Molecular probe, preparation method thereof and application of molecular probe in nitroreductase detection field

Abstract

The invention particularly relates to a molecular probe, a preparation method thereof and application of the molecular probe in the field of nitroreductase detection. The invention provides a molecular probe compound Cy647 capable of being used for detecting nitroreductase in living cells, the chemical structural formula of the molecular probe compound Cy647 is as follows: # imgabs0 #, the fluorescence emission wavelength of the molecule is in a near infrared region, and the molecular probe compound Cy647 has a good fluorescence imaging effect; the fluorescent probe disclosed by the invention has high selectivity for capturing nitroreductase, and a unique product Cy468 with a determined structure is generated, so that quantitative detection is more accurate.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular probes, and particularly relates to a molecular probe, a preparation method thereof, and an application in the field of nitroreductase detection. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] As an important class of enzymes related to oxidative stress, nitroreductase plays an indispensable role in cells and is widely involved in many important physiological and pathological processes, especially in inflammatory responses, apoptosis, and oxidative damage. In the development process of diseases such as tumors, nitroreductase plays a special role. Due to rapid proliferation and relatively insufficient angiogenesis, tumor tissues are often in a hypoxic microenvironment. This environment can induce a significant up-regulation of the expression level of nitroreductase in tumor cells. Since traditional chemical detection methods often require complex sample pretreatment processes and are also prone to changes or even loss of intracellular enzyme activity, thereby affecting the accuracy of detection results.

[0004] Therefore, it is of great significance to design a specific detection probe molecule for nitroreductase. As far as the inventor knows, molecular probes have high selectivity and can effectively avoid interference from other substances in cells; moreover, they have extremely high sensitivity and can detect nitroreductase in living cells, providing a powerful tool for in-depth study of the mechanism of action of nitroreductase in cell physiological and pathological processes. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the technical problem to be solved by the present invention is to provide a molecular probe for detecting nitroreductase in biological cells, a preparation method thereof, and an application.

[0006] In the first aspect of the present invention, a molecular probe is provided, and its chemical structure is shown as the following formula: .

[0007] The chemical name of the above molecular probe is (E)-1-ethyl-2-[2-(7-[(4-nitrobenzyloxy)carbonyl]-2,3,4,5,6,7,8,9,13,14-decahydro-12H-1,10-ethanobenzo[k][1,4,7,10]tetraazacyclotridec-15-yl)vinyl]benzo[cd]indol-1-ium chloride, named Cy647.

[0008] In the second aspect, the present invention also provides a preparation method of the molecular probe described in the first aspect, and the synthetic route of the preparation method is as follows: ; The preparation method includes the following steps: (1) 2-[(E)-2-[(E)-2-chloro-3-[(E)-2-(1-ethylbenzo[cd]indol-2(1H)-ylidene)ethylidene]cyclohex-1-en-1-yl]vinyl]-1-ethylbenzo[cd]indol-1-ium iodide (named Cy527) reacts with 1,4,7,10-tetraazacyclododecane to obtain an intermediate product (E)-2-[2-(2,3,4,5,6,7,8,9,13,14-decahydro-12H-1,10-ethanobenzocyclo[k][1,4,7,10]tetraazacyclotridecen-15-yl)vinyl]-1-ethylbenzo[cd]indol-1-ium chloride (named Cy468); (2) (E)-2-[2-(2,3,4,5,6,7,8,9,13,14-decahydro-12H-1,10-ethanobenzocyclo[k][1,4,7,10]tetraazacyclotridecen-15-yl)vinyl]-1-ethylbenzo[cd]indol-1-ium chloride (Cy468) reacts with 4-nitrobenzyl chloroformate to obtain a molecular probe (E)-1-ethyl-2-[2-(7-[(4-nitrobenzyloxy)carbonyl]-2,3,4,5,6,7,8,9,13,14-decahydro-12H-1,10-ethanobenzocyclo[k][1,4,7,10]tetraazacyclotridec-15-yl)vinyl]benzo[cd]indol-1-ium chloride (Cy647).

[0009] The specific reaction of the above step (1) is as follows: The starting reactant Cy527 reacts with 1,4,7,10-tetraazacyclododecane under the action of potassium carbonate and chloroform. The reaction temperature is 75-85 °C, and the reaction time is 22-26 h; after the reaction, the solvent is removed to obtain a solid part. The above solid part is added to a mixed mobile phase to dissolve and purified by silica gel column chromatography to obtain the intermediate product Cy468.

[0010] Further, the dosage ratio of Cy527, 1,4,7,10-tetraazacyclododecane, potassium carbonate, and chloroform is 3-4 mmol: 7-9 mmol: 7-8 mmol: 20-40 mL.

[0011] Further, the solvent is removed by means of reduced pressure, vacuum concentration, or evaporation to dryness. The obtained solid part is first dissolved in dichloromethane, and excess potassium carbonate can be removed by washing with water multiple times. After washing with water, it is concentrated again, added to a mixed mobile phase to dissolve, and the mixed mobile phase is obtained by mixing dichloromethane and methanol in a volume ratio of 24-27:1.

[0012] The specific reaction of the above step (2) is as follows: the intermediate product Cy468 is dissolved in dichloromethane, and 4-nitrobenzyl chloroformate and potassium carbonate are added to react in an ice bath for 1.5 to 2.5 hours; after the reaction is completed, the solvent is removed, a mixed mobile phase is added to dissolve, and then purified by silica gel column chromatography to obtain Cy647.

[0013] Furthermore, the dosage ratio of the intermediate product Cy468, dichloromethane, 4-nitrobenzyl chloroformate, and potassium carbonate is 1~2 mmol: 10~30 mL: 6~10 mmol: 5~8 mmol.

[0014] Furthermore, after the reaction is completed, the steps of removing the solvent and purifying by silica gel column chromatography are as follows: the solvent can be removed by reducing pressure, vacuum concentration or evaporation to obtain a solid portion; the solid portion is first dissolved by adding dichloromethane, and then washed with water for multiple times to remove excess potassium carbonate, and then concentrated again after washing with water, and dissolved by adding a mixed mobile phase, and then purified by silica gel column chromatography to obtain Cy647, wherein the mixed mobile phase is a mixture of dichloromethane and methanol in a volume ratio of 8 to 12:1.

[0015] The third aspect is the application of the molecular probe described in the first aspect in the field of nitroreductase detection.

[0016] The molecular probe Cy647 is converted back into the intermediate product Cy468 under the action of nitroreductase. The reaction principle is as follows: The molecular probe Cy647 provided by the present invention has a strong absorption peak at an ultraviolet absorption wavelength of 615 nm. After binding to nitroreductase, the intensity of the absorption peak decreases. Those skilled in the art can confirm whether nitroreductase exists in the environment to be tested by detecting the change in the absorption peak intensity at the ultraviolet absorption wavelength of 615 nm; in addition, the molecular probe Cy647 itself has fluorescence intensity at an emission wavelength of 730 nm. After binding to nitroreductase, Cy468 is generated, and the fluorescence emission intensity gradually shifts to 470 nm. Those skilled in the art can detect the fluorescence intensity at emission wavelengths of 730 nm and 470 nm to detect nitroreductase in the environment to be tested.

[0017] The present invention also mixes nitroreductase with common components in the living cell environment to investigate the detection specificity of the above molecular probe. The investigation results show that the detection background in living cells hardly interferes with the molecular probe Cy647, and this molecular probe has good selectivity for nitroreductase in the environment. The present invention also adds it to tumor cells to verify its detection ability. The verification results show that Cy647 can be normally converted into Cy468 in living tumor cells. The above conversion can realize the detection of the content of nitroreductase through fluorescence imaging observation, and at the same time does not affect the survival state of cells, and can be used as a molecular probe for nitroreductase in living cells.

[0018] Therefore, an implementation manner of the above application in the third aspect is as follows: adding the above molecular probe Cy647 into a biological sample to be detected, and determining the expression level of nitroreductase in the sample to be detected by detecting the fluorescence intensity at the ultraviolet absorption wavelength of 615 nm or the fluorescence emission wavelengths of 730 nm and 470 nm; compared with the control sample, when the absorption peak intensity at the ultraviolet absorption wavelength of 615 nm decreases, the fluorescence intensity at the fluorescence emission wavelength of 730 nm decreases, and the fluorescence intensity at the fluorescence emission wavelength of 470 nm increases, it indicates that there is overexpressed nitroreductase in the sample to be detected, and the biological sample includes but is not limited to cell samples, tissue samples or organ samples.

[0019] Another implementation manner of the above application is as follows: adding the above molecular probe Cy647 into a tissue sample to be detected, incubating for a period of time, and observing and recording the fluorescence intensity at the emission wavelengths of 730 nm and 470 nm through a fluorescence imaging device.

[0020] Another implementation manner is as follows: using the above molecular probe Cy647 as a contrast agent, injecting it into an experimental sample through intravenous injection, and observing and recording the fluorescence intensity at the emission wavelength of 730 nm through a fluorescence imaging device.

[0021] Compared with the prior art, the beneficial effects of the present invention are: (1) The molecular probe Cy647 of the present invention uses 4-nitrophenyl as the reaction site, a near-infrared molecular probe that twists the intramolecular charge transfer mechanism, so that the probe will have an obvious fluorescence signal readout for nitroreductase. The emission wavelength of the molecular product is in the near-infrared region, with less fluorescence background interference, and can produce good fluorescence imaging effects.

[0022] (2) The molecular probe Cy647 of the present invention has high selectivity for capturing nitroreductase, and generates a unique product Cy468 with a determined structure. The quantitative detection can be made more accurate through the fluorescence intensity changes at the two fluorescence emission wavelengths of 730 nm and 470 nm.

[0023] (3) In addition to being detected through the fluorescence pathway, this molecular probe can also be detected by changes in the ultraviolet absorption spectrum. Since the change in the ultraviolet color before and after the reaction is obvious, it can be detected by the naked eye, which is convenient and fast.

[0024] (4) Since the long wavelength of the molecular probe Cy647 is in the infrared region, it has less harm to biological cells and can perform fluorescence imaging without affecting the normal physiological functions of cells, which further promotes the exploration of the role of small biological molecules in living organisms. The application of the present invention in the detection of living cells further promotes the exploration of the role of small biological molecules in living organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0026] Figure 1 is the mass spectrum of the molecular probe Cy647 in Example 1; Figure 2 is the 1H NMR spectrum of the molecular probe Cy647 in Example 1; Figure 3 is the 13C NMR spectrum of the molecular probe Cy647 in Example 1; Figure 4 is the mass spectrum of the product Cy468 in Example 1; Figure 5 is the 1H NMR spectrum of the product Cy468 in Example 1; Figure 6 is the 13C NMR spectrum of the product Cy468 in Example 1; Figure 7 is the mechanism diagram of the molecular probe Cy647 detecting nitroreductase in Example 1; Figure 8 is the change diagram of the ultraviolet spectrum of the molecular probe Cy647 with time in Example 1; Figure 9 is the fluorescence diagram of the short wavelength region of the molecular probe Cy647 changing with time in Example 1; Figure 10 is the fluorescence diagram of the long wavelength region of the molecular probe Cy647 changing with time in Example 1; Figure 11 is the ratio-type linear diagram of the molecular probe Cy647 detecting nitroreductase in Example 1; Figure 12 is the selectivity diagram of the molecular probe Cy647 for detecting nitroreductase in Example 1; Figure 13 is the confocal fluorescence imaging diagram of the molecular probe Cy647 on HepG-2 cells in Example 1; Among them, a1, a2, and a3 are the bright-field, confocal laser fluorescence green channel imaging, and confocal laser fluorescence red channel imaging of HepG-2 cells incubated with the molecular probe Cy647 for 0.5 h in sequence; b1, b2, and b3 are the bright-field, confocal laser fluorescence green channel imaging, and confocal laser fluorescence red channel imaging of HepG-2 cells incubated with the molecular probe Cy647 for 1 h in sequence; c1, c2, and c3 are the bright-field, confocal laser fluorescence green channel imaging, and confocal laser fluorescence red channel imaging of HepG-2 cells incubated with the molecular probe Cy647 for 3 h in sequence; d1, d2, and d3 are the bright-field, confocal laser fluorescence green channel imaging, and confocal laser fluorescence red channel imaging of HepG-2 cells incubated with the molecular probe Cy468 for 0.5 h in sequence; Figure 14 It is the detection diagram of the cytotoxicity of the molecular probe Cy647 to HepG-2 cells in Example 1. Detailed implementation manners

[0027] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0028] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or their combinations.

[0029] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0030] The sources of experimental equipment and reagents involved in the following research are as follows: HepG-2 cells: Shanghai Fuheng Biotechnology Co., Ltd., human liver cancer cells, product number: FH0076.

[0031] High performance liquid chromatography - mass spectrometry: Agilent 1260 mass spectrometry system (Agilent, USA), equipped with a degassing device, quaternary pump, and auto sampler. High performance liquid chromatography separation was carried out using a Hypersil GOLD C18 column (2.1 mm × 50 mm, 1.8 μm, Agilent, USA).

[0032] Fluorescence detection: FLS - 1000 fluorescence spectrometer. For green light detection, the excitation wavelength was 390 nm, the excitation and emission slit widths were both 20.0 nm, the voltage was 900 V, and the scanning speed was 1200 nm / min; for red light detection, the excitation wavelength was 630 nm, the excitation and emission slit widths were both 20.0 nm, the voltage was 900 V, and the scanning speed was 1200 nm / min.

[0033] Fluorescence imaging observation: Conducted using a LSM / 880 NLO (Zeiss, Germany) confocal imaging system and observed with a 40× objective lens.

[0034] Separation and purification of the compound: Achieved based on a thin - layer chromatography silica gel column, where the packing material was 300 - 400 mesh.

[0035] Example 1 In this example, a molecular probe for detecting nitroreductase in biological cells was provided, and the structure of the probe was shown as the following formula: In this example, a preparation method of the above - mentioned molecular probe was also provided, and its synthetic route was as follows: Specifically, the steps of the preparation method were as follows: (1) Add 3.4 mmol of Cy527 and 8.7 mmol of 1,4,7,10 - tetraazacyclododecane into a 100 mL round - bottom flask. Under the action of 7.2 mmol of potassium carbonate and 30 mL of chloroform, react at 80 °C for 24 hours. After the reaction, let it stand and cool to room temperature, concentrate it under vacuum using a rotary evaporator. Wash the solid obtained from the reaction 3 times with 250 mL of dichloromethane, then concentrate it under vacuum using a rotary evaporator again, and then elute it with a dichloromethane / methanol (volume ratio 25:1) mixed mobile phase for silica gel column chromatography purification to obtain the intermediate product (Cy468).

[0036] (2) Dissolve 1.6 mmol of the intermediate Cy468 in 20 mL of dichloromethane, add 8.0 mmol of 4-nitrobenzyl chloroformate and 5.1 mmol of potassium carbonate, and react in an ice bath for 2 h. After the reaction is completed, transfer the mixture to a separatory funnel, wash it 3 times with water, and concentrate it under vacuum using a rotary evaporator. The crude product is obtained, and the crude product is purified by column chromatography with a mixed mobile phase of dichloromethane / methanol (volume ratio 10:1) to obtain the desired product, which is the molecular probe (Cy647).

[0037] The mass spectrum of the molecular probe (Cy647) is as Figure 1 shown, and the 1H NMR spectrum is as Figure 2 shown, and the 13C NMR spectrum is as Figure 3 shown; the specific data of the 1H NMR spectrum and the 13C NMR spectrum are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.25 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H), 7.97(t, J = 7.4 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 22.9, 10.6 Hz,2H), 7.75 – 7.71 (m, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.47 – 7.43 (m, 1H), 7.39(d, J = 8.0 Hz, 1H), 6.78 (d, J = 7.0 Hz, 1H), 5.87 (d, J = 12.8 Hz, 1H),5.36 – 5.31 (m, 1H), 5.24 – 5.18 (m, 1H), 5.14 (d, J = 13.7 Hz, 2H), 5.05 (d,J = 13.9 Hz, 1H), 4.81 (s, 2H), 4.71 – 4.61 (m, 1H), 4.30 (d, J = 11.9 Hz,4H), 3.96 (ddd, J = 42.7, 30.2, 13.8 Hz, 7H), 3.86 – 3.76 (m, 2H), 3.75 –3.62 (m, 2H), 2.68 – 2.61 (m, 1H), 2.53 (s, 1H), 1.36 (s, 3H), 1.26 (s, 2H). 1313C NMR (126 MHz, CDCl3) δ 172.86, 155.55, 155.47, 155.02, 147.60, 143.63, 141.86, 130.62, 129.48, 129.07, 128.83, 128.33, 127.99, 127.35, 126.28, 123.88, 123.80, 123.45, 121.79, 118.89, 107.62, 103.38, 66.04, 65.96, 65.82, 59.87, 59.25, 49.96, 42.62, 37.64, 31.55, 31.02, 26.89, 23.23, 22.62, 12.92. The product Cy468 was obtained by the transformation of nitroreductase, and its mass spectrum is as Figure 4 shown, and the 1H NMR spectrum is as Figure 5 shown, and the 13C NMR spectrum is as Figure 6 shown; the specific data of the 1H NMR spectrum and the 13C NMR spectrum are as follows: 1 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 7.2 Hz, 1H), 7.81 (t, J = 9.5 Hz, 2H), 7.73 – 7.67 (m, 1H), 7.64 (s, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.36 – 7.29 (m, 1H), 6.71 (t, J = 8.9 Hz, 1H), 5.97 (t, J = 11.3 Hz, 1H), 4.50 – 4.40 (m, 1H), 4.22 (d, J = 13.4 Hz, 1H), 4.05 (d, J = 15.8 Hz, 2H), 3.97 (dd, J = 14.4, 7.2 Hz, 2H), 3.86 – 3.77 (m, 2H), 3.74 (d, J = 12.3 Hz, 1H), 3.15 (d, J = 20.0 Hz, 3H), 2.99 (s, 1H), 2.73 (t, J = 26.0 Hz, 6H), 2.54 (d, J = 14.8 Hz, 3H), 1.97 (d, J = 32.8 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H). 1313C NMR (126 MHz, CDCl3) δ 171.35, 158.46, 147.39, 142.33, 135.78, 131.17, 130.70, 129.19, 129.01, 127.48, 126.62, 124.99, 122.92, 117.84, 107.26, 102.41, 99.33, 60.71, 56.79, 55.12, 51.00, 47.36, 46.72, 45.49, 42.70, 37.52, 30.48, 24.61, 22.88, 12.80. Performance verification I. Verification of the detection ability of the molecular probe Cy647 for nitroreductase The ability of the molecular probe Cy647 to detect nitroreductase was verified as follows: (1) Prepare a DMSO solution of the molecular probe Cy647 with a concentration of 1 mM; (2) In a 2 mL mixed system of 10% DMSO-PBS, add 10 μM of the molecular probe Cy647 and 10 μg / mL of nitroreductase and incubate for different times, and perform the following detections: a. With the ultraviolet absorption wavelength as the abscissa and the absorbance as the ordinate, obtain the ultraviolet absorption spectrum of the molecular probe Cy647 for detecting nitroreductase. It can be seen that Figure 8 as time increases, the intensity of the absorption peak at 615 nm gradually decreases; b. Subsequently, the fluorescence response properties of the molecular probe Cy647 to nitroreductase were studied. With the emission wavelength as the abscissa and the fluorescence intensity as the ordinate, obtain the fluorescence emission spectrum of the molecular probe Cy647 for detecting nitroreductase. It can be seen that Figure 9 and 10 as time increases, the fluorescence emission peak at 470 nm gradually increases, and at the same time, the fluorescence emission intensity at 730 nm continuously decreases.

[0038] (3) In the above mixed system, add 10 μM of the molecular probe Cy647, and then add nitroreductase solutions with different concentrations respectively, so that the final concentration of nitroreductase in the reaction system is 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL. After incubating for 40 minutes, measure the fluorescence intensity ratio of the above solutions at 470 nm and 730 nm.

[0039] With the nitroreductase concentration on the abscissa and the fluorescence intensity ratio at 470 nm and 730 nm correspondingly on the ordinate, a linear graph of the fluorescence intensity ratio (green light / red light) of the probe Cy647 against the nitroreductase concentration is obtained. Through Figure 11 It can be seen that as the nitroreductase concentration increases, the fluorescence ratio (F 470 / F 730 ) has a good linear relationship with the nitroreductase concentration. Through calculation, the detection limit of the molecular probe Cy647 for nitroreductase is 13 ng / mL.

[0040] The above research results show that as the molecular probe Cy647 binds to nitroreductase during the incubation process, the ultraviolet absorption peak and fluorescence absorption spectrum of the above compound will change accordingly, which proves that the molecular probe Cy647 does respond to nitroreductase in the environment and can be observed based on both ultraviolet and fluorescence methods, confirming the feasibility of Cy647 as a molecular probe for nitroreductase.

[0041] II. Selectivity verification of the molecular probe Cy647 for nitroreductase To verify the selectivity of the molecular probe Cy647 for detecting nitroreductase in different targets, the following experiment was designed: With different targets on the abscissa and the fluorescence intensity change on the ordinate, a selectivity graph of the molecular probe for detecting nitroreductase is obtained. The targets include anion solutions, biothiols, and metal ion solutions, as Figure 12 shown, Figure 12 in which the targets corresponding to the bar data from left to right are: 1: blank, 2: Na + , 3: K + , 4: Ca 2+ , 5: Fe 2+ , 6:Fe 3+ , 7: ClO - , 8: H2O2, 9: O 2﹣ , 10: ·OH, 11: ONOO - , 12: Vitamin C, 13: DTT (DL-dithiothreitol), 14: BSA, 15: glutathione (GSH), 16: arginine (Arg), 17: serine (Ser), 18: cysteine (Cys), 19: carboxylesterase (CES1), 20: tyrosinase, 21: azoreductase, 22: nitroreductase (NTR).

[0042] Through Figure 12It can be seen that the molecular probe Cy647 has strong selectivity for nitroreductase.

[0043] III. Application of Molecular Probe Cy647 in Cell Imaging To verify the imaging ability of the molecular probe Cy647 in tumor cells, in this example, HepG-2 cells were used as the experimental model, and the steps were as follows: (1) Prepare a DMSO standard solution of the molecular probe Cy647 with a concentration of 1 mM. (2) Cell culture: The resuscitated HepG-2 cells were cultured. The culture medium contained 10% fetal bovine serum, 1% double antibody, 89% DMEM, and was cultured in an environment of 37 °C, 5% CO2, and 1% O2 for 24 h to obtain well-growing cells for use. (3) Place the live HepG-2 cells into the culture medium for cultivation. A total of 4 groups were cultured, and the inoculation amount in each group of the culture medium was 2×10 7 ~9×10 7 cells / mL, and cultivated for 24 h. They were divided into four groups: a, b, c, and d. Group a of HepG-2 cells was added with 10 μM of the molecular probe Cy647 and incubated for 30 min; Group b of HepG-2 cells was added with 10 μM of the molecular probe Cy647 and incubated for 60 min; Group c of HepG-2 cells was added with 10 μM of the molecular probe Cy647 and incubated for 3 h; Group d was added with 10 μM of the molecular probe Cy468 and incubated for 30 min. Confocal laser fluorescence imaging of HepG-2 was performed. The excitation wavelength E x of the green channel = 405 nm, and the emission wavelength E m = 410 - 550 nm; the excitation wavelength E x of the red channel = 594 nm, and the emission wavelength E m = 620 - 750 nm; the confocal maps of the four groups of cells were obtained, as shown in Figure 13 . It can be seen from Figure 13 a2 and a3 that after incubating the cells with Cy647 for 30 min, there was no fluorescence in the green channel, and the fluorescence intensity in the red channel was high, indicating the presence of Cy647 in the cells. After incubating for 1 h, at this time, a weak fluorescence appeared in the green channel, indicating that a small amount of Cy468 was produced. Continuing to incubate for 3 h, the green fluorescence intensity was high, and the red fluorescence in the red channel decreased significantly, indicating that most of the Cy647 was converted into Cy468. As a control, after incubating the cells with Cy468 for 30 min, the cell imaging effects were as shown in Figure 13 d2 and d3, with high green light intensity and weak red light intensity, proving that Cy647 can be converted into Cy468 in hypoxic cells. IV. Effect of Molecular Probe Cy647 on Cell Viability Detect the effect of the molecular probe Cy647 on cell viability. The specific steps are as follows: Add the molecular probe Cy647 with concentrations of 0 µM, 10 µM, 20 µM, 30 µM, 40 µM, and 50 µM respectively to the cell culture medium, and culture in an incubator at 37 °C and 5% CO2 for 24 h. Add 20 µL of 4-methylthiazolyl tetrazolium (MTT) at 5 mg / mL to the cell culture medium and culture for 4 h. Evaluate the cell viability by the MTT colorimetric method. Take the cell group without adding the molecular probe Cy647 as 100% viability, and draw a relative bar chart for the relevant data of the experimental groups with different concentrations of the molecular probe Cy647 added. The results are as Figure 14 shown. Through Figure 14 it can be seen that adding 0 - 50 µM of the molecular probe Cy647 to the cell culture medium has a relatively small effect on cell viability, and the viability is above 90%.

[0044] Example 2 In this example, another preparation method of the molecular probe Cy647 is provided. The steps are as follows: (1) Add 3.0 mmol of Cy527 and 7.0 mmol of 1,4,7,10-tetraazacyclododecane to a 100 mL round-bottom flask. Under the action of 7.0 mmol of potassium carbonate and 20 mL of chloroform, react at 75 °C for 26 hours. After the reaction is completed, let it stand and cool to room temperature, concentrate it under vacuum with a rotary evaporator, wash the solid obtained from the reaction 3 times with 250 mL of dichloromethane, then concentrate it under vacuum with a rotary evaporator, and then elute with a mixed mobile phase of dichloromethane / methanol (volume ratio 24:1) for silica gel column chromatography purification to obtain the intermediate product (Cy468).

[0045] (2) Dissolve 1.0 mmol of the intermediate product Cy468 in 10 mL of dichloromethane, add 6.0 mmol of 4-nitrobenzyl chloroformate and 5.0 mmol of potassium carbonate, and react in an ice bath for 1.5 h. After the reaction is completed, transfer the mixture to a separatory funnel, wash it 3 times with water, and concentrate it under vacuum with a rotary evaporator. That is, the crude product is obtained. The crude product is eluted with a mixed mobile phase of dichloromethane / methanol (volume ratio 8:1) for column chromatography purification to obtain the required product, which is the molecular probe (Cy647).

[0046] Example 3 In this example, another preparation method of the molecular probe Cy647 is provided. The steps are as follows: (1) Add 4.0 mmol of Cy527 and 9.0 mmol of 1,4,7,10-tetraazacyclododecane into a 100 mL round-bottom flask. Under the action of 8.0 mmol of potassium carbonate and 40 mL of chloroform, react at 85 °C for 22 hours. After the reaction is completed, let it stand and cool to room temperature, concentrate it under vacuum using a rotary evaporator. Wash the solid obtained from the reaction 3 times with 250 mL of dichloromethane, then concentrate it under vacuum using a rotary evaporator. Then add a mixed mobile phase of dichloromethane / methanol (volume ratio of 27:1) for elution and purify it by silica gel column chromatography to obtain the intermediate product (Cy468).

[0047] (2) Dissolve 2.0 mmol of the intermediate product Cy468 in 30 mL of dichloromethane, add 10.0 mmol of 4-nitrobenzyl chloroformate and 8.0 mmol of potassium carbonate, and react in an ice bath for 1.5 h. After the reaction is completed, transfer the mixture to a separatory funnel, wash it 3 times with water, and concentrate it under vacuum using a rotary evaporator. The crude product is obtained. Subject the crude product to column chromatography purification with a mixed mobile phase of dichloromethane / methanol (volume ratio of 12:1) for elution to obtain the required product, which is the molecular probe (Cy647).

[0048] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A molecular probe, characterized in that Its chemical structure is shown below: 。 2. The method for preparing a molecular probe according to claim 1, characterized in that: The synthetic route of the preparation method is as follows: ； The preparation method comprises the following steps: (1) Cy527 reacts with 1,4,7,10-tetraazacyclododecane to obtain the intermediate product Cy468; (2) Cy468 reacts with 4-nitrobenzyl chloroformate to obtain the molecular probe Cy647.

3. The method for preparing a molecular probe according to claim 2, characterized in that: The specific reaction of step (1) is as follows: the starting reactant Cy527 reacts with 1,4,7,10-tetraazacyclododecane in the presence of potassium carbonate and chloroform, the reaction temperature is 75-85°C, and the reaction time is 22-26 h; after the reaction is completed, the solvent is removed to obtain a solid part, the solid part is added to a mixed mobile phase to dissolve, and the solid part is purified by silica gel column chromatography to obtain an intermediate product Cy468.

4. The method for preparing a molecular probe according to claim 3, characterized in that: The dosage ratio of Cy527, 1,4,7,10-tetraazacyclododecane, potassium carbonate and chloroform is 3-4 mmol: 7-9 mmol: 7-8 mmol: 20-40 mL.

5. The method for preparing a molecular probe according to claim 3, characterized in that: The solvent is removed by reducing pressure, vacuum concentration or evaporation to obtain a solid portion, which is first dissolved in dichloromethane, washed with water for multiple times to remove excess potassium carbonate, concentrated again after washing with water, and dissolved in a mixed mobile phase, wherein the mixed mobile phase is a mixture of dichloromethane and methanol in a volume ratio of 24-27:

1.

6. The method for preparing a molecular probe according to claim 2, characterized in that: The specific reaction of step (2) is as follows: the intermediate product Cy468 is dissolved in dichloromethane, and 4-nitrobenzyl chloroformate and potassium carbonate are added to react in an ice bath for 1.5 to 2.5 hours; after the reaction is completed, the solvent is removed, a mixed mobile phase is added to dissolve, and then purified by silica gel column chromatography to obtain Cy647.

7. The method for preparing a molecular probe according to claim 6, characterized in that: The dosage ratio of the intermediate product Cy468, dichloromethane, 4-nitrobenzyl chloroformate, and potassium carbonate is 1-2 mmol: 10-30 mL: 6-10 mmol: 5-8 mmol; Alternatively, in step (2), the steps of removing the solvent after the reaction and purifying by silica gel column chromatography are as follows: the solvent is removed by reducing pressure, vacuum concentration or evaporation to obtain a solid portion; the solid portion is first dissolved in dichloromethane, washed with water for multiple times to remove excess potassium carbonate, concentrated again after washing with water, dissolved in a mixed mobile phase, and purified by silica gel column chromatography to obtain Cy647, wherein the mixed mobile phase is a mixture of dichloromethane and methanol in a volume ratio of 8 to 12:

1.

8. Use of the molecular probe according to claim 1 in the field of nitroreductase detection.

9. The use of the molecular probe according to claim 8 in the field of nitroreductase detection, characterized in that: The molecular probe Cy647 is used as a molecular probe of nitroreductase and is applied in living cells.

10. The use of the molecular probe according to claim 8 in the field of nitroreductase detection, characterized in that: The method for using the molecular probe for nitroreductase detection is as follows: adding the molecular probe Cy647 to a biological sample to be tested, and determining the expression of nitroreductase in the sample to be tested by detecting the fluorescence intensity at an ultraviolet absorption wavelength of 615 nm or at fluorescence emission wavelengths of 730 nm and 470 nm; compared with the control sample, when the absorption peak intensity at an ultraviolet absorption wavelength of 615 nm decreases, the fluorescence intensity at a fluorescence emission wavelength of 730 nm decreases, and the fluorescence intensity at a fluorescence emission wavelength of 470 nm increases, it indicates that overexpressed nitroreductase exists in the sample to be tested, and the biological sample includes but is not limited to a cell sample, a tissue sample, or an organ sample; Alternatively, the molecular probe Cy647 is added to the tissue sample to be tested, and after incubation for a period of time, the fluorescence intensity is observed and recorded at emission wavelengths of 730 nm and 470 nm by a fluorescence imaging device; Alternatively, the molecular probe Cy647 is used as a contrast agent and injected into the experimental sample via intravenous injection, and the fluorescence intensity is observed and recorded at an emission wavelength of 730 nm using a fluorescence imaging device.

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