Fluorescent probe for rapid detection of nitroreductase, preparation method and application thereof

Abstract

The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe for rapid detection of nitroreductase, a preparation method thereof, and an application thereof. The present invention provides a fluorescent probe for rapid detection of nitroreductase, a preparation method thereof, and an application thereof. The fluorescent probe is named DM-CN. Due to the quenching effect of the nitro group, DM-CN is almost non-fluorescent. However, when DM-CN reacts with NTR in the presence of NADH, the 4-nitrobenzene substrate is reduced to the corresponding amine by NTR, the ether bond is broken, and then the phenol oxygen undergoes an intramolecular nucleophilic attack on the nitrile group to form iminocoumarin, and then the fluorescence emission is significantly enhanced, thereby achieving specific recognition of nitroreductase. The fluorescent probe DM-CN has the characteristics of high specificity, high sensitivity, high fluorescence intensity response, short response time, and little damage to organisms, and can be used for the detection of nitroreductase in A549 cells and zebrafish.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, in particular to a fluorescent probe for rapidly detecting nitroreductase, and a preparation method and application thereof. Background Art

[0002] The rapid proliferation of tumor cells leads to an imbalance between oxygen consumption and supply, resulting in significantly lower oxygen concentrations in tumor tissue than in normal tissue. Hypoxia is closely associated with various physiological activities in tumors, including the overexpression of multiple intracellular enzymes, such as nitroreductase (NTR), azoreductase, and quinone reductase. In solid tumors, the concentration of NTR is positively correlated with the degree of hypoxia: lower oxygen concentrations correlate with higher NTR concentrations. Elevated NTR levels in cells are closely associated with the development of various diseases, such as stroke, inflammatory diseases, tumors, and myocardial ischemia. Therefore, measuring nitroreductase levels can indirectly assess the status of the tumor region.

[0003] Currently, a variety of methods have been developed to detect tumor cell hypoxia, such as magnetic resonance imaging, oxygen electrodes, endogenous marker (hypoxia-inducible factors, HIFs) monitoring, and bioluminescent labeling. Among these methods, fluorescence imaging technology has become one of the most powerful tools for monitoring changes in cellular NTR levels due to its significant advantages such as ease of operation, high sensitivity, non-invasiveness, real-time imaging, and high spatiotemporal resolution. In recent years, researchers have developed a variety of NTR fluorescent probes with nitroaromatic structures as recognition groups. Although most of these probes have high selectivity, long emission wavelengths, and excellent biological application potential, they still have some shortcomings, such as: (1) high detection limit; (2) weak fluorescence intensity; (3) long response time. Therefore, it is of great significance to design a near-infrared fluorescent probe for rapid detection of nitroreductase. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned existing technologies and provide a fluorescent probe for rapid detection of nitroreductase, as well as its preparation method and application. The fluorescent probe has the characteristics of high specificity, high sensitivity, high fluorescence intensity response, short response time, and little damage to organisms.

[0005] The present invention provides a fluorescent probe for rapid detection of nitroreductase, the structural formula of the fluorescent probe is:

[0006] .

[0007] The present invention also provides a method for preparing the fluorescent probe for rapid detection of nitroreductase, comprising the following steps:

[0008] S1. K2CO3, 4-nitrobenzyl bromide and dry DMF were mixed, and 8-hydroxyjulolidine-9-carboxaldehyde was added, and the mixture was heated and stirred for reaction. After the reaction, the mixture was extracted, dried and evaporated under reduced pressure to obtain a crude product, and the crude product was purified by column chromatography to obtain an intermediate product;

[0009] S2. Benzimidazole-2-acetonitrile, the intermediate product and anhydrous ethanol are mixed, and piperidine is added dropwise to obtain a mixed solution. The mixed solution is refluxed and heated to react, and the precipitated product is filtered to obtain a crude product. The crude product is purified by recrystallization to obtain a fluorescent probe.

[0010] According to the preparation method provided by the present invention, the molar volume ratio of K2CO3, 4-nitrobenzyl bromide, dry DMF and 8-hydroxyjulolidine-9-carboxaldehyde in S1 is 1.5-2.5 mmol: 1-2 mmol: 10-20 mL: 1-2 mmol.

[0011] According to the preparation method provided by the present invention, the stirring reaction time in S1 is 2 to 6 hours.

[0012] According to the preparation method provided by the present invention, the extraction agent in S1 is ethyl acetate, the number of extractions is 3 to 5 times, the drying desiccant is anhydrous Na2SO4, and the column chromatography purification uses petroleum ether / ethyl acetate as eluent, and the elution ratio is 1.5 to 2.5:0.5 to 1.

[0013] According to the preparation method provided by the present invention, the molar volume ratio of the benzimidazole-2-acetonitrile, the intermediate product, anhydrous ethanol and piperidine in S2 is 0.1-0.5 mmol: 0.1-0.5 mmol: 5-15 mL: 0.1-0.2 mL.

[0014] According to the preparation method provided by the present invention, the temperature of the reflux heating reaction in S2 is 85° C., the time of the reflux heating reaction is 2 to 6 hours, and the recrystallization solvent of the crude product is methanol.

[0015] The present invention also provides an application of the fluorescent probe for rapid detection of nitroreductase, wherein the fluorescent probe for rapid detection of nitroreductase is used for preparing a reagent for detecting nitroreductase in A549 cells and zebrafish.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The present invention provides a fluorescent probe for rapid detection of nitroreductase, as well as its preparation method and application. The fluorescent probe, named DM-CN, is nearly non-fluorescent due to the quenching effect of the nitro group. However, when DM-CN reacts with NTR in the presence of NADH, the 4-nitrobenzene substrate is reduced by NTR to the corresponding amine, cleaving the ether bond. Subsequently, the phenolic oxygen undergoes intramolecular nucleophilic attack on the nitrile group to form an iminocoumarin. This significantly enhances fluorescence emission, thereby achieving specific recognition of nitroreductase. This fluorescent probe, DM-CN, exhibits high specificity, high sensitivity, a high fluorescence intensity response, a short response time, and minimal damage to organisms. It can be used to detect nitroreductase in A549 cells and zebrafish. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 1 is the H NMR spectrum of the probe DM-CN;

[0020] Figure 2 is the mass spectrum of probe DM-CN;

[0021] Figure 3 (a) HPLC (b) HRMS mechanism verification of probe DM-CN;

[0022] Figure 4 (a) UV absorption spectrum and (b) fluorescence spectrum of the reaction between probe DM-CN and nitroreductase;

[0023] Figure 5 is the kinetic response diagram of the reaction between the probe DM-CN and different concentrations of NTR;

[0024] Figure 6 The fluorescence spectra of probe DM-CN and 0-9 μg / L NTR and the linear fitting graph are shown;

[0025] Figure 7 This is the fluorescence response graph of the probe DM-CN to different types of interfering substances, including NTR (8 μg / mL), HSA 10 mg / mL, and the concentration of other analytes were 500 μmol / L;

[0026] Figure 8This is the paper chromatogram of different interferents of the probe DM-CN. The left side is a photo taken under fluorescent light, and the right side is a photo taken under 365nm UV light. 1-12 are Blank, Vc, Cys, GSH, Arg, VB6, Glu, Ser, DTT, HAS, NADH, NTR;

[0027] Figure 9 The probe DM-CN was applied to A549 cells and cultured for 8 h under normoxia (21% O2), hypoxia (12%, 8%, 1% O2 oxygen concentration), and 2% oxygen concentration + dicoumarol, followed by laser confocal imaging with 10 μmol / L DM-CN for 30 min.

[0028] Figure 10 The probe DM-CN (10 μmol / L) was used to monitor A549 cells in real time under 2% oxygen concentration;

[0029] Figure 11 This is a fluorescence inverted microscope image of 6-8 dpf zebrafish embryos incubated with the probe DM-CN 40 μmol / L for 30 min;

[0030] Figure 12 This is a fluorescence inverted microscope image of 8 dpf zebrafish embryos treated with DM-CN for 30 min and then incubated with 40 μmol / L DM-CN for 30 min.

[0031] Figure 13 This is the visualization of NTR released from a fresh wound after incubation with the probe DM-CN (40 μmol / L) for 30 min. DETAILED DESCRIPTION

[0032] Example 1

[0033] This embodiment provides a fluorescent probe DM-CN for rapid detection of nitroreductase and a preparation method thereof, comprising the following steps:

[0034] S1. Place 2.25 mmol of K2CO3, 1.5 mmol of 4-nitrobenzyl bromide, and 15 mL of dry DMF (N,N-dimethylformamide) in a 50 mL round-bottom flask, then add 1.5 mmol of 8-hydroxyjulolidine-9-carbaldehyde. Stir at room temperature for 4 h and monitor the reaction in real time by thin layer chromatography (TLC).

[0035] After the reaction, the mixture was extracted with ethyl acetate three times, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent at a ratio of 2:1 to obtain a light yellow solid intermediate product DM-1.

[0036] ;

[0037] S2. Place 0.3 mmol of benzimidazole-2-acetonitrile, 0.3 mmol of DM-1, and 10 mL of anhydrous ethanol in a 25 mL round-bottom flask, add 0.1 mL of piperidine dropwise, and reflux the mixed solution to 85°C for 4 h. A large amount of solid precipitates, and the crude product is filtered off. The crude product is purified by recrystallization from methanol to obtain an orange solid DM-CN.

[0038] .

[0039] The reaction equations for the steps provided in this embodiment are:

[0040] .

[0041] The results of the fluorescent probe structure characterization data are as follows: Figure 1 As shown:

[0042] 1 H NMR (400 MHz, DMSO- d 6), δ (ppm): 8.26 (s, 1H), 8.22 (d, J = 8.7 Hz,2H), 7.86 - 7.80 (m, 4H), 7.19 - 7.15 (m, 2H), 4.99 (s, 3H), 3.29 - 3.26 (m,4H), 2.71 (t, J = 6.3 Hz, 4H), 1.91 - 1.83 (m, 4H).

[0043] The mass spectrometry results of the probe DM-CN are as follows Figure 2 As shown;

[0044] from Figure 3 As can be seen from (a), the DM-CN in the product prepared in this example is almost non-fluorescent due to the quenching effect of the nitroaromatic group. Figure 3As can be seen in (b), when DM-CN reacts with NTR in the presence of NADH (reduced nicotinamide adenine dinucleotide), the 4-nitrobenzene substrate is reduced by NTR to the corresponding amine, the ether bond is broken, and then the phenol oxygen undergoes intramolecular nucleophilic attack on the nitrile group to form iminocoumarin. Then, the fluorescence emission is significantly enhanced, thereby achieving specific recognition of nitroreductase. The reaction mechanism is as follows:

[0045] .

[0046] Example 2

[0047] This embodiment provides a fluorescent probe DM-CN for rapid detection of nitroreductase and a preparation method thereof, comprising the following steps:

[0048] S1. Place 1.5 mmol of K2CO3, 1 mmol of 4-nitrobenzyl bromide, and 10 mL of dry DMF in a 50 mL round-bottom flask, then add 1 mmol of 8-hydroxyjulolidine-9-carboxaldehyde. Stir at room temperature for 2 h and monitor the reaction in real time by thin-layer chromatography (TLC).

[0049] After the reaction, the mixture was extracted with ethyl acetate four times, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent at a ratio of 1.5:0.5 to obtain a light yellow solid intermediate product DM-1.

[0050] S2. Place 0.1 mmol of benzimidazole-2-acetonitrile, 0.1 mmol of DM-1, and 5 mL of anhydrous ethanol in a 25 mL round-bottom flask, add 0.1 mL of piperidine dropwise, and reflux the mixed solution to 85°C for 2 h. A large amount of solid precipitates, and the crude product is filtered off. The crude product is purified by recrystallization from methanol to obtain an orange solid DM-CN.

[0051] Example 3

[0052] This embodiment provides a fluorescent probe DM-CN for rapid detection of nitroreductase and a preparation method thereof, comprising the following steps:

[0053] S1. Place 2.5 mmol of K2CO3, 2 mmol of 4-nitrobenzyl bromide, and 20 mL of dry DMF in a 50 mL round-bottom flask, then add 2 mmol of 8-hydroxyjulolidine-9-carbaldehyde. Stir at room temperature for 6 h and monitor the reaction in real time by thin layer chromatography (TLC).

[0054] After the reaction, the mixture was extracted with ethyl acetate five times, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether / ethyl acetate as eluent at a ratio of 2.5:1 to obtain a light yellow solid intermediate product DM-1.

[0055] S2. Place 0.5 mmol of benzimidazole-2-acetonitrile, 0.5 mmol of DM-1, and 15 mL of anhydrous ethanol in a 25 mL round-bottom flask, add 0.2 mL of piperidine dropwise, and reflux the mixed solution to 85 °C for 6 h. A large amount of solid precipitates, and the crude product is filtered off. The crude product is purified by recrystallization from methanol to obtain an orange solid DM-CN.

[0056] Example 4

[0057] In this example, the probe DM-CN prepared in Example 1 was subjected to spectral testing.

[0058] The probe DM-CN was prepared into a 1 mM stock solution in dimethyl sulfoxide, the nitroreductase was prepared into a 500 μg / mL stock solution in deionized water, and the NADH was prepared into a 1 mM stock solution.

[0059] To investigate the response of the probe DM-CN to nitroreductase, 10 μM probe DM-CN, 500 μM NADH, 8 μg / mL nitroreductase, and 10 mM phosphate buffer (pH = 6.0) were diluted to 500 μL with high-purity water, and the UV-visible absorption spectrum and fluorescence emission spectrum were measured.

[0060] The results of the test are as follows Figures 4-6 As shown, the absorption spectrum of the probe DM-CN exhibits two absorption peaks at 450 nm and 500 nm. The addition of NTR significantly increases the 450 nm peak and the 500 nm peak. The probe solution itself exhibits no significant fluorescence, but the addition of NTR significantly enhances the fluorescence at 550 nm. The fluorescence intensity of the reaction system increases with increasing NTR concentration and exhibits a good linear relationship with NTR concentration within the range of 1 to 8 μg / mL.

[0061] Example 5

[0062] In this example, the probe DM-CN prepared in Example 1 was subjected to a selectivity test.

[0063] 10 μM of the probe DM-CN was mixed with inorganic ions (K + Mg 2+ , Ca 2+ 、HSO3 - 、SO3 2- , HS -), glucose, Vc, VB6, HSA (human serum albumin), Glu (glutamic acid), Arg (arginine), Ser (serine), Asp (aspartic acid), Pro (proline), ROS (HClO, 1 O2, H2O2, •OH) and biothiols (GSH (glutathione), Cys (cysteine), Hcy (homocysteine) and DTT (dithiothreitol)) were mixed with HSA at 10 mg / mL and the concentrations of other analytes were 500 μmol / L, and their fluorescence responses to NTR were tested.

[0064] The test results are as follows Figures 7-8 As shown in the figure, no significant increase in fluorescence intensity was observed when these potentially interfering substances were added to the DM-CN solution. However, when 8 μg / mL of NTR was added to the DM-CN solution, the fluorescence intensity was significantly enhanced. This indicates that DM-CN has significantly higher selectivity for NTR than for other tested analytes, which can be attributed to the specific reduction of the probe DM-CN by NTR.

[0065] In addition, the probe DM-CN has been successfully used in test strips to detect NTR, making the detection system simpler and more practical.

[0066] Example 6

[0067] This example provides a cell imaging application of the probe DM-CN prepared in Example 1.

[0068] A549 cells were placed in 35 mm confocal culture dishes and cultured in an incubator (temperature: 37 °C, 5% CO2). After the cells adhered to the wall, they were further incubated under different hypoxic conditions and divided into four groups for culture: oxygen concentrations of 21%, 12%, 8%, and 2% for 8 h.

[0069] The cells were washed three times with PBS, and 10 μM of the probe DM-CN was added to the cells under the four culture conditions. The culture was continued for 30 minutes, and the cell culture medium was discarded. For the inhibitor group, the cells were pre-treated with dicoumarol for 30 minutes and then incubated with 10 μmol / L of the DM-CN probe for 30 minutes.

[0070] All groups were rinsed three times with 20 mmol / L PBS (pH 7.4), and fluorescence imaging of cells under three culture conditions was performed using a confocal laser scanning microscope (FluoViewTM FV1000, Olympus, Japan). Figure 9 shown.

[0071] Figure 9The probe DM-CN was applied to A549 cells and cultured for 8 h under normoxia (21% O2), hypoxia (12%, 8%, 1% O2 oxygen concentration), and 2% oxygen concentration + dicoumarol, followed by laser confocal imaging with 10 μmol / L DM-CN for 30 min. Figure 10 This is a graph showing real-time monitoring of A549 cells by the probe DM-CN (10 μmol / L) under 2% oxygen concentration conditions; Figure 9 and Figure 10 It can be found that the fluorescence intensity of cells cultured under 2% oxygen content and cells cultured under 21% oxygen content was significantly stronger after interacting with the probe.

[0072] It was demonstrated that under hypoxic conditions, the content of cellular nitroreductase changed, and the probe could effectively detect the changes of nitroreductase in A549 cells.

[0073] Example 7

[0074] This example provides zebrafish injury imaging using the probe DM-CN prepared in Example 1.

[0075] This example designs an in vivo inflammation detection method using translucent zebrafish embryos with a broken tail to monitor the degree of hypoxia caused by wound inflammation.

[0076] Zebrafish embryos at 3 to 8 dpf (days post-fertilization) were incubated with 40 μmol / L DM-CN probe in E3 embryo medium at 28°C for 30 min to allow the probe to fully penetrate the zebrafish tissues. The embryos were then washed three times with PBS (pH = 7.4) to remove the remaining probe and anesthetized with tricaine.

[0077] For inhibitor experiments, zebrafish were incubated with dicoumarol (500 μmol / L, 1000 μmol / L) for another 30 min before adding the probe.

[0078] For the tail amputation experiment, after the probe was incubated for 30 min, the tail was amputated and the tail of the 8 dpf zebrafish embryo was removed with a thin blade. The embryo was then kept in water for 15 min and then subjected to fluorescence imaging. The fluorescence images were obtained using an inverted fluorescence microscope.

[0079] like Figure 11 、 Figure 12 and Figure 13 As shown in the figure, strong fluorescence was clearly observed from the tail of the 8 dpf zebrafish embryo, while no fluorescence signal was detected from the tail of the control zebrafish embryo. Based on the results of the interference experiment, ROS substances that may be produced in fresh wounds caused by trauma did not trigger a fluorescent reaction with the DM-CN probe.

[0080] This suggests that external damage may lead to the formation of a local hypoxic environment, triggering the release of a large number of NTRs, which in turn causes the probe DM-CN to produce a fluorescent signal.

[0081] When the fluorescent probe DM-CN prepared by the present invention reacts with NTR in the presence of NADH, the 4-nitrobenzene substrate is reduced by NTR to the corresponding amine, cleaving the ether bond. Subsequently, the phenolic oxygen undergoes intramolecular nucleophilic attack on the nitrile group to form an iminocoumarin, which significantly enhances fluorescence emission, thereby achieving specific recognition of nitroreductase. When 8 μg / mL of NTR is added to the DM-CN solution, the fluorescence intensity is significantly enhanced. DM-CN has significantly higher selectivity for NTR than other test analytes. Under hypoxic conditions, the cellular nitroreductase content changes, and this probe can effectively detect changes in nitroreductase in A549 cells. External damage to zebrafish leads to the formation of a localized hypoxic environment, triggering the release of a large amount of NTR, which in turn causes the probe DM-CN to produce a fluorescent signal, facilitating the detection of nitroreductase content in zebrafish and indirectly assessing the tumor area.

[0082] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A fluorescent probe for rapid detection of nitroreductase, characterized in that: The structural formula of the fluorescent probe is: 。 2. A method for preparing a fluorescent probe for rapid detection of nitroreductase according to claim 1, characterized in that: The following steps are involved: S1. K2CO3, 4-nitrobenzyl bromide and dry DMF were mixed, and 8-hydroxyjulolidine-9-carboxaldehyde was added, and the mixture was heated and stirred for reaction. After the reaction, the mixture was extracted, dried and evaporated under reduced pressure to obtain a crude product, and the crude product was purified by column chromatography to obtain an intermediate product; S2. Benzimidazole-2-acetonitrile, the intermediate product and anhydrous ethanol are mixed, and piperidine is added dropwise to obtain a mixed solution. The mixed solution is refluxed and heated to react, and the precipitated product is filtered to obtain a crude product. The crude product is purified by recrystallization to obtain a fluorescent probe.

3. The preparation method according to claim 2, characterized in that The molar volume ratio of K2CO3, 4-nitrobenzyl bromide, dry DMF and 8-hydroxyjulolidine-9-carbaldehyde in S1 is 1.5-2.5 mmol: 1-2 mmol: 10-20 mL: 1-2 mmol.

4. The preparation method according to claim 2, characterized in that The stirring reaction time in S1 is 2 to 6 h.

5. The preparation method according to claim 2, characterized in that The extraction agent used in S1 is ethyl acetate, the number of extractions is 3 to 5 times, the drying agent is anhydrous Na2SO4, and the column chromatography purification uses petroleum ether / ethyl acetate as eluent, and the elution ratio is 1.5 to 2.5:0.5 to 1.

6. The preparation method according to claim 2, characterized in that The molar volume ratio of the benzimidazole-2-acetonitrile, the intermediate product, anhydrous ethanol and piperidine in S2 is 0.1-0.5 mmol: 0.1-0.5 mmol: 5-15 mL: 0.1-0.2 mL.

7. The preparation method according to claim 2, characterized in that The temperature of the reflux heating reaction in S2 is 85° C., the time of the reflux heating reaction is 2 to 6 h, and the recrystallization solvent of the crude product is methanol.

8. A use of the fluorescent probe for rapid detection of nitroreductase as claimed in claim 1, characterized in that: The fluorescent probe for rapidly detecting nitroreductase is used to prepare a reagent for detecting nitroreductase in A549 cells and zebrafish.

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