflux for 12 hours. After the reaction mixture was cooled to room temperature, water was added to the reaction solution, and a large amount of yellow solid was generated. The intermediate 3 was obtained by dichloromethane / petroleum ether column chromatography;

[0013] S4: Add intermediate 3, a BODIPY derivative with an azide group, sodium ascorbate and copper sulfate solution into a reaction bottle, add triethylamine dropwise, and stir at room temperature for 48 hours. After the reaction is completed, separate and purify the final product with dichloromethane / methanol.

[0014] Furthermore, the amino compound is a C4-C20 long-chain alkylamine; the 4' position of the 4-aminoazobenzene derivative is H or -N((CH2) m CH3)2 group.

[0015] The third object of the present invention is to provide the use of the perylene imide-based FRET hypoxia enzyme fluorescent probe as described above in near-infrared imaging of tumor hypoxia.

[0016] The present invention selects perylene imide derivatives as the luminescent subject for the first time, introduces an azo group into the amide of perylene through chemical modification to adjust the luminescent properties of the molecule, introduces a BODIPY energy acceptor group into the bend of perylene, constructs an anaerobic enzyme recognition group with intramolecular FRET properties, and realizes accurate recognition of anaerobic enzymes through the dual effects of switch response and FRET. The probe molecule reacts with the anaerobic enzyme, and the azo group is oxidized, thereby affecting the strong change in the luminescent intensity of the molecular subject. Through this selective recognition change, the concentration of anaerobic enzymes in cells or tissues is expressed, thereby realizing the diagnosis of cancer in the organism.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the hypoxia enzyme material prepared by the present invention modifies the azo group onto the perylene imide derivative for the first time, introduces the azo group onto the perylene imide derivative through simple steps, and prepares a series of hypoxia enzyme perylene imide fluorescent materials with good photostability, obvious selective recognition of hypoxia enzymes, and strong anti-interference ability, which have strong absorption and fluorescence response to azoreductase in cells, and can realize dual signal detection of hypoxia enzymes in tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the UV absorption spectrum of the probe molecule PDI-8.

[0020] Figure 2 This is the fluorescence spectrum diagram of the probe molecule PDI-8 recognizing and enhancing hypoxia enzymes.

[0021] Figure 3 The bar graph shows the selective recognition of the probe molecule PDI-8 to hypoxia enzyme (1-Na + , 2-K + , 3-Ca 2+ 、4-Mg 2+ , 5-H202, 6-GSH, 7-DTT, 8-Arg, 9-Try, 10-Cys, 11-His, 12-Leu, 13-Glu, 14-SOD, 15-HSA, 16-BSA, 17-GOx, 18-AZR).

[0022] Figure 4 These are fluorescence imaging images of the probe molecule PDI-8 used for liver cells (4a - imaging of the probe molecule and normal cells, 4b - imaging of the probe molecule and cancer cells under non-hypoxic conditions, 4c - imaging of the probe molecule and hypoxic cells under hypoxic conditions). DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Embodiment 1:

[0026] Step 1:

[0027]

[0028] Under nitrogen protection, add 3.2 g (12 mmol) of compound 1-bromoperylene tetracarboxylic anhydride PDI-1, 1.8 g (12 mmol) of 6-aminododecane, and 120 ml of anhydrous ethanol to a 250 mL two-necked bottle, and react in an oil bath at 85°C for 24 hours. The reaction progress is detected by thin layer chromatography. After the reaction is completed, the solvent is removed under reduced pressure, and the reaction solution is poured into 150 ml of ice water, and the pH is adjusted to weak acidity with dilute hydrochloric acid, and the mixture is filtered under reduced pressure and recrystallized from ethanol to obtain compound PDI-2, 8.3 g, with a yield of 60%. δ8.95 (s, 1H), 8.35-8.29 (d, J = 9.0 Hz, 3H), 8.20-8.16 (d, J = 9.0 Hz, 3H), 3.24 (m, 12H), 1.54-1.30 (m, 8H), 0.92 (m, 6H).

[0029] Step 2

[0030]

[0031] Under nitrogen protection, compound PDI-2 (4 g, 6.3 mmol) and 4-aminoazobenzene (1.9 g, 7 mmol) were dissolved in 100 ml of acetic acid / imidazole and then slowly heated to 125-130 ° C and kept warm for 36 hours. The reaction progress was tracked by chromatographic detection. After the reaction, the reaction solution was poured into 50 ml of ice water, and a large amount of red solid precipitated. The solution was filtered off under reduced pressure and dried. Column chromatography was performed using dichloromethane / methanol as the mobile phase to obtain 4.6 g of red solid PDI-3 with a yield of 79%. 1 H NMR (300MHz, CDCl3): δ8.95(s,1H),8.35-8.29(d,J=9.0Hz,3H),8.20-8.16(d,J=9.0Hz,3H),8.02-7.96(d,J=9.0H z, 4H), 7.36-7.33 (d, J = 9.0Hz, 4H), 4.26 (m, 2H), 3.24 (m, 1H), 2.88-1.42 (m, 24H), 1.44-1.30 (m, 8H), 1.24 (m, 12H).

[0032] Step 3:

[0033]

[0034] Under nitrogen protection, compound PDI-3 (5g, 5.4mmol), 5-hydroxypentyne 1g, 11mmol), potassium carbonate (4g) were dissolved in 100mL ethylene glycol monomethyl ether and heated under reflux for 12 hours. After the reaction was cooled to room temperature, 100mL water was added to the reaction solution, and a large amount of yellow solid was generated. A red solid of 3g was obtained by dichloromethane / petroleum ether column chromatography. Yield: 62%. 1H NMR (300MHz, CDCl3): δ8.95(s,1H),8.35-8.29(d,J=9.0Hz,3H),8.20-8.16(d,J=9.0Hz,3H),8.02-7.96(d,J=9.0Hz,4H),7.36-7.33(d ,J=9.0Hz,4H),4.26(m,2H),4.16(m,2H),3.24(m,1H),2.93(s,1H),2.46(m,2H),1.88-1.42(m,26H),1.44-1.30(m,8H),1.24(m,12H).

[0035] Step 4:

[0036]

[0037] Under nitrogen protection, 1 mL of triethylamine was added dropwise to a chloroform / ethanol / H2O (v / v / v=8:2:1) solution containing compounds BODIPY-1 (1.1 mmol, 0.5 g), PDI-4 (0.9 mmol, 0.8 g), sodium ascorbate (0.023 mmol, 0.0045 g) and CuSO4 (0.019 mmol, 0.003 g), and the mixture was stirred at room temperature for 48 hours. After the reaction was completed, the solvent was removed under reduced pressure, and a red solid was obtained by suction filtration. PDI-51.1 g was purified by column chromatography using dichloromethane / methanol (1:5), and the yield was 90%. 1 H NMR (300MHz, CDCl3): δ8.95 (s, 1H), 8.35-8.29 (d, J = 9.0Hz, 3H), 8.20-8.16 (d, J =9.0Hz,3H),8.02-7.96(d,J=9.0Hz,4H),7.52-7.50(d,J=6.0Hz,2H),7.36-7.3 3(d,J=9.0Hz,4H),6.93-6.91(d,J=6.0Hz,2H),6.02(s,2H),4.45-4.05(m,4H), 4.26(m,2H),2.88-1.42(m,30H),1.64(s,6H),1.44-1.30(m,8H),1.24(m,12H).

[0038] Embodiment 2:

[0039]

[0040] 3.0 g of red solid PDI-3 was obtained, with a yield of 78%. 1H NMR (300MHz, CDCl3): δ8.95(s,1H),8.35-8.29(d,J=9.0Hz,3H),8.20-8.16(d,J=9.0Hz,3H),8.02-7.96(d,J=9.0H z, 4H), 7.36-7.33 (d, J = 9.0Hz, 4H), 4.26 (m, 2H), 3.24 (m, 1H), 2.88-1.42 (m, 22H), 1.44-1.30 (m, 4H), 1.24 (m, 6H).

[0041] Step 2:

[0042]

[0043] A large amount of yellow solid was generated. Yield: 84%. 1 H NMR (300MHz, CDCl3): δ8.95(s,1H),8.35-8.29(d,J=9.0Hz,3H),8.20-8.16(d,J=9.0Hz,3H),8.02-7.96(d,J=9.0Hz,4H),7.36-7.33( d,J=9.0Hz,4H),4.26(m,2H),4.16(m,2H),3.24(m,1H),2.93(s,1H),2.46(m,2H),1.88-1.42(m,22H),1.44-1.30(m,4H),1.24(m,6H).

[0044] Step 3:

[0045]

[0046] 4.6 g of red solid PDI-5 was obtained, with a yield of 90%. 1 H NMR (300MHz, CDCl3): δ8.95 (s, 1H), 8.35-8.29 (d, J = 9.0Hz, 3H), 8.20-8.16 (d, J=9.0Hz, 3H), 8.02-7.96 (d, J=9.0Hz, 4H), 7.52-7.50 (d, J=6.0Hz, 2H), 7.36-7. 33(d,J=9.0Hz,4H),6.93-6.91(d,J=6.0Hz,2H),6.02(s,2H),4.45-4.05(m,4H) ,4.26(m,2H),2.88-1.42(m,22H),1.64(s,6H),1.44-1.30(m,4H),1.24(m,6H).

[0047] The material prepared in Example 1 was tested for fluorescence and ultraviolet spectrum: the material was configured into 10 -5 ~10 -6 mol / L concentration of dichloromethane solution, using ultraviolet absorption spectrometer and fluorescence spectrometer to measure the absorption spectrum and emission spectrum of the material respectively, such as Figure 1-2 shown.

[0048] The reaction conditions designed by the invention are mild, the reaction is efficient, the operation is simple, and the applicability is wide, which provides a new idea for enriching and developing infrared hypoxia enzyme detection.

[0049] Example 3: Fluorescence recognition experiment:

[0050] 2 μg / mL of fluorescent probe PDI-8 was mixed with metal ions (Na + , K + , Ca 2+ Mg 2+ , 2mM), redox molecules (AA, H2O2, GSH, DTT, 2mM), amino acids (Arg, Try, Cys, His, Leu, Glu, 2mM) and proteins (BSA, HSA, 2mg / mL; SOD, GOx, 2μg / mL) and 2μM AZR were reacted at 37℃ for half an hour, and then the fluorescence changes were detected. Figure 3 It can be found that except for AZR, the fluorescence signals of other solutions did not change significantly. The results show that the probe CHR-C has a specific response ability to hypoxia-related azoreductase.

[0051] Example 4: Fluorescence cell imaging experiment:

[0052] The present invention further investigates the use of PDI-8 for cell fluorescence imaging. After cancer cells were inoculated in a 35 mm culture dish, they were incubated in 1% oxygen and normoxic environments for 24 hours, then reacted with 2 μg / mL probe PDI-8 for a period of time and stained with AZR for cell nuclei, and finally imaged using a confocal fluorescence microscope, with cells not treated with the probe as controls, and the excitation wavelength was 405 nm. Figure 4 As shown, regardless of whether the cells are in normoxic or hypoxic state, when there is no reaction with the probe PDI-8, no red light can be observed after 405nm laser irradiation ( Figure 4 a); When the cells in hypoxic state are not bound to the probe, only a very dim red light is emitted Figure 4 b); when cells in a hypoxic state react with the hypoxia probe and then irradiate with laser, obvious red fluorescence can be observed ( Figure 4 c). The results show that the probe constructed in this case can indeed be used for hypoxia imaging.

[0053] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A FRET hypoxia enzyme fluorescent probe based on perylene diimide, characterized in that: The material uses perylene imide as the main luminescent unit, azo derivatives as the modifying group, and BODIPY as the intramolecular energy donor to construct an intramolecular FRET molecule with the following structural formula: Wherein, R1 is H or -N((CH2) m CH3)2, m is an integer from 2 to 10; R2 is a long chain alkyl group from C4 to C20.

2. The method for preparing the perylene imide-based FRET hypoxia enzyme fluorescent probe according to claim 1, characterized in that: The steps include: S1: Add 1-bromoperylenetetramethylenedianhydride and an amino compound in a molar ratio of 1:1 into a reaction bottle, use ethanol as a solvent, heat to 85°C in an oil bath, and react for 48 hours. After the reaction is completed, separate and purify to obtain intermediate 1; S2: Add intermediate 1 and 4-aminoazobenzene derivative to a reaction bottle, add glacial acetic acid / imidazole, raise the temperature to 125-135°C and react for 8 hours. After the reaction is completed, separate and purify to obtain intermediate 2; S3: Under nitrogen protection, the intermediate 2, 5-hydroxypentyne and potassium carbonate were dissolved in ethylene glycol monomethyl ether and heated under reflux for 12 hours. After the reaction mixture was cooled to room temperature, water was added to the reaction solution, and a large amount of yellow solid was generated. The intermediate 3 was obtained by dichloromethane / petroleum ether column chromatography; S4: Add intermediate 3, a BODIPY derivative with an azide group, sodium ascorbate and copper sulfate solution into a reaction bottle, add triethylamine dropwise, and stir at room temperature for 48 hours. After the reaction is completed, separate and purify the final product with dichloromethane / methanol.

3. The method for preparing the perylene imide-based FRET hypoxia enzyme fluorescent probe according to claim 2, characterized in that: The amino compound is a C4-C20 long-chain alkylamine.

4. The method for preparing the perylene imide-based FRET hypoxia enzyme fluorescent probe according to claim 2, characterized in that: The 4' position of the 4-aminoazobenzene derivative is H or -N((CH2) m CH3)2 group.

5. Use of the perylene imide-based FRET hypoxia enzyme fluorescent probe as claimed in claim 1 in near-infrared imaging of tumor hypoxia.

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