Formaldehyde fluorescent probe and its preparation method and application

Abstract

The present invention belongs to the technical field of fluorescent probes, and specifically relates to a formaldehyde fluorescent probe based on formaldehyde-induced catalytic hydrolysis of aminocarboxylic acid hydroxylamine esters, a preparation method thereof, and applications thereof. The preparation of the formaldehyde fluorescent probe specifically comprises the following steps: S1: a naphthalimide derivative A is refluxed with triphosgene and 4-dimethylaminopyridine in a toluene solution to obtain an intermediate B; S2: the intermediate B is reacted with a hydroxylamine derivative C in a dichloromethane solution at room temperature to obtain a probe compound I; the molar ratio of the naphthalimide derivative A, triphosgene, and 4-dimethylaminopyridine in S1 is 1:1.5:3; the molar ratio of the intermediate B to the hydroxylamine derivative C in S2 is 1:1.3; the fluorescent probe of the present invention can selectively and rapidly react with formaldehyde to generate a product with strong fluorescence, and exhibits high selectivity and sensitivity to formaldehyde.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a formaldehyde fluorescent probe based on formaldehyde-induced catalytic hydrolysis of aminocarboxylic acid hydroxylamine ester, and a preparation method and application thereof. Background Art

[0002] Formaldehyde is the simplest reactive carbonyl compound and is widely used in industrial fields such as detergents, plastics, wood processing, and pharmaceutical synthesis. Formaldehyde is toxic. As early as 2004, the International Agency for Research on Cancer (IARC), an agency of the World Health Organization, designated formaldehyde as a carcinogen. Long-term exposure to formaldehyde can cause strong irritation to the human eyes, nose, skin, and respiratory tract, leading to chronic respiratory diseases, as well as nasopharyngeal cancer, colon cancer, brain tumors, diabetes, leukemia, Alzheimer's disease, and cardiovascular disease. Therefore, it is of great significance to develop corresponding fluorescent probes with high selectivity, high sensitivity, and fast response that can detect intracellular formaldehyde levels.

[0003] Traditional formaldehyde detection methods include high-performance liquid chromatography, spectrophotometry, electrochemical detection, gas chromatography, and mass spectrometry. These methods are costly, have low sensitivity, and require destructive pretreatment of biological samples, making them inconvenient for imaging and tracing formaldehyde in living cells and more complex biological samples, resulting in significant limitations. Small molecule fluorescent probes, however, have been increasingly used in various fields in recent years due to their advantages such as strong selectivity, high sensitivity, fast response time, and low cost. Therefore, the development of corresponding formaldehyde fluorescent probes with high selectivity, high sensitivity, fast response, and minimal interference with biological systems is of great significance. Summary of the Invention

[0004] In order to solve the above technical problems in this field, the present invention provides the following technical solutions.

[0005] The first aspect of the present invention discloses a formaldehyde fluorescent probe, characterized in that the fluorescent probe has a structure shown in the following formula I:

[0006]

[0007] Among them, R 1 R is independently selected from hydrogen atom, linear or branched alkyl group; 2 、R 3 、R 4 、R 5 、R 6 is independently selected from a hydrogen atom, a nitro group, and a linear or branched alkyl group.

[0008] Preferably, the formaldehyde fluorescent probe is as shown in the following formula I-1:

[0009]

[0010] The second aspect of the present invention discloses a method for preparing the formaldehyde fluorescent probe represented by the above formula I, which is achieved by the following preparation route:

[0011]

[0012] The preparation of the formaldehyde fluorescent probe represented by the above formula I specifically includes the following steps:

[0013] S1: Naphthalimide derivative A reacts with triphosgene and 4-dimethylaminopyridine in a toluene solution under reflux to obtain intermediate B;

[0014] S2: Intermediate B reacts with hydroxylamine derivative C in dichloromethane solution at room temperature to obtain probe compound I.

[0015] Preferably, the molar ratio of the naphthalimide derivative A, triphosgene and 4-dimethylaminopyridine in S1 is 1:1.5:3.

[0016] Preferably, the molar ratio of the intermediate B to the hydroxylamine derivative C in S2 is 1:1.3.

[0017] The third aspect of the present invention discloses the application of the above-mentioned formaldehyde fluorescent probe for detecting formaldehyde in cells.

[0018] The reaction principle of the detection is that formaldehyde reacts with aminocarboxylic acid hydroxylamine ester to form amino alcohol intermediate D, and then the hydroxyl group attacks the carbonyl group of the aminocarboxylic acid ester, releasing the 4-aminonaphthalimide fluorescent product, producing a fluorescence enhancement response, and simultaneously generating hydroxylamine carbonate lactone E, which is hydrolyzed to release formaldehyde, carbon dioxide and hydroxylamine F.

[0019]

[0020] The specific detection method includes the following steps:

[0021] (1) adding the formaldehyde fluorescent probe of the present invention into the cells to be tested,

[0022] (2) Using a flow cytometer, the changes in fluorescence intensity in the cells before and after the addition of the formaldehyde fluorescent probe of the present invention are read.

[0023] Preferably, the fluorescent probe can be used in living cells; the living cells are human fibrosarcoma cells.

[0024] Preferably, the fluorescent probe can be used for cells in a state of ferroptosis; and monitor changes in formaldehyde concentration in cells in a state of ferroptosis.

[0025] The present invention achieves the following beneficial technical effects:

[0026] (1) High sensitivity

[0027] The fluorescent probe of the present invention can selectively and rapidly react with formaldehyde to produce a product with strong fluorescence. Compared with other common aldehydes, amino acids and metal ions, the formaldehyde fluorescent probe of the present invention shows higher selectivity and sensitivity for formaldehyde.

[0028] (2) Strong anti-interference ability

[0029] The formaldehyde fluorescent probe of the present invention can resist the interference of formaldehyde, 2-ethylacrolein, 3-methyl-2-butenal, methylglyoxal, acetaldehyde, isovaleraldehyde, n-butyraldehyde, propionaldehyde, benzaldehyde, glyoxal, aluminum ions, calcium ions, cadmium ions, cobalt ions, ferrous ions, ferric ions, magnesium ions, zinc ions, nickel ions, alanine, arginine, serine, cysteine, glutathione, hydrogen peroxide, and glucose.

[0030] (3) Can be used under physiological conditions

[0031] The formaldehyde fluorescent probe of the present invention can be used under physiological conditions, and is less interfered with by common aldehydes and other substances in organisms. It can be used to detect intracellular formaldehyde levels and has potential practical application value.

[0032] (4) Significant preparation advantages

[0033] The preparation method of the fluorescent probe of the present invention is simple and has a high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the fluorescent probe I-1 of Example 1 1 HNMR spectrum.

[0035] Figure 2 is the fluorescent probe I-1 of Example 1 13 C NMR spectrum.

[0036] Figure 3 The mass spectrum of fluorescent probe Ⅰ-1 in Example 1 is

[0037] Figure 4 The figure shows the changes in the fluorescence spectrum of the fluorescent probe I-1 of Example 1 with a concentration of 1 μmol / L in PBS (10 mM, pH = 7.4) as different amounts of formaldehyde are added. In the figure, from bottom to top, the fluorescence spectra are the formaldehyde concentrations of 0, 5, 10, 20, 30, 40, 50, 100, and 200 μmol / L, respectively; the reaction time is 120 minutes.

[0038] Figure 5 This is a graph showing the change in fluorescence intensity at 550 nm over time of the fluorescent probe Ⅰ-1 of Example 1 at a concentration of 1 μmol / L and 50 μmol / L formaldehyde in PBS (10 mM, pH=7.4) within 120 minutes.

[0039] Figure 6 This is a columnar fluorescence data diagram of the selectivity of the fluorescent probe I-1 of Example 1 at a concentration of 1 μmol / L against 50 μmol / L of different interfering analytes (wherein the concentration of glutathione is 200 μmol / L) in PBS (10 mM, pH = 7.4). From left to right in the figure are: 1. blank; 2. formaldehyde; 3. 2-ethylacrolein; 4. 3-methyl-2-butenal; 5. methylglyoxal; 6. acetaldehyde; 7. isovaleraldehyde; 8. n-butyraldehyde; 9. propionaldehyde; 10. benzaldehyde; 11. acetaldehyde Dialdehyde; 12. aluminum ion; 13. calcium ion; 14. cadmium ion; 15. cobalt ion; 16. ferrous ion; 17. ferric ion; 18. magnesium ion; 19. zinc ion; 20. nickel ion; 21. alanine; 22. arginine; 23. serine; 24. cysteine; 25. glutathione; 26. hydrogen peroxide; 27. glucose; Since the reaction of the probe Ⅰ-1 of the present invention with the analyte formaldehyde for 60 minutes is sufficient to achieve the fluorescence intensity required for the fluorescence test, 60 minutes is selected as the reaction time.

[0040] Figure 7 This is a graph showing changes in cell activity after incubation of fluorescent probe Ⅰ-1 in mouse macrophages (RAW264.7) at different probe concentrations for 5 hours.

[0041] Figure 8 This is a graph showing the changes in intracellular formaldehyde concentration at different incubation times using fluorescent probe Ⅰ-1 in human fibrosarcoma cells (HT-1080) using flow cytometry to monitor the ferroptosis inducer Erastin. DETAILED DESCRIPTION

[0042] The present invention is further illustrated by way of examples below, but the invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications. The raw materials and equipment used in the examples are well known to those skilled in the art and are all commercially available, easily obtained, or prepared.

[0043] Example 1 Synthesis of fluorescent probe I-1:

[0044]

[0045] 25.4 mg of the starting naphthalimide derivative A1 (0.1 mmol) and 4-dimethylaminopyridine (36.7 mg, 0.3 mmol) were dissolved in 3 mL of toluene. The mixture was then cooled to 0°C and a toluene solution of triphosgene (45.5 mg, 0.15 mmol) was slowly added dropwise. The mixture was refluxed for 6 hours to yield intermediate B1. A dichloromethane solution of hydroxylamine derivative C1 (20 mg, 0.13 mmol) was then added to the reaction system. The reaction was continued at room temperature for 12 hours until the reaction was complete. The toluene and dichloromethane in the reaction solution were removed by vacuum distillation on a rotary evaporator to obtain the crude product. The product was purified by silica gel chromatography using a 100:1 volume ratio of dichloromethane to methanol to afford 15 mg of a yellow solid (34.6% yield). 1 H NMR(600MHz,DMSO-d6)δ11.40(s,1H),10.15(s,1H),8.60(t,J=1.8Hz,1H),8 .55(d,J=6.6Hz,1H),8.52(t,J=7.8Hz,2H),8.14(dd,J=8.4,1.2Hz,1H),8.0 6(d,J=7.8Hz,1H),7.96(dd,J=8.4,1.8Hz,1H),7.93–7.89(t,J=7.8Hz,1H), 7.68(t,J=7.8Hz,1H),4.06–3.99(m,2H),1.67(m,2H),0.94(t,J=7.2Hz,3H); 13 C NMR (151MHz, DMSO) δ163.5,163.0,153.9,147.8,143.4,140.2,131.2,131.0,129.9,128.3,126. 5,126.1,124.4,122.3,122.1,118.5,117.7,112.0,106.2,41.2,20.9,11.4; ESI-HRMS:m / z[m+Na + ]calcdfor C 22 H 18 N4NaO6 + :457.1119,found:457.1122.

[0046] Example 2 Fluorescence spectrum changes of fluorescent probe I-1 in response to different concentrations of formaldehyde

[0047] Take the fluorescent probe Ⅰ-1 prepared in Example 1 and dissolve it in DMSO to prepare a fluorescent probe mother solution with a concentration of 1 mmol / L; add a formaldehyde solution with a mass fraction of 37% into distilled water to prepare a formaldehyde mother solution with a concentration of 100 mmol / L. According to the concentrations of the fluorescent probe and formaldehyde, calculate the required PBS aqueous solution (10 mM, pH = 7.4) and add it to a 1 cm × 1 cm quartz cuvette (volume 3.5 mL), take 3 μL of the fluorescent probe mother solution and add it to the PBS aqueous solution, and then add formaldehyde mother solutions of different concentrations (0-200 μmol / L) to prepare a test solution with a probe concentration of 1 μmol / L, totaling 3 mL. After 120 minutes of reaction, the fluorescence spectrum changes of the fluorescent probe Ⅰ-1 reacting with different concentrations of formaldehyde were tested using a fluorescence spectrometer (excitation wavelength was 425 nm). The fluorescence spectrum changes are as follows Figure 4 As shown in Figure 2, it can be seen that with the gradual increase of formaldehyde concentration, the fluorescence peak of the probe solution at 550 nm gradually increases.

[0048] Example 3 Fluorescence spectrum changes of fluorescent probe I-1 in reaction with formaldehyde over time

[0049] Calculate the required PBS aqueous solution (10mM, pH=7.4) based on the concentration of the fluorescent probe and formaldehyde and add it to a 1cm×1cm quartz cuvette (volume 3.5mL). Take 3μL of the fluorescent probe stock solution in Example 2 and add it to the PBS aqueous solution. Then add 1.5μL of the formaldehyde stock solution prepared in Example 2 to prepare a test solution with a probe concentration of 1μmol / L and a formaldehyde concentration of 50μmol / L, totaling 3mL. Use 425nm as the excitation wavelength to test the changes in the fluorescence spectrum over time. Figure 5 As shown, as time increases, the fluorescence peak at 550 nm gradually increases and reaches a maximum value around 120 minutes.

[0050] Example 4 Study on the Selectivity of Fluorescent Probe I-1 for Different Interfering Analytes

[0051] The required PBS aqueous solution (10 mM, pH = 7.4) was calculated according to the concentration of the fluorescent probe and different interfering analytes and added to a 1 cm × 1 cm quartz cuvette (volume 3.5 mL). 3 μL of the fluorescent probe mother solution in Example 2 was added to the PBS aqueous solution, and then 50 μmol / L of different analytes (wherein the glutathione concentration was 200 μmol / L) were added: formaldehyde, 2-ethylacrolein, 3-methyl-2-butenal, methylglyoxal, acetaldehyde, isovaleraldehyde, n-butyraldehyde, propionaldehyde, benzaldehyde, glyoxal, aluminum ion, calcium ion, cadmium ion, cobalt ion, ferrous ion, ferric ion, magnesium ion, zinc ion, nickel ion, alanine, arginine, serine, cysteine, glutathione, hydrogen peroxide, and glucose to prepare a test solution with a probe concentration of 1 μmol / L and a different interfering analyte concentration of 50 μmol / L, totaling 3 mL. At the same time, a blank test sample with only the probe added was retained. Since the fluorescence test brightness requirement can be met when the reaction time is 60 minutes, 60 minutes is selected as the test time of the fluorescent probe I-1. After 60 minutes of reaction, the fluorescence emission intensity of different samples at 550nm (excitation wavelength is 425nm) is tested using a fluorescence spectrometer. Figure 6 As shown in the figure, the fluorescence of the formaldehyde test solution increased significantly compared to the blank test solution, while the fluorescence of other analytes did not increase much. The experimental results show that the fluorescent probe I-1 has good selectivity for formaldehyde.

[0052] Example 5 Changes in cell activity after incubation of fluorescent probe I-1 in mouse macrophages (RAW264.7) at different probe concentrations for 5 hours.

[0053] The fluorescent probe I-1 prepared in Example 1 was dissolved in DMSO to prepare a fluorescent probe stock solution with a concentration of 100 mmol / L. The first group of cells was incubated for 5 hours without adding any test substance. The second group of cells was added with the fluorescent probe stock solution to a probe concentration of 1 μmol / L and incubated for 5 hours. The third group of cells was added with the fluorescent probe stock solution to a probe concentration of 5 μmol / L and incubated for 5 hours. The fourth group of cells was added with the fluorescent probe stock solution to a probe concentration of 10 μmol / L and incubated for 5 hours. The fifth group of cells was added with the fluorescent probe stock solution to a probe concentration of 20 μmol / L and incubated for 5 hours. The sixth group of cells was added with the fluorescent probe stock solution to a probe concentration of 50 μmol / L and incubated for 5 hours. The seventh group of cells was added with the fluorescent probe stock solution to a probe concentration of 100 μmol / L and incubated for 5 hours. Then, 10 μL of CCK8 solution was added and the cells were incubated for 1 hour. The fluorescence intensity of the four groups of cells was then analyzed using a flow cytometer. like Figure 7As shown in the figure, with the increase of probe concentration, the cell survival rate gradually decreased. The experimental results show that fluorescent probe Ⅰ-1 has a good cell survival rate within the concentration range of 20μM.

[0054] Example 6 Fluorescent probe I-1 was used to monitor changes in intracellular formaldehyde concentrations in human fibrosarcoma cells (HT-1080) at different incubation times using flow cytometry with the ferroptosis inducer Erastin.

[0055] The fluorescent probe I-1 prepared in Example 1 was dissolved in DMSO to prepare a fluorescent probe stock solution with a concentration of 10 mmol / L. The ferroptosis inducer erastin was added to 1 mL of DMSO to prepare an erastin stock solution with a concentration of 10 mmol / L. The ferroptosis inhibitor Fer-1 was added to 1 mL of DMSO to prepare a Fer-1 stock solution with a concentration of 10 mmol / L. 2 μL of the fluorescent probe stock solution was added to the first group of cells at a probe concentration of 10 μmol / L and incubated for 30 minutes. 2 μL of the erastin stock solution was added to the second group of cells at an erastin concentration of 10 μmol / L. After incubation for 4 hours, the cells were washed with HBSS and then 2 μL of the fluorescent probe stock solution was added to the cells at a probe concentration of 10 μmol / L. The cells were incubated for 30 minutes. In the third group, 2 μL of erastin stock solution and 2 μL of Fer-1 stock solution were added to the cells, with erastin concentrations of 10 μmol / L and Fer-1 concentrations of 10 μmol / L. After incubation for 4 hours, the cells were washed with HBSS, and 2 μL of the fluorescent probe stock solution was added to the cells, with the probe concentration of 10 μmol / L, for incubation for 30 minutes. In the fourth group, 2 μL of erastin stock solution was added to the cells, with erastin concentrations of 10 μmol / L. After incubation for 8 hours, the cells were washed with HBSS, and 2 μL of the fluorescent probe stock solution was added to the cells, with the probe concentration of 10 μmol / L, for incubation for 30 minutes. In the fifth group, 2 μL of erastin stock solution and 2 μL of Fer-1 stock solution were added to the cells, with erastin concentrations of 10 μmol / L and Fer-1 concentrations of 10 μmol / L. After incubation for 8 hours, the cells were washed with HBSS, and 2 μL of the fluorescent probe stock solution was added to the cells, with the probe concentration of 10 μmol / L, for incubation for 30 minutes. After the cells were incubated, samples were prepared and the fluorescence intensity of the four groups of cells was analyzed using flow cytometry. The excitation wavelength used was 425 nm and the emission wavelength was 550 nm. Figure 8 As shown, after 4 hours of incubation with erastin, the intracellular formaldehyde concentration decreased compared to the blank control group. The fluorescence intensity gradually increased with the increase in erastin incubation time, while the fluorescence intensity decreased after the addition of Fer-1. These results demonstrate that fluorescent probe I-1 can be used to detect intracellular formaldehyde concentrations in HT-1080 cells undergoing ferroptosis.

[0056] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A formaldehyde fluorescent probe, characterized in that The formaldehyde fluorescent probe is shown in the following formula I-1: 。 2. A method for preparing a formaldehyde fluorescent probe as shown in formula I-1 as claimed in claim 1, characterized in that: This is achieved through the following preparation route: 。 3. The method for preparing a formaldehyde fluorescent probe according to claim 2, wherein: The following steps are involved: 0.1 mmol of the raw material naphthalene imide derivative A1 and 0.3 mmol of 4-dimethylaminopyridine were dissolved in toluene, then cooled to 0°C, and a toluene solution containing 0.15 mmol of triphosgene was slowly added dropwise. The mixture was refluxed for 6 hours to obtain intermediate B1. A dichloromethane solution containing 0.13 mmol of hydroxylamine derivative C1 was further added to the reaction system, and the reaction was continued at room temperature for 12 hours. After the reaction was completed, the toluene and dichloromethane in the reaction solution were removed by reduced pressure distillation using a rotary evaporator to obtain a crude product. The product was purified by silica gel chromatography using dichloromethane and methanol in a volume ratio of 100:1 as eluent to obtain the formaldehyde fluorescent probe shown in Formula I-1.

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