A fluorescent probe for nitroreductase, its preparation method and application

Abstract

This invention belongs to the field of fluorescent probe technology, specifically relating to a nitroreductase fluorescent probe, its preparation method, and its application. To develop an NTR fluorescent probe with high selectivity, high sensitivity, short response time, and long excitation and emission wavelengths, this invention utilizes pentamethine cyanine dye and a "ester group → carboxyl group" conversion strategy at the central site of this dye to develop a nitroreductase fluorescent probe, Cy5-NTR. In the presence of NADPH, the nitro group of the probe is first catalyzed by the overexpression of NTR by cancer cells, resulting in a single-electron transfer and the generation of a nitro anion radical. This radical is then further reduced to an amino group, followed by a 1,6-rearrangement and elimination reaction, releasing the strongly fluorescent centrally carboxyl-substituted pentamethine cyanine dye Cy5-COO. This probe can not only distinguish cancer cells / tissues from normal cells / tissues with high contrast under hypoxic conditions, but also provide in situ real-time imaging of tumors in tumor-bearing mice.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a nitroreductase fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Nitroreductase (NTR) is an endogenous flavoprotein enzyme with reduction function, which widely exists in bacteria, eukaryotes and mammals, and can reduce metabolic aromatic nitro compounds or nitro-substituted heterocyclic compounds under the action of coenzyme nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NADH / NADPH) electron donor. Hypoxia is a common feature of solid tumors. At present, several methods are used to evaluate the degree of tumor hypoxia, such as oxygen pressure measurement, blood flow velocity measurement and hypoxia marker detection, etc. However, these methods are too cumbersome in actual operation. NTR is usually overexpressed in hypoxic tumor cells, which can regulate the growth and apoptosis of tumor cells, and plays an important role in tumor invasion, metastasis and drug resistance. Therefore, NTR is widely used to evaluate the degree of tumor hypoxia, and thus quantitative detection of NTR is of great significance for the diagnosis and treatment of cancer. At present, the most effective method for detecting NTR activity is the fluorescent probe method. However, most of the reported nitroreductase fluorescent probes have problems such as long reaction time, poor sensitivity and short excitation and emission wavelength, etc. Therefore, it is necessary to develop NTR fluorescent probes with high selectivity, high sensitivity, short response time and long excitation and emission wavelength. SUMMARY

[0003] The application is based on pentamethine cyanine dye, and a nitroreductase (NTR) fluorescent probe Cy5-NTR is developed by using the conversion strategy of the center site "ester group -> carboxyl group" of the dye. In the presence of NADPH, the nitro group of the probe is first catalyzed by NTR overexpressed in cancer cells to undergo single electron transfer to generate a nitro anion radical, which is then further reduced to an amino group, followed by 1,6-rearrangement and elimination reaction, thereby releasing the strong fluorescent center site carboxyl-substituted pentamethine cyanine dye Cy5-COO, resulting in a significant increase in solution fluorescence. The probe can detect NTR with high sensitivity and selectivity, and the detection limit can reach 0.001 μg / mL. Cell and in vivo experiments show that the probe has low toxicity and good biological compatibility, can not only distinguish cancer cells / tissues and normal cells / tissues with high contrast under hypoxic conditions, but also can in situ real-time image the tumor of tumor-bearing mice, and thus has great application potential in fluorescence-guided tumor surgery.

[0004] To achieve the above-mentioned purposes, the application adopts the following technical solutions:

[0005] A nitroreductase fluorescent probe has the following structural formula:

[0006]

[0007] A preparation method of a nitroreductase fluorescent probe, comprising the following steps:

[0008]

[0009] Step 1, in an N2 environment, DMF is dissolved in anhydrous dichloromethane, then oxalyl chloride is gradually added, and the reaction is stirred at room temperature; after the reaction is completed, the solvent is fully evaporated under reduced pressure to obtain compound 1 as a white solid, which is directly used in the next reaction without purification;

[0010] Step 2, compound 1 and monomethyl malonate are dissolved in anhydrous dichloromethane to perform a reflux reaction; after the solvent is evaporated under reduced pressure, compound 2 is obtained; then acetic anhydride, 1,2,3,3-tetramethyl-3H-indole iodide and anhydrous sodium acetate are sequentially added to perform a stirring reaction; then the reaction solution is diluted with water and extracted with dichloromethane; the organic layers are combined and dried over anhydrous sodium sulfate, distilled under reduced pressure and separated and purified by column chromatography to obtain compound Cy5-COOM as a dark blue solid;

[0011] Step 3, compound Cy5-COOM is dissolved in a mixed solution of MeOH and NaOH to perform a stirring reaction; after cooling, the reaction solution is diluted with water and extracted with dichloromethane; the organic layers are combined and dried over anhydrous sodium sulfate, distilled under reduced pressure and separated and purified by column chromatography to obtain compound Cy5-COO as a dark blue solid;

[0012] Step 4, Cy5-COO, p-nitrobenzyl bromide and potassium carbonate are dissolved in anhydrous N,N-dimethylformamide to perform a stirring reaction; after the reaction is completed, the reaction solution is cooled, diluted with water and extracted with dichloromethane; the organic phases are combined and dried over anhydrous sodium sulfate, distilled under reduced pressure and separated and purified by column chromatography to obtain compound Cy5-NTR as a dark blue solid.

[0013] Further, in step 1, the molar ratio of DMF to oxalyl chloride is 30:35, and the stirring reaction time is 2 h.

[0014] Further, in step 2, the molar ratio of compound 1 to monomethyl malonate is 2:1, the reflux reaction temperature is 40°C, and the reflux reaction time is overnight; the molar ratio of compound 2, 1,2,3,3-tetramethyl-3H-indole iodide and anhydrous sodium acetate is 1:2:3, the stirring reaction temperature is 90°C, and the stirring reaction time is 4 h; the developing agent for column chromatography separation and purification is dichloromethane / methanol = 10 / 1 (v / v).

[0015] Further, in step 3, the stirring reaction temperature is 43°C, and the stirring reaction time is 3 h; the developing agent for column chromatography separation and purification is 10-50% methanol / dichloromethane (v / v).

[0016] Further, the molar ratio of Cy5-COO, p-nitrobenzyl bromide and potassium carbonate in step 4 is 1:3:2, the stirring reaction temperature is 48℃, the time is 3h, and the developing agent for column chromatography separation and purification is CH2Cl2 / MeOH=15:1(v / v).

[0017] The application of the nitroreductase fluorescent probe is used for distinguishing normal cells / tissues and cancer cells / tissues, and is used for quickly imaging tumors of tumor-bearing mice.

[0018] Compared with the prior art, the application has the following advantages:

[0019] The fluorescent probe method is one of the most effective methods for detecting NTR activity, however, most of the reported nitroreductase fluorescent probes have problems of long reaction time, poor sensitivity and short excitation and emission wavelength. The NTR fluorescent probe developed in the application can detect NTR with high sensitivity and high selectivity in the far infrared to near infrared region, the response time is less than 5 minutes in vitro, and the detection limit is 0.001 μg / mL. In addition, due to the high sensitivity of the probe, the probe can not only distinguish cancer cells / tissues and normal cells / tissues with high contrast under hypoxic conditions, but also can realize rapid imaging (1 minute) of tumors, so it has great application potential in fluorescence-guided tumor surgery. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 NMR and HRMS graphs of the compound Cy5-COO;

[0021] Figure 2 NMR and HRMS graphs of the compound Cy5-COOM;

[0022] Figure 3 NMR and HRMS graphs of the compound Cy5-NTR;

[0023] Figure 4 In (A), it is the ultraviolet-visible absorption spectrum before and after the reaction of Cy5-NTR (2 μM) and NTR (1.0 μg / mL, containing 0.5 mM NADPH) under the condition of PBS (10 mM, pH=7.4) at 37℃; (B) is a fluorescence spectrum change diagram after the reaction of Cy5-NTR (5 μM) and APN (2.5 μg / mL, containing 0.5 mM NADPH) under the condition of PBS (10 mM, pH=7.4) at 37℃, and a diagram of the change of fluorescence intensity at 662 nm with time;

[0024] Figure 5The HPLC and HRMS chromatograms before and after the reaction of Cy5-NTR with NTR (containing NADPH) are shown. HPLC conditions: C18 column (2.1×100 mm), mobile phase: MeCN / H2O (3:7 to 9:1, v / v, containing 0.1% formic acid), flow rate: 0.2 mL / min.

[0025] Figure 6 In the figure, (A) shows the fluorescence spectra of Cy5-NTR (2 μM) before and after reaction with increased concentrations of APN (0-0.5 μg / mL, containing 0.5 mM NADPH) under the conditions of 37℃, PBS (10 mM, pH=7.4); (B) shows the linear correlation between fluorescence intensity at 662 nm and NTR concentration.

[0026] Figure 7 To determine the reaction conditions at 37°C, PBS (10 mM, pH = 7.4), Cy5-NTR (2 μM) in DMEM, or with (B) NADPH (500 μM), (C) GSH (1 mM), (D) Cys (200 μM), (E) H2O2 (100 μM), and (F) O2, respectively. ·- UV-Vis absorption spectrum changes after reaction with (100μM), (G)APN (50ng / mL), and (H)GGT (50U / L) (30 minutes);

[0027] Figure 8 Cell viability of A549 cells after treatment with different concentrations (0 μM, 2.0 μM, 4.0 μM, 6.0 μM, 8.0 μM and 10.0 μM) of Cy5-NTR for 24 hours;

[0028] Figure 9 Cell images of cancer cells (HepG2 and A549 cells) and normal cells (BEAS-2B and LO2 cells) loaded with Cy5-NTR (2.0 μM), respectively; for cancer cells, cells were incubated with the probe for 60 minutes, or pre-incubated with an inhibitor (DIC, 100 μM, 60 minutes) before incubating with the probe for 60 minutes; for normal cells, cells were incubated with the probe only for 60 minutes; images were collected at wavelengths of 640–750 nm (λ). ex =633nm), scale bar: 20μm;

[0029] Figure 10 In the image, (A) shows confocal images of HepG2 tumor tissue and right leg muscle tissue loaded with Cy5-NTR (2.0 μM), respectively. For the former, the tissue was incubated with the probe for 60 minutes, or pre-incubated with an inhibitor (DIC, 100 μM, 60 minutes) before incubating with the probe for 60 minutes. For the latter, the tissue was incubated with the probe only for 60 minutes. The collection wavelength was 640-750 nm (λ).ex =633nm), scale bar: 20μm; (B) is the fluorescence quantitative chromatogram of (A);

[0030] Figure 11 In vivo imaging of HepG2 tumor-bearing mice after intratumoral injection of Cy5-NTR (10 μM, 100 μL); using a 610 nm excitation filter and a 700 nm emission filter. Detailed Implementation

[0031] To further illustrate the technical solution of the present invention, the present invention will be further described below through embodiments.

[0032] Example 1

[0033] A fluorescent probe for nitroreductase (NTR) has the following structural formula:

[0034]

[0035] A method for preparing a nitroreductase (NTR) fluorescent probe includes the following steps:

[0036] Step 1: Under N2 environment, DMF (2.3 mL, 30 mmol) and anhydrous dichloromethane (15 mL) were added to a dry flask, followed by the gradual addition of oxaloyl chloride (3.0 mL, 35 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was evaporated completely under reduced pressure to obtain compound 1 (3.6 g, 94.7%), a white solid, which was used directly in the next reaction without purification.

[0037] Step 2: Compound 1 (1.2 g, 10 mmol) and monomethyl malonate (0.59 g, 5 mmol) were dissolved in anhydrous dichloromethane (20 mL) and refluxed overnight at 40 °C. After evaporating the solvent under reduced pressure, compound 2 was obtained. Acetic anhydride (10 mL), 1,2,3,3-tetramethyl-3H-indole iodide (3.01 g, 10 mmol) and anhydrous sodium acetate (1.23 g, 15 mmol) were added sequentially to the solution. The mixture was stirred at 90 °C for 4 hours. The solution was then diluted with water and extracted with dichloromethane. The organic layers were combined and purified by drying with anhydrous sodium sulfate, vacuum distillation, and column chromatography (dichloromethane / methanol = 10 / 1) to obtain compound Cy5-COOM (1.1 g, 38.7%), which was a dark blue solid. 1H NMR(600Hz,CD3Cl)δ8.52(d,J=14.4Hz,2H),7.45(t,J=7.2Hz,4H),7.33(t,J =8.4Hz,4H),7.03(d,J=15.0Hz,4H),3.99(s,6H),3.93(s,3H),1.79(s,12H); 13 C NMR (150MHz, CD3Cl) δ177.7,167.1,142.5,141.2,128.9,126.4,122.3,111.9,102.1,52.0,50.2,34.3,28.2; ESI-MS[M] + :calcd for441.2537,Found 441.2526.

[0038] Step 3: The compound Cy5-COOM (852 mg, 1.5 mmol) was dissolved in a mixed solution of MeOH (40 mL) and NaOH (2 mM, 60 mL), and the mixture was stirred at 43 °C for 3 hours. After cooling, the mixture was 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 (10-50% methanol / dichloromethane, v / v) to obtain the compound Cy5-COO (318 mg, 38.3%), which was a dark blue solid. 1 H NMR (600Hz, CD3OD) δ8.29(s,2H),7.45(m,8H),6.90(s,2H),3.72(s,6H),1.76(s,12H); 13 C NMR (150MHz, CD3OD) δ142.7,141.4,128.4,125.5,121.9,111.0,101.7,60.1,49.4,30.6,26.7,19.5,13.1; ESI-MS[M] + :calcdfor427.2380,Found 427.2389.

[0039] Step 4: Cy5-COO (554 mg, 1.0 mmol), p-nitrobenzyl bromide (645 mg, 3.0 mmol), and potassium carbonate (276 mg, 2.0 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL). The mixture was stirred at 48 °C for 3 hours. After the reaction was completed, the mixture was cooled, diluted with water, and extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and purified by vacuum distillation and column chromatography (dichloromethane / methanol = 15 / 1, v / v) to obtain compound Cy5-NTR (390 mg, yield 56.6%), which was a dark blue solid. 1HNMR(600Hz,CD3Cl)δ8.64(d,J=14.4Hz,2H),8.31(d,J=9.0Hz,2H),7.67(d,J=8.4Hz,2H),7.46(q,J =7.2Hz,4H),7.35(q,J=7.8Hz,4H),7.11(d,J=13.8Hz,2H),5.46(s,2H),4.04(s,6H),1.74(s,12H); 13 C NMR (150MHz, CD3Cl) δ177.9,166.1,147.8,143.7,142.6,141.2,128.9,128.5,126.5,123.9,122.3,111.9,102.5,65.0,53.5,50.3,27.9; ESI-MS[M] + :calcd for 562.2700,Found 562.2697.

[0040] Example 2

[0041] 1. Study of photophysical properties

[0042] We first tested the photophysical properties of the probe Cy5-NTR and NTR (containing 0.5 mM NADPH) before and after reaction in PBS (10 mM, pH = 7.4), and the results are as follows: Figure 4 As shown. By Figure 4 The ultraviolet-visible absorption spectrum shown in (A) is as follows: Figure 4 As shown in the fluorescence spectrum in (B), the maximum absorption peak of Cy5-NTR is located at around 610 nm. When it interacts with NTR (containing 0.5 mM NADPH), the absorption peak red-shifts to 635 nm. When excited at 633 nm, the probe Cy5-NTR itself only has weak fluorescence. When it interacts with NTR (containing 0.5 mM NADPH), the fluorescence intensity at 662 nm gradually increases and reaches saturation within 5 minutes.

[0043] Depend on Figure 5 The product generated by the reaction of Cy5-NTR with NTR (containing 0.5 mM NADPH) was confirmed by high performance liquid chromatography-mass spectrometry (HPLC-MS) to be a carboxyl-substituted Cy5 dye.

[0044] Depend on Figure 6 As shown in (A) and (B), when NTR (containing 0.5 mM NADPH) is gradually added to the probe Cy5-NTR, the fluorescence intensity of the probe at 662 nm gradually increases and is linearly correlated with the concentration of NTR. The detection limit of the probe for NTR is calculated to be 0.001 μg / mL.

[0045] To further verify the selectivity of the probe for NTR, the Cy5-NTR of the probe was tested in DMEM medium or in media containing NADPH, GSH, Cys, H2O2, and O2, respectively. ·- The stability of APN and GGT in PBS, as shown in the results. Figure 7 As shown, the probe remained stable in the aforementioned substances within a 30-minute detection period. These results demonstrate that the Cy5-NTR probe is a highly selective and sensitive NTR fluorescent probe, with potential applications in real-time imaging of hypoxia-related diseases.

[0046] 2. Cell and tissue imaging studies

[0047] First, we tested the biosafety of the Cy5-NTR probe at the cellular level using the CCK8 assay, and the results are as follows: Figure 8 As shown, when the probe concentration is less than 10 μM, the cell survival rate is greater than 80%, proving that the probe has low toxicity at the working concentration.

[0048] Considering that cancer cells express nitroreductase in large quantities under hypoxic conditions, we pre-placed cancer cells (including HepG2 and A549 cells) and normal cells (including LO2 and BEAS-2B cells) in a hypoxic environment (1% O2), and then incubated the Cy5-NTR probe separately. The results are as follows: Figure 9 As shown, cancer cells exhibit a distinct red fluorescent signal compared to normal cells. When cancer cells were pre-incubated with the inhibitor DIC and then incubated with the probe in a hypoxic environment, almost no fluorescent signal was observed in the cancer cells, indicating that the red fluorescent signal was indeed caused by the overexpression of NTR.

[0049] Next, we evaluated the probe's ability to distinguish between cancerous and normal tissues, and the results are as follows: Figure 10 As shown. By Figure 10 As shown in (A), similar to cell experiments, tumor tissue sections loaded with Cy5-NTR exhibited a bright fluorescence signal in the red channel under hypoxic conditions, which could be suppressed by DIC; normal tissue sections loaded with Cy5-NTR showed a negligible fluorescence signal in the red channel. Figure 10 The fluorescence quantitative data in (B) show that the average fluorescence intensity of normal tissue is 14 times that of normal tissue, which is far greater than the clinically acceptable threshold of 2.

[0050] 3. In vivo imaging research

[0051] Given the excellent differentiation effect of the Cy5-NTR probe between cancer cells / tissues and normal cells / tissues, we finally used Cy5-NTR for real-time imaging of tumors in HepG2 tumor-bearing mice, and the results were as follows: Figure 11As shown, Cy5-NTR was injected in situ into the tumor and leg of mice, respectively. The fluorescence intensity in the tumor area gradually increased and reached its maximum at 1 minute, while no fluorescence signal was observed in the normal tissue area. The fluorescence density ratio (T / N) of the two could reach 10. Therefore, Cy5-NTR can provide high-contrast in situ real-time imaging of tumor-bearing mice, providing a potential tool for tumor surgical navigation.

[0052] In summary, this invention utilizes pentamethine cyanine dye and its central "ester group → carboxyl group" conversion strategy to construct an NTR fluorescent probe. This probe exhibits high stability in various biologically relevant substances, and can sense NTR with high selectivity and sensitivity, with a detection limit of 0.001 μg / mL. Importantly, taking advantage of the characteristic of cancer cells overexpressing APN under hypoxic conditions, this probe can not only distinguish between cancer cells / tissues and normal cells / tissues at the cellular and tissue levels, but also perform high-contrast imaging of tumors in tumor-bearing mice at the in vivo level. Therefore, this probe has great application potential in surgical navigation.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nitroreductase fluorescent probe, characterized in that, The structural formula is: 。 2. A method for preparing the nitroreductase fluorescent probe according to claim 1, characterized by, The method comprises the following steps: Step 1, under the N2 environment, DMF is dissolved in anhydrous dichloromethane, and then oxalyl chloride is gradually added, and the reaction is stirred at room temperature; after the reaction is completed, the solvent is fully evaporated under reduced pressure to obtain compound 1 in the form of a white solid, which is directly used in the next reaction without purification; Step 2, compound 1 and monomethyl malonate are dissolved in anhydrous dichloromethane to perform a reflux reaction; after the solvent is evaporated under reduced pressure, compound 2 is obtained; then, acetic anhydride, 1,2,3,3-tetramethyl-3H-indole iodide and anhydrous sodium acetate are sequentially added to perform a stirring reaction; then, the reaction solution is diluted with water and extracted with dichloromethane; the organic layers are combined and dried over anhydrous sodium sulfate, distilled under reduced pressure and separated and purified by column chromatography to obtain compound Cy5-COOM in the form of a dark blue solid; The compound 1 is: The compound 2 is: The compound Cy5-COOM is: ; Step 3, compound Cy5-COOM is dissolved in a mixed solution of MeOH and NaOH to perform a stirring reaction; after cooling, the reaction solution is diluted with water and extracted with dichloromethane; the organic layers are combined and dried over anhydrous sodium sulfate, distilled under reduced pressure and separated and purified by column chromatography to obtain compound Cy5-COO in the form of a dark blue solid; The compound Cy5-COO is: ; Step 4, Cy5-COO, p-nitrobenzyl bromide and potassium carbonate are dissolved in anhydrous N,N-dimethylformamide to perform a stirring reaction; after the reaction is completed, the reaction solution is cooled, diluted with water and extracted with dichloromethane; the organic phases are combined and dried over anhydrous sodium sulfate, distilled under reduced pressure and separated and purified by column chromatography to obtain compound Cy5-NTR in the form of a dark blue solid.

3. The method for preparing a nitroreductase fluorescent probe according to claim 2, characterized in that, In step 1, the molar ratio of DMF to oxalyl chloride is 30:35, and the stirring reaction time is 2 h.

4. The method for preparing a nitroreductase fluorescent probe according to claim 2, characterized in that, In step 2, the molar ratio of compound 1 to monomethyl malonate is 2:1, the reflux reaction temperature is 40℃, the reflux reaction time is overnight, the molar ratio of compound 2, 1,2,3,3-tetramethyl-3H-indole iodide and anhydrous sodium acetate is 1:2:3, the stirring reaction temperature is 90℃, the stirring reaction time is 4 h, and the developing agent used in column chromatography separation and purification is dichloromethane / methanol = 10 / 1, v / v.

5. The method for preparing a nitroreductase fluorescent probe according to claim 2, characterized in that, In step 3, the stirring reaction temperature is 43℃, the stirring reaction time is 3 h, and the developing agent used in column chromatography separation and purification is 10-50% methanol / dichloromethane, v / v.

6. The method for preparing a nitroreductase fluorescent probe according to claim 2, characterized in that, In step 4, the molar ratio of Cy5-COO, p-nitrobenzyl bromide and potassium carbonate is 1:3:2, the stirring reaction temperature is 48℃, the stirring reaction time is 3 h, and the developing agent used in column chromatography separation and purification is CH2Cl2 / MeOH = 15:1, v / v.

7. Use of the nitroreductase fluorescent probe according to claim 1, characterized in that, A product for preparing normal cell / tissue and cancer cell / tissue.

8. Use of the nitroreductase fluorescent probe according to claim 1, characterized in that, A product for preparing a fast imaging tumor of a tumor-bearing mouse.

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