A nitroreductase-responsive fluorescent probe, and a preparation method and application thereof

Abstract

This invention provides a nitroreductase-responsive fluorescent probe, its preparation method, and its application, belonging to the field of bioanalysis and detection technology. The nitroreductase-responsive fluorescent probe of this invention has the structure shown in Formula I; this invention also provides a preparation method and application of this nitroreductase-responsive fluorescent probe. The nitroreductase-responsive fluorescent probe of this invention can react with nitroreductase and emit green fluorescence in the presence of nicotinamide adenine dinucleotide, exhibiting high specificity and sensitivity, and can realize the detection of changes in nitroreductase content in hypoxic cells and sperm.

Description

Technical Field

[0001] This invention belongs to the field of bioanalysis and detection technology, specifically relating to a nitroreductase-responsive fluorescent probe, its preparation method, and its application. Background Technology

[0002] Clinical infertility is a common problem faced by couples of reproductive age, affecting a significant portion of the population, with male factors accounting for a large proportion of infertility cases. According to clinical guidelines, semen quality analysis is an essential part of the initial examination of infertile couples; this assessment covers multiple semen parameters, such as semen volume, sperm density, motility, and morphology. Despite this, a large number of infertile men are still classified as having idiopathic infertility or whose specific cause cannot be determined. This unexplained fertilization failure remains a major challenge in the medical field. Unfortunately, reliable etiological indicators are currently lacking in clinical practice to effectively assess sperm samples from infertile men.

[0003] Sperm can synthesize adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) and anaerobic glycolysis, providing energy for fertilization. Sperm are exposed to constantly changing environmental factors such as pH and O2 levels. Therefore, maintaining a stable energy supply under external pressure is crucial for successful fertilization.

[0004] Nitroreductase (NTR) is a flavin reductase that reduces nitro aromatic compounds to hydroxylamine or amino compounds in the presence of nicotinamide adenine dinucleotide (NADH). Numerous studies have shown that hypoxia can lead to increased expression of NTR, and oxygen levels also affect sperm energy metabolism. Therefore, changes in NTR can indicate the metabolic activity of hypoxic sperm, and developing an effective method for detecting NTR is of great significance in the detection of infertile sperm.

[0005] Developing a novel, highly specific nitroreductase-responsive fluorescent probe that leverages the non-invasive and adjustable properties of small molecule fluorescent probes to prepare kits for detecting nitroreductases in human sperm and living cells has become a major research need. Summary of the Invention

[0006] The purpose of this invention is to provide a nitroreductase-responsive fluorescent probe, its preparation method, and its application. This fluorescent probe has high specificity and sensitivity for recognizing substrates and can detect changes in nitroreductase content in hypoxic cells and sperm.

[0007] The technical solution adopted to achieve the above objective is to provide a nitroreductase-responsive fluorescent probe, which has the structure shown in Formula I:

[0008]

[0009] This invention also provides a method for preparing a nitroreductase-responsive fluorescent probe, comprising the following steps:

[0010] (1) Dissolve 2,4-dichloropurine and 1-bromopropane in an organic solvent and react them under the action of a catalyst to obtain the first intermediate;

[0011] (2) Dissolve the first intermediate and dimethylamine in an organic solvent and react them under the action of a catalyst to obtain the second intermediate;

[0012] (3) The second intermediate was subjected to the Vilsmeier reaction to obtain the third intermediate;

[0013] (4) The third intermediate and 4-methylpyridine are dissolved in an organic solvent and refluxed under the action of a catalyst to obtain the fourth intermediate;

[0014] (5) The fourth intermediate and p-nitrobenzyl bromide were dissolved in an organic solvent and refluxed to obtain the nitroreductase-responsive fluorescent probe as shown in Formula I; its synthetic route is as follows:

[0015]

[0016] Preferably, in step (1), the organic solvent is dimethyl sulfoxide, the reaction temperature is 70-90℃, and the time is 5-7h; in step (2), the dimethylamine is a dimethylamine aqueous solution with a mass fraction of 35-45%, the organic solvent is N,N-dimethylformamide, the reaction temperature is 90-110℃, and the time is 45-50h; the catalyst in both steps (1) and (2) is potassium carbonate.

[0017] More preferably, the reaction temperature in step (1) is 80°C and the reaction time is 6h; in step (2), the dimethylamine is a 40% dimethylamine aqueous solution, the reaction temperature is 100°C and the reaction time is 48h.

[0018] More preferably, the molar ratio of 2,4-dichloropurine, 1-bromopropane and catalyst in step (1) is 1:6:1.5.

[0019] More preferably, in step (2), the molar ratio of the first intermediate, dimethylamine and catalyst is 1:8:3.

[0020] Preferably, step (3) includes the following steps: N,N-dimethylformamide is cooled at -2 to 2°C for 10 to 30 minutes, phosphorus oxychloride is added dropwise, and the mixture is stirred at -2 to 2°C for 25 to 35 minutes, then heated to 75 to 85°C to obtain Vilsmeier reagent; the second intermediate is dissolved in N,N-dimethylformamide and then added dropwise to Vilsmeier reagent, and the mixture is stirred at 75 to 85°C for 22 to 26 hours to obtain the third intermediate.

[0021] More preferably, step (3) includes the following steps: after cooling N,N-dimethylformamide at 0°C for 30 min, phosphorus oxychloride is added dropwise, and after stirring at 0°C for 30 min, the temperature is raised to 80°C to obtain Vilsmeier reagent; after dissolving the second intermediate with N,N-dimethylformamide, it is added dropwise to Vilsmeier reagent, and the mixture is stirred at 80°C for 24 h to obtain the third intermediate.

[0022] More preferably, in step (3), the molar ratio of N,N-dimethylformamide in the second intermediate, phosphorus oxychloride and Vilsmeier reagent is 1:10:15.

[0023] Preferably, in step (4), the organic solvent is N,N-dimethylformamide, the catalyst is potassium tert-butoxide, and the reaction time is 2-4 h; in step (5), the organic solvent is toluene, and the reaction time is 8-12 h.

[0024] Preferably, the reaction time in step (4) is 3 hours; the reaction time in step (5) is 12 hours.

[0025] More preferably, in step (4), the molar ratio of the third intermediate, 4-methylpyridine and the catalyst is 1:2:3.

[0026] More preferably, the molar ratio of the fourth intermediate and p-nitrobenzyl bromide in step (5) is 1:1.5.

[0027] The present invention also provides the application of the above-mentioned nitroreductase-responsive fluorescent probe in the preparation of a sperm nitroreductase detection kit.

[0028] The present invention also provides the application of the above-mentioned nitroreductase-responsive fluorescent probe in the preparation of a cell hypoxia detection kit.

[0029] The present invention also provides a sperm nitroreductase detection kit, comprising a nitroreductase-responsive fluorescent probe.

[0030] The present invention also provides a cell hypoxia detection kit, comprising a nitroreductase-responsive fluorescent probe.

[0031] The beneficial effects of the above-mentioned technical solution in this invention are as follows: During the use of the sperm nitroreductase detection kit, the nitroreductase-responsive fluorescent probe needs to react with nitroreductase in the presence of nicotinamide adenine dinucleotide and emit green fluorescence; the detection reagent for the level of nitroreductase in sperm is a detection reagent at the level of live sperm fluorescence imaging, and the methods for detecting sperm nitroreductase at the level of live sperm fluorescence imaging include laser confocal imaging technology and flow cytometry detection technology. Furthermore, during the use of the sperm nitroreductase detection kit and the cell hypoxia detection kit, the concentration of the nitroreductase-responsive fluorescent probe is 5 μM.

[0032] The present invention has the following beneficial effects:

[0033] (1) This invention is the first to develop a purine fluorescent backbone as a fluorescent probe for hypoxia recognition, wherein the recognition substrate is nitroreductase; the probe can react with nitroreductase to produce green fluorescence in the presence of nicotinamide adenine dinucleotide; the probe can emit green fluorescence after co-incubation with hypoxic cells and sperm, realizing bioimaging of hypoxic cells and sperm, with significant selectivity and detection sensitivity.

[0034] (2) The kit provided by this invention was successfully used for flow cytometry staining of sperm samples from infertile patients, realizing the detection of sperm samples; compared with sperm samples from normal subjects, the expression of nitroreductase in sperm from infertile patients did not increase after hypoxia; this invention is the first to use nitroreductase activity as a detection indicator for male infertility, providing an effective theoretical reference for male infertility in sperm pathophysiology. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the mechanism by which a nitroreductase-responsive fluorescent probe responds to nitroreductase.

[0036] Figure 2 The images show the fluorescence spectra of the nitroreductase-responsive fluorescent probe after reaction with nitroreductase; where A is the fluorescence spectrum of the nitroreductase-responsive fluorescent probe reacting with different substrates; and B is the fluorescence spectrum of the nitroreductase-responsive fluorescent probe reacting with the fourth intermediate after reaction with nitroreductase.

[0037] Figure 3 High-resolution mass spectra of the nitroreductase-responsive fluorescent probe after reaction with nitroreductase; where A is the high-resolution mass spectrum of the nitroreductase-responsive fluorescent probe after reaction with nitroreductase; and B is the fluorescence spectrum of the fourth intermediate.

[0038] Figure 4This chart shows the reaction trends of different concentrations of nitroreductase-responsive fluorescent probes with different concentrations of nitroreductase. Specifically, A represents the fluorescence spectra of the nitroreductase-responsive fluorescent probes with different reaction times; B represents the fluorescence spectra of the nitroreductase-responsive fluorescent probes with different concentrations of nitroreductase; C represents the fluorescence spectra of the nitroreductase-responsive fluorescent probes with different concentrations of nitroreductase; D represents the enzyme kinetic curves of the nitroreductase-responsive fluorescent probes with different concentrations of nitroreductase; E represents the enzyme kinetic curves of the nitroreductase-responsive fluorescent probes with different concentrations of nitroreductase; and F represents the enzyme kinetic curves of the nitroreductase-responsive fluorescent probes with different concentrations of nitroreductase.

[0039] Figure 5 The graph shows the fluorescence intensity values ​​of the reaction between nitroreductase-responsive fluorescent probes and different bioanalytes.

[0040] Figure 6 This figure shows the cytotoxicity of different concentrations of nitroreductase-responsive fluorescent probes on human liver cancer cells.

[0041] Figure 7 The results show the biocompatibility of the nitroreductase-responsive fluorescent probe; where A is a confocal microscopy image of human alveolar basal epithelial cells with lung cancer under different oxygen levels and the action of nitroreductase inhibitors; B is a quantitative analysis of the fluorescence intensity in Figure A.

[0042] Figure 8 The images show confocal imaging results of a nitroreductase-responsive fluorescent probe in hypoxic cells; where A is a confocal microscopy image of a nitroreductase-responsive fluorescent probe in human dental pulp mesenchymal stem cells under hypoxic conditions; and B is a fluorescence intensity map of the area traversed by the straight line in image A.

[0043] Figure 9 The figures show the experimental results of nitroreductase-responsive fluorescent probes in normal donor sperm samples under different oxygen levels and the action of nitroreductase inhibitors; where A is a confocal microscopy image; and B is a quantitative analysis of fluorescence intensity in figure A.

[0044] Figure 10 The figures show the experimental results of a nitroreductase-responsive fluorescent probe in normal donor sperm samples after hydrogen peroxide treatment; where A is a confocal microscopy image; and B is a quantitative analysis of the fluorescence intensity in image A.

[0045] Figure 11 Flow cytometry clustering of nitroreductase-responsive fluorescent probes in sperm samples from infertile patients;

[0046] Figure 12A statistical chart showing the number of positive cells in sperm samples from infertile patients;

[0047] Figure 13 A statistical graph showing the positive fluorescence intensity in sperm samples from infertile patients;

[0048] Figure 14 The receiver operating characteristic (ROC) curve is a test curve that uses the increase in green fluorescence intensity after staining with a nitroreductase-responsive fluorescent probe as the detection standard. Detailed Implementation

[0049] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0050] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0051] Example 1: Nitroreductase-responsive fluorescent probe and its preparation method

[0052] A nitroreductase-responsive fluorescent probe has the structure shown in Formula I:

[0053]

[0054] This embodiment also discloses a method for preparing a nitroreductase-responsive fluorescent probe, including the following steps:

[0055] (1) 2,6-Dichloropurine (6 g, 31.75 mmol, 1.0 eq) and potassium carbonate (6.58 g, 47.13 mmol, 1.5 eq) were added to a 100 mL round-bottom flask, along with 30 mL of dimethyl sulfoxide and 1-bromopropane (23.43 g, 190.48 mmol, 6.0 eq). The mixture was stirred and heated at 80 °C for 6 h. After the reaction was complete, deionized water was added, and the mixture was extracted with ethyl acetate. The organic phase was collected, and the solvent was removed by vacuum distillation. The residue was separated by column chromatography to obtain a pale yellow oil. Natural crystallization yielded the first intermediate (3.4552 g of white solid, yield 47.09%) (PE:EA = 4:1). The NMR data of the first intermediate are as follows:

[0056] 1 H NMR (400MHz, Chloroform-d) δ (TMS, ppm) 8.10 (s, 1H), 4.22 (t, J = 7.2Hz, 2H), 1.94 (h, J = 7.4Hz, 2H), 0.97 (t, J = 7.4Hz, 3H).

[0057] 13 C NMR(101MHz,Chloroform-d)δ(TMS,ppm)153.17,152.85,151.69,145.83,130.73,46.24,23.17,11.11.

[0058] (2) The first intermediate (3 g, 12.98 mmol, 1.0 eq) and potassium carbonate (4.9923 g, 38.94 mmol, 3.0 eq) were added to a 100 mL round-bottom flask. 30 mL of N,N-dimethylformamide was added, and the flask was placed in an ice-water bath. After cooling, a 40% dimethylamine aqueous solution (4.6825 g, 103.89 mmol, 8.0 eq) was added, and the mixture was stirred in the ice-water bath for 1 h. The mixture was then transferred to a heating reactor and reacted at 100 °C for 48 h. After the reaction was complete, the cooled reaction solution was poured into ice water and allowed to stand overnight. A white flocculent precipitate formed. The precipitate was filtered under reduced pressure the next day and washed with ice water to obtain the second intermediate (3.1042 g of white solid, yield 96.30%). The second intermediate did not require further purification and could be used for the next reaction. The NMR data for the second intermediate are as follows:

[0059] 1 H NMR(400MHz,Chloroform-d)δ(TMS,ppm)7.41(d,J=1.2Hz,1H),4.01–3.95(m,2H ),3.44(s,6H),3.16(d,J=1.3Hz,6H),1.85(d,J=7.9Hz,2H),0.94–0.89(m,3H).

[0060] 13 C NMR(101MHz,Chloroform-d)δ(TMS,ppm)159.36,154.71,153.29,135.68,135.66,113.64,44.72,37.23,23.02,11.28.

[0061] (3) Add 30.19 mL of N,N-dimethylformamide (28.62 g, 391.5 mmol, 15 eq) to a 100 mL round-bottom flask, stir in an ice-water bath at 0 °C for 30 min, and then add 23.89 mL dropwise. POCl3 (40.02 g, 261 mmol, 10 eq) was stirred in an ice-water bath at 0 °C for 30 min to obtain Vilsmeier's reagent. Then, a solution of N,N-dimethylformamide containing the second intermediate (8.3376 g, 26.1 mmol, 1.0 eq) was added dropwise to Vilsmeier's reagent and transferred to a heated reactor. The reaction was carried out at 80 °C for 24 h. After cooling to room temperature, pure water was added to the reaction mixture, and the mixture was extracted with dichloromethane. The organic phase was collected, and the solvent was removed by vacuum distillation. Column chromatography was used to separate the third intermediate (3.2865 g of yellow-green flocculent precipitate, yield 45.57%) (PE:EA = 2:1). The NMR data for the third intermediate are as follows:

[0062] 1 H NMR (400MHz, Chloroform-d) δ (TMS, ppm) 9.69 (s, 1H), 4.42–4.38 (m, 2H), 3.40 (s, 6H), 3.20 (s, 6H), 1.80 (q, J = 7.3Hz, 2H), 0.88 (t, J = 7.4Hz, 3H).

[0063] 13 C NMR(101MHz,Chloroform-d)δ(TMS,ppm)182.26,160.48,155.72,155.12,141.10,117.08,44.53,37.17,23.13,11.16.

[0064] (4) The third intermediate (82.90 g, 0.3 mmol, 1.0 eq) and potassium tert-butoxide (101.0 mg, 0.9 mmol, 3.0 eq) were added to a 25 mL round-bottom flask. After adding 6 mL of N,N-dimethylformamide, 4-methylpyridine (58.3 μL, 0.6 mmol, 2.0 eq) was added. The mixture was refluxed under nitrogen protection for 3 h. After the reaction was completed, pure water was added, and the mixture was extracted with dichloromethane. The organic phase was collected and separated by column chromatography to obtain the fourth intermediate (44.0 mg yellow solid, yield 41.73%) (PE:EA = 2:1). The NMR data of the fourth intermediate are as follows:

[0065] 1H NMR(400MHz,Chloroform-d)δ(TMS,ppm)8.60–8.56(m,2H),7.56(d,J=15.8Hz,1H),7.38(d,J=5.6Hz,2H),7.14( d,J=15.8Hz,1H),4.14(t,J=7.1Hz,2H),3.51(s,6H),3.18(s,6H),1.84(p,J=7.3Hz,2H),0.94(t,J=7.4Hz,3H).

[0066] 13 C NMR(TMS,ppm)(101MHz,Chloroform-d)δ159.29,154.51,154.44,150.25,143.99,142.1 4,129.23,120.91,118.40,114.67,43.26,38.14,37.19,29.69,23.54,11.35.HRMS[M+H] + Calcd.352.2245 for C 19 H 26 N7 + Found: 352.2242.

[0067] (5) The fourth intermediate (44.0 mg, 0.13 mmol, 1.0 eq) and p-nitrobenzyl bromide (42.1 mg, 0.20 mmol, 1.5 eq) were added to a 25 mL round-bottom flask, and 2 mL of toluene was added. The mixture was refluxed overnight under nitrogen protection for 12 h. After the reaction was completed, the toluene was removed by vacuum distillation, and an appropriate amount of ethyl acetate was added and stirred at room temperature for 2 h. The precipitated solid was filtered under vacuum, washed three times with ethyl acetate, and dried to obtain the nitroreductase-responsive fluorescent probe as shown in Formula I (50.0 mg of purple solid, yield 67.18%). The obtained product does not require further purification and can be used directly. The NMR data of the nitroreductase-responsive fluorescent probe are as follows:

[0068] 1 H NMR(400MHz,Chloroform-d)δ(TMS,ppm)9.23(d,J=6.5Hz,2H),8.15(d,J=8.1Hz,2H),7.90–7.83(m,4H),7.51–7.35 (m,2H),6.36(s,2H),4.13(d,J=8.0Hz,2H),3.44(s,6H),3.12(s,6H),1.74(q,J=7.3Hz,2H),0.88(t,J=7.4Hz,3H).

[0069] 13 C NMR(101MHz,Chloroform-d)δ(TMS,ppm)158.57,157.46,152.48,147.80,147.70,144.89,143.94,134. 00,133.91,130.86,128.49,128.00,127.20,121.03,65.79,47.27,42.18,41.06,27.47,14.97.HRMS[M] + Calcd.487.2565 for C 26 H 31 N8O2 + Found: 487.2559.

[0070] Its synthetic route is as follows:

[0071]

[0072] Example 2: Mechanism of nitroreductase-responsive fluorescent probe to nitroreductase

[0073] The response mechanism of the nitroreductase-responsive fluorescent probe prepared in Example 1 to nitroreductase was investigated, and the specific process is as follows:

[0074] The reaction mechanism of the probe PPy was determined by high-resolution mass spectrometry (HRMS) and fluorescence spectroscopy. The fluorescence spectra (λ) of the nitroreductase-responsive fluorescent probe (labeled PPy), PPy + nitroreductase (NADH), PPy + NADH + nitroreductase (NTR), and the fourth intermediate of the compound (labeled PPy-N) were measured. ex =405nm), the result is as follows Figure 2 As shown, the theoretical product of the nitroreductase fluorescent probe (PPy) and nitroreductase is the fourth intermediate (PPy-N).

[0075] from Figure 2 As can be seen, at 37℃, the spectrum of the probe PPy (5 μM) reacting with nitroreductase (10 μg / mL) in the presence of NADH (500 μM) overlaps with that of the fourth intermediate PPy-N; high-resolution mass spectrometry also indicates the formation of compound PPy-N. Figure 3 This demonstrates that the mechanism of the reaction between the nitroreductase-responsive fluorescent probe PPy and nitroreductase in this invention is consistent with expectations. Figure 1 ).

[0076] Example 3: Fluorescence spectroscopy and selectivity analysis of nitroreductase-responsive fluorescent probes.

[0077] The nitroreductase-responsive fluorescent probe prepared in Example 1 was analyzed, and the results are as follows: Figures 4-5 As shown; Figure 4 A and Figure 4 D shows the fluorescence spectrum and enzyme kinetic curve of the reaction between the nitroreductase-responsive fluorescent probe PPy and nitroreductase over time. Figure 4 B and Figure 4 E represents the fluorescence spectrum and enzyme kinetic curve of the reaction between PPy probes of different concentrations and nitroreductase. Figure 4 C and Figure 4 F represents the fluorescence spectrum and enzyme kinetic curve of the reaction between the nitroreductase-responsive fluorescent probe PPy and different concentrations of nitroreductase, respectively (λ). ex =405nm, λ em =540nm), the results show that the nitroreductase-responsive fluorescent probe PPy of the present invention has good responsiveness to nitroreductase and is a suitable detection tool.

[0078] At the same time, such as Figure 5 As shown, the nitroreductase-responsive fluorescent probe PPy was co-incubated with other analytes that may exist in organisms, and no enhancement of green fluorescence was observed, indicating that the nitroreductase-responsive fluorescent probe PPy of the present invention can specifically respond to nitroreductase.

[0079] Example 4: Biocompatibility Analysis of Nitroreductase-Responsive Fluorescent Probes

[0080] The biocompatibility and confocal imaging performance of the nitroreductase-responsive fluorescent probe prepared in Example 1 were analyzed to evaluate the probe's response to nitroreductase in vivo. The specific procedures are as follows:

[0081] First, its cell compatibility was studied and analyzed, and the results were as follows: Figure 6 As shown, after co-culturing with different concentrations of nitroreductase-responsive fluorescent probe PPy (0–10 μM) for 24 h, the survival rate of human liver cancer cells (HepG2) was greater than 80%, indicating that at the concentration used in this invention (5 μM), there is low cytotoxicity.

[0082] Subsequently, a hypoxia model was established in human alveolar basal epithelial cells (A549) of lung cancer: (1) Normoa group: cells were cultured in a conventional constant temperature incubator (37℃, 5% CO2) and then directly stained with the probe PPy; (2) Hypoxia group: cells were cultured in a conventional constant temperature incubator and then placed in a hypoxia incubator (37℃, 5% CO2, 3% O2) for 1 h and then stained with the nitroreductase-responsive fluorescent probe PPy; (3) Inhibitor group: cells were added with the nitroreductase inhibitor dicumarol (10 μM) and then incubated in a conventional constant temperature incubator for 1 h and then transferred to a hypoxia incubator for 1 h and then stained with the nitroreductase-responsive fluorescent probe PPy; after 1 h of staining, the original culture medium of each group was discarded, new culture medium was added, and the cells were directly placed in a laser confocal microscope for imaging (λ). ex =405nm, λ em =500~600nm), the results are as follows Figure 7 As shown, Figure 7 In B, **** represents P < 0.0001. From Figure 7 As can be seen in A and 7B, a significant enhancement of green fluorescence signal was observed in the hypoxia group, and the fluorescence intensity of hypoxic cells with added nitroreductase inhibitors was on par with that in the normoxic group. The results indicate that the nitroreductase-responsive fluorescent probe of the present invention has excellent ability to respond to nitroreductase at the cellular level.

[0083] Simultaneously, hypoxic human dental pulp mesenchymal stem cells (hDMSCs) were co-stained with the nitroreductase-responsive fluorescent probe of this invention and commercially available mitochondrial deep red dye (MTDR) and then subjected to laser confocal imaging (product (green): λ). ex =405nm, λ em =500~600nm; Raw material channel (red): λ ex =488nm, λ em =700~750nm; MTDR channel (yellow): λ ex =633nm, λ em =650~720nm), the results are as follows Figure 8 As shown in Figures 8A and 8B, consistent with the expected mechanism, the nitroreductase-responsive fluorescent probe PPy carries a positive charge before responding to nitroreductase (red channel), enabling it to target mitochondria and exhibiting a good co-localization coefficient (0.86) with commercially available mitochondrial dyes. However, after responding to nitroreductase (green channel), the positive charge disappears, the mitochondrial localization effect decreases, and the co-localization coefficient decreases (0.62).

[0084] Example 5: The ability of nitroreductase-responsive fluorescent probes in hypoxic sperm imaging to detect nitroreductase activity.

[0085] In the embodiments described below, the studies involved were approved by the Ethics Committee of West China Second University Hospital, Sichuan University, and each participant signed an informed consent form. The inclusion and exclusion criteria used were selected according to the guidelines of the World Health Organization (WHO).

[0086] Sperm staining was performed in the same manner as in Example 4. After obtaining clinical donation samples, the cells were washed three times by centrifugation with phosphate-buffered saline (PBS, pH = 7.4) and then subjected to hypoxia treatment for staining. The results are as follows: Figure 9 As shown, Figure 9 In B, **** represents P < 0.0001. From Figure 9 As can be seen, the hypoxia imaging results are consistent with those in Example 4, indicating that the probe of the present invention is suitable for imaging sperm nitroreductase.

[0087] Example 6: Nitroreductase-responsive fluorescent probes were used as nitroreductase detection kits for the detection of a sperm oxidative stress model.

[0088] Establishment of the oxidative stress model: After washing, clinical sperm samples were added to a sperm suspension with an equal volume of commercially available 30% hydrogen peroxide solution, resulting in a final concentration of 15%. After 15 min of hydrogen peroxide treatment, the liquid was removed by centrifugation, and PBS solution was added before hypoxia treatment and staining. Laser confocal imaging results are shown below. Figure 10 As shown, Figure 10 In B, **** represents P < 0.0001. From Figure 10 The results show that sperm treated with hydrogen peroxide exhibited lower green fluorescence enhancement values ​​after hypoxic culture and probe staining, significantly different from the untreated group. This suggests that nitroreductase activity may differ in diseased sperm.

[0089] Example 7: Nitroreductase-responsive fluorescent probes were used as nitroreductase detection kits for the detection of sperm samples from patients with idiopathic infertility.

[0090] In this embodiment, the semen control sample was collected from a previously confirmed fertile healthy donor; idiopathic infertility refers to the inability to conceive normally or the termination of pregnancy despite normal semen analysis.

[0091] After simultaneously hypoxic treatment of healthy donor samples and infertile patient samples, they were stained using the kit of this invention and then analyzed by flow cytometry.

[0092] Kit staining procedure: After washing, the samples were resuspended in phosphate-buffered saline (PBS) at pH 7.4 and incubated for 1 hour each in a 37°C incubator (normal oxygen, 21% O2) and a 37°C hypoxic incubator (hypoxia, 3% O2). Then, the PBS was removed, and fresh PBS containing 5 μM of the nitroreductase-responsive fluorescent probe PPy was added. The samples were then incubated for another 1 hour under either normal or hypoxic conditions. Finally, the nitroreductase-responsive fluorescent probe was removed, and the samples were resuspended in fresh PBS for instrument detection. Results are as follows: Figures 11-14 As shown, Figures 12-13 In the middle, **** represents P<0.0001, and ns represents not significant.

[0093] from Figures 11-13 As can be seen, normal sperm samples showed a significant enhancement of green fluorescence signal after hypoxia (Q1: 405+, 488-), while sperm samples from infertile patients showed almost no green fluorescence signal and positive cells. Using the kit of this invention, a total of 8 normal samples and 8 patient samples were tested. The enhancement value of the green fluorescence signal (ΔMFI inQ1) was used as the detection index to plot the receiver operating characteristic (ROC) curve. Figure 14 The area under the curve (AUC) was 0.9750. These results indicate that the indicator detected by this kit (the enhancement value of green fluorescence after nitroreductase response) can serve as a potential indicator of sperm pathogenesis in fertilization failure and embryonic maldevelopment.

[0094] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.

Claims

1. A nitroreductase-responsive fluorescent probe, characterized in that, The nitroreductase-responsive fluorescent probe has the structure shown in Formula I: 。 2. The method for preparing the nitroreductase-responsive fluorescent probe according to claim 1, characterized in that, The synthesis route is as follows: 。 3. The method for preparing the nitroreductase-responsive fluorescent probe as described in claim 2, characterized in that, Includes the following steps: (1) Dissolve 2,4-dichloropurine and 1-bromopropane in an organic solvent and react them under the action of a catalyst to obtain the first intermediate; (2) Dissolve the first intermediate and dimethylamine in an organic solvent and react them under the action of a catalyst to obtain the second intermediate; (3) The second intermediate was subjected to the Vilsmeier reaction to obtain the third intermediate; (4) The third intermediate and 4-methylpyridine are dissolved in an organic solvent and refluxed under the action of a catalyst to obtain the fourth intermediate; (5) Dissolve the fourth intermediate and p-nitrobenzyl bromide in an organic solvent and reflux to obtain the nitroreductase-responsive fluorescent probe as shown in Formula I.

4. The method for preparing the nitroreductase-responsive fluorescent probe as described in claim 3, characterized in that, In step (1), the organic solvent is dimethyl sulfoxide, the reaction temperature is 70~90℃, and the time is 5~7 h; in step (2), the dimethylamine is a dimethylamine aqueous solution with a mass fraction of 35~45%, the organic solvent is N,N-dimethylformamide, the reaction temperature is 90~110℃, and the time is 45~50 h; the catalyst in both steps (1) and (2) is potassium carbonate.

5. The method for preparing the nitroreductase-responsive fluorescent probe as described in claim 3, characterized in that, Step (3) includes the following steps: N,N-dimethylformamide is cooled at -2~2℃ for 10~30 min, phosphorus oxychloride is added dropwise, and the mixture is stirred at -2~2℃ for 25~35 min, then heated to 75~85℃ to obtain Vilsmeier reagent; the second intermediate is dissolved in N,N-dimethylformamide and then added dropwise to Vilsmeier reagent, and the mixture is stirred at 75~85℃ for 22~26 h to obtain the third intermediate.

6. The method for preparing the nitroreductase-responsive fluorescent probe as described in claim 3, characterized in that, In step (4), the organic solvent is N,N-dimethylformamide, the catalyst is potassium tert-butoxide, and the reaction time is 2-4 h; in step (5), the organic solvent is toluene, and the reaction time is 8-12 h.

7. The application of the nitroreductase-responsive fluorescent probe according to claim 1 in the preparation of a sperm nitroreductase detection kit.

8. The application of the nitroreductase-responsive fluorescent probe according to claim 1 in the preparation of a cell hypoxia detection kit.

9. A sperm nitroreductase detection kit, characterized in that, Includes the nitroreductase-responsive fluorescent probe as described in claim 1.

10. A cell hypoxia detection kit, characterized in that, Includes the nitroreductase-responsive fluorescent probe as described in claim 1.

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