Water-soluble coumarin Fe 3+ Fluorescent probes, methods of making and use in imaging zebrafish

Abstract

The application discloses water-soluble coumarin Fe 3+ The application discloses a fluorescent probe, a preparation method thereof and application of the fluorescent probe in zebrafish imaging, and a structural formula of the fluorescent probe is shown in the following formula. 3+ The fluorescent probe has the advantages of simple synthesis, novel structure, good water solubility, good selectivity, sensitivity, low detection limit and the like, and has the capability of observing Fe 3+ in a zebrafish body.

Description

Technical Field

[0001] This invention relates to the field of fluorescent molecular probes. More specifically, this invention relates to a water-soluble coumarin Fe... 3+ Fluorescent probes, their preparation methods, and their applications in zebrafish imaging. Background Technology

[0002] Fe 3+ It is one of the essential trace elements for the human body, usually in the form of Fe. 3+ or Fe 2+ It exists in the form of living organisms or in the natural environment, in which Fe 3+ Fe plays an irreplaceable and vital role in human life systems, widely participating in various life processes and constituting many enzymes and proteins within living organisms. These include the synthesis of RNA and DNA within cells, cellular and oxygen metabolism, enzyme catalysis, and proton transfer. Both excess and deficiency of Fe can cause serious functional disorders in the human body. Furthermore, research has found that Fe... 3+ One reason for the abnormal content is that there may be excessive metal ions in the environmental water. Water is the basic guarantee for maintaining normal human life activities, and metal pollution entering the water cycle will directly cause great harm to the human body.

[0003] Scientists have now developed various methods for using Fe 3+ Analytical methods for ion detection include flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption spectrometry (GFAAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS), and colorimetry. Although all these methods can effectively and sensitively quantify Fe in samples... 3+ However, these methods typically suffer from drawbacks such as expensive equipment, time-consuming processes, complex operation, and unsuitability for real-time and field detection. In contrast, fluorescent probes offer advantages such as high selectivity, high sensitivity, low equipment dependence, simple operation, and low cost. Moreover, they can be combined with fluorescence microscopy imaging techniques to achieve non-destructive in-situ detection at the in vivo level, making fluorescent probes one of the most promising methods. Summary of the Invention

[0004] This invention provides water-soluble coumarin Fe 3+ This fluorescent probe is simple to synthesize, has a novel structure, and good water solubility, and is applied to the detection of Fe. 3+ Ions have advantages such as good selectivity, sensitivity, and low detection limit.

[0005] To achieve these objectives and other advantages of the present invention, a water-soluble coumarin Fe is provided. 3+ The fluorescent probe has the following structural formula:

[0006]

[0007] Where R is

[0008] This invention also provides water-soluble coumarin Fe 3+ The method for preparing the fluorescent probe, and the preparation of the above-mentioned water-soluble coumarin Fe 3+ The fluorescent probe involves the following steps:

[0009] Step 1: Using p-tolueneacetic acid and 2,4-dihydroxybenzaldehyde as starting materials, dissolve them in acetic anhydride, add triethylamine as a catalyst to react and obtain acetylated coumarin nuclei; add the acetylated coumarin nuclei to methanol, slowly add concentrated hydrochloric acid, and remove the acetyl group in the acidic environment to obtain benzopyranone coumarin intermediate (1).

[0010] Step 2: Using benzopyranone coumarin intermediate (1), bromopropyne and potassium carbonate as raw materials, and acetonitrile as solvent, an alkynyl-containing intermediate (2) is obtained by nucleophilic substitution.

[0011] Step 3: After dissolving the azide amino acid in tert-butanol, add the alkynyl-containing intermediate (2), then add copper sulfate pentahydrate, sodium ascorbate, and pure water. Use click chemistry to synthesize water-soluble coumarin Fe. 3+ Fluorescent probe.

[0012] Preferably, step one is as follows: p-tolueneacetic acid and 2,4-dihydroxybenzaldehyde are dissolved in acetic anhydride as a solvent, and triethylamine is added as a catalyst for reaction. The reaction temperature is 130-150℃, and the reaction is carried out for 6-8 hours. After the reaction is completed, the mixture is cooled and recrystallized to obtain acetylated coumarin nuclei. The acetylated coumarin nuclei are added to methanol, and concentrated hydrochloric acid is slowly added dropwise. The temperature is raised to 60-65℃ and the reaction is carried out for 3-4 hours. After the reaction is completed, a white transparent solid is filtered to obtain benzopyranone coumarin intermediate (1).

[0013] Preferably, the reaction temperature in step two is 55-60℃, and the reaction time is 3-4 hours. After the reaction is completed, the mixture is filtered, concentrated, dissolved in ethyl acetate, washed with saturated ammonium chloride and saturated brine in sequence, and the organic layer is dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain the alkynyl-containing intermediate (2).

[0014] Preferably, the azide amino acid in step three is azide-leucine, azide-valine, azide-aminobutyric acid, or azide-threonine, corresponding to the synthesized water-soluble coumarin Fe. 3+ The fluorescent probes have the following structural formulas:

[0015]

[0016] Preferably, the azide amino acid is prepared by the following steps: sodium azide is dissolved in water and DCM is added as a solvent. Trifluoromethanesulfonic anhydride is added dropwise at 0°C. After 3-4 hours, potassium carbonate, amino acid, and anhydrous copper sulfate are added and reacted for 3-4 hours. The organic solvent is removed, the pH is adjusted to 2, and the mixture is extracted with ethyl acetate. The organic layer is dried with anhydrous sodium sulfate and then evaporated to dryness to obtain the azide amino acid. The molar volume ratio of sodium azide:DCM:trifluoromethanesulfonic anhydride:potassium carbonate:amino acid:anhydrous copper sulfate is 9.2 mmol:15 mL:18.4 mmol:7.2 mmol:9.2 mmol:0.92 mmol.

[0017] Wherein, the amino acid is L-leucine, L-valine, γ-aminobutyric acid or L-threonine, and the corresponding azide amino acids are azide-leucine, azide-valine, azide-aminobutyric acid or azide-threonine, respectively.

[0018] Preferably, the click chemistry method in step three is as follows: Azide amino acids are dissolved in tert-butanol, then an alkynyl-containing intermediate (2), copper sulfate pentahydrate, sodium ascorbate, and pure water are added. The mixture is heated to 55-60℃ and reacted for 4-5 hours. After the reaction is completed under TLC monitoring, saturated brine is added for washing, followed by extraction with ethyl acetate, drying with anhydrous sodium sulfate, and purification by silica gel column chromatography after vacuum concentration to obtain water-soluble coumarin Fe. 3+ Fluorescent probe; wherein the column chromatography silica gel column purification uses a gradient elution of dichloromethanol = 40:1; 20:1; 10:1.

[0019] Preferably, in step one, the molar ratio of p-tolueneacetic acid: 2,4-dihydroxybenzaldehyde: triethylamine is (1.5-2): 1: (1.5-2); the volume molar ratio of acetic anhydride: methanol: concentrated hydrochloric acid: 2,4-dihydroxybenzaldehyde is 2.5-3 mL: 2-2.5 mL: 90-100 μL: 1 mmol.

[0020] In step two, the molar volume ratio of benzopyranone coumarin intermediate (1): potassium carbonate: bromopropyne: acetonitrile is 1-1.2 mmol: 4-4.8 mmol: 2.4 mmol: 15 mL;

[0021] In step three, the molar ratio of azide amino acid: alkyne-containing intermediate: copper sulfate pentahydrate: sodium ascorbate is 1:1:0.4:0.85, and the molar volume ratio of alkyne-containing intermediate (2) to pure water is 1 mmol:2 mL.

[0022] This invention also provides water-soluble coumarin Fe 3+ Application of fluorescent probes in zebrafish imaging.

[0023] The present invention has at least the following beneficial effects:

[0024] 1. This invention allows for the preparation of water-soluble coumarin Fe through simple steps. 3+ A fluorescent probe that reacts with Fe 3+ Upon ion contact, the fluorescence intensity rapidly decreased, exhibiting a "turn-off" mode, thus achieving the desired fluorescence intensity for Fe. 3+ Fluorescent recognition of ions provides high detection sensitivity.

[0025] 2. The fluorescent probe of this invention is effective against Fe. 3+ The ions exhibit good selectivity compared to other common cations, especially Fe. 2+

[0026] The fluorescence signal remained essentially unchanged after ion interaction, exhibiting high anti-interference ability, short reaction time, and low detection limit; it also allows for the observation of Fe in zebrafish. 3+ The ability to detect Fe in living organisms 3+ Its potential.

[0027] 3. The fluorescent probe of this invention has high water solubility, with a water content of 40%-75%, and high biocompatibility.

[0028] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0029] Figure 1 The water-soluble coumarin Fe in Example 5 of this invention 3+ Fluorescent probes (3A-3D) for Fe 3+ Water percentage diagram for ion selective recognition

[0030] Figure 2 The water-soluble coumarin Fe in Example 6 of this invention 3+ Fluorescent probes (3A-3D) for Fe 3+ Fluorescence emission spectrum of ion-selective recognition;

[0031] Figure 3 The water-soluble coumarin Fe in Example 7 of this invention 3+ Fluorescent probe (3B) in Fe 3+ Graph showing the changes in fluorescence emission intensity in the presence of ions and other cations;

[0032] Figure 4 The water-soluble coumarin Fe in Examples 8 and 9 of this invention 3+ Fluorescent probe (3B) at different concentrations of Fe 3+ Fluorescence emission spectrum changes and detection limit calculation in the presence of ions;

[0033] Figure 5The water-soluble coumarin Fe in Example 10 of this invention 3+ Graph showing the change in fluorescence emission intensity of fluorescent probe needle (3B) at different pH values;

[0034] Figure 6 The water-soluble coumarin Fe in Example 11 of this invention 3+ Fluorescence emission intensity variation of fluorescent probe (3B) in the presence of chelating agent EDTA;

[0035] Figure 7 The water-soluble coumarin Fe in Example 12 of this invention 3+ Imaging of the fluorescent probe (3B) in zebrafish;

[0036] Figure 8 MS, 1H-NMR and 13C-NMR spectra of the benzopyranone coumarin intermediate (1) obtained in Examples 1-4 of this invention;

[0037] Figure 9 MS, 1H-NMR and 13C-NMR spectra of the alkyne-containing intermediate (2) obtained in Examples 1-4 of the present invention;

[0038] Figure 10 The water-soluble coumarin Fe obtained in Example 1 of this invention 3+ MS spectrum, 1H-NMR spectrum, and 13C-NMR spectrum of fluorescent probe (3A);

[0039] Figure 11 The water-soluble coumarin Fe obtained in Example 2 of this invention 3+ MS, 1H-NMR, and 13C-NMR spectra of the fluorescent probe (3B);

[0040] Figure 12 The water-soluble coumarin Fe obtained in Example 3 of this invention 3+ MS spectrum, 1H-NMR spectrum, and 13C-NMR spectrum of the fluorescent probe (3C);

[0041] Figure 13 The water-soluble coumarin Fe obtained in Example 4 of this invention 3+ MS, 1H-NMR, and 13C-NMR spectra of the fluorescent probe (3D). Detailed Implementation

[0042] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0043] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0044] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0045] This invention provides water-soluble coumarin Fe 3+ The fluorescent probe has the following structural formula:

[0046]

[0047] Where R is

[0048] Water-soluble coumarin Fe 3+ The synthesis route of the fluorescent probe is as follows:

[0049]

[0050] Example 1

[0051] This invention prepares water-soluble coumarin Fe with structural formula 3A. 3+ The preparation method of the fluorescent probe is as follows:

[0052] Step 1: Accurately weigh 0.60 g (4.3 mmol) of 2,4-dihydroxybenzaldehyde and 0.98 g (6.5 mmol) of p-tolueneacetic acid. Use 12 mL of acetic anhydride as solvent, heat to 100 °C, and after the mixture dissolves, add 906 μL (6.5 mmol) of triethylamine. Heat to 150 °C and react for 6 h. After the reaction is completed by TLC, cool at room temperature. The acetylated coumarin core recrystallizes and precipitates. Filter to obtain pale yellow crystals. Add the above product to 10 mL of methanol, slowly add 90 μL of concentrated hydrochloric acid (the concentration of concentrated hydrochloric acid is 12 mol / L according to national standards), heat to 65 °C and react for 4 h. The pale yellow crystals gradually turn into a white transparent solid. After the reaction is completed, filter to obtain benzopyranone coumarin intermediate (1).

[0053] Step 2: Add the above-mentioned benzopyranone coumarin intermediate (1) (0.3 g, 1.2 mmol) and potassium carbonate (0.66 g, 4.8 mmol) to 15 mL of acetonitrile, add bromopropyne (206 μL, 2.4 mmol) dropwise, heat to 60 °C, and react for 4 h. After the reaction is complete, filter, concentrate, dissolve in ethyl acetate, wash with saturated ammonium chloride and saturated brine in sequence, retain the organic layer, dry with anhydrous sodium sulfate, concentrate under reduced pressure to obtain the alkynyl-containing intermediate (2);

[0054] Step 3: Dissolve sodium azide (0.6 g, 9.2 mmol) in a small amount of water (1.5 mL) and add DCM (15 mL) as solvent. Add trifluoromethanesulfonic anhydride (3 mL, 18.4 mmol) dropwise at 0 °C. After 4 h, add potassium carbonate (1 g, 7.2 mmol), L-leucine (9.2 mmol), and anhydrous copper sulfate (0.92 mmol) and react for 4 h. Remove the organic solvent and adjust the pH to 2. Extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, and evaporate to dryness to obtain azide-leucine. 1 mmol of azidoleucine was dissolved in tert-butanol, and then an alkynyl-containing intermediate (2) (0.3 g, 1 mmol), copper sulfate pentahydrate (0.112 g, 0.4 mmol), sodium ascorbate (0.168 g, 0.85 mmol), and 2 mL of pure water were added. The mixture was heated to 60 °C and reacted for 5 h. After the reaction was monitored by TLC, the mixture was washed directly with saturated brine, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethanol = 40:1; 20:1; 10:1 gradient elution) to obtain water-soluble coumarin Fe with structural formula 3A. 3+ Fluorescent probe.

[0055] Example 2

[0056] This invention prepares water-soluble coumarin Fe with structural formula 3B. 3+ The preparation method of the fluorescent probe is as follows:

[0057] Step 1: Accurately weigh 0.60 g (4.3 mmol) of 2,4-dihydroxybenzaldehyde and 0.98 g (6.5 mmol) of p-tolueneacetic acid. Use 12 mL of acetic anhydride as solvent, heat to 100 °C, and after the mixture dissolves, add 906 μL (6.5 mmol) of triethylamine. Heat to 150 °C and react for 6 h. After the reaction is completed by TLC, cool at room temperature. The acetylated coumarin core recrystallizes and precipitates. Filter to obtain pale yellow crystals. Add the above product to 10 mL of methanol, slowly add 90 μL of concentrated hydrochloric acid (the concentration of concentrated hydrochloric acid is 12 mol / L according to national standards), heat to 65 °C and react for 4 h. The pale yellow crystals gradually turn into a white transparent solid. After the reaction is completed, filter to obtain benzopyranone coumarin intermediate (1).

[0058] Step 2: Add the above-mentioned benzopyranone coumarin intermediate (1) (0.3 g, 1.2 mmol) and potassium carbonate (0.66 g, 4.8 mmol) to 15 mL of acetonitrile, add bromopropyne (206 μL, 2.4 mmol) dropwise, heat to 60 °C, and react for 4 h. After the reaction is complete, filter, concentrate, dissolve in ethyl acetate, wash with saturated ammonium chloride and saturated brine in sequence, retain the organic layer, dry with anhydrous sodium sulfate, concentrate under reduced pressure to obtain the alkynyl-containing intermediate (2);

[0059] Step 3: Dissolve sodium azide (0.6 g, 9.2 mmol) in a small amount of water (1.5 mL) and add DCM (15 mL) as solvent. Add trifluoromethanesulfonic anhydride (3 mL, 18.4 mmol) dropwise at 0 °C. After 4 h, add potassium carbonate (1 g, 7.2 mmol), L-valine (9.2 mmol), and anhydrous copper sulfate (0.92 mmol) and react for 4 h. Remove the organic solvent and adjust the pH to 2. Extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, and evaporate to dryness to obtain azide-valine amino acid. 1 mmol of valine azide was dissolved in tert-butanol, and then an alkynyl-containing intermediate (2) (0.3 g, 1 mmol), copper sulfate pentahydrate (0.112 g, 0.4 mmol), sodium ascorbate (0.168 g, 0.85 mmol), and 2 mL of pure water were added. The mixture was heated to 60 °C and reacted for 5 h. After the reaction was monitored by TLC, the mixture was washed directly with saturated brine, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethanol = 40:1; 20:1; 10:1 gradient elution) to obtain water-soluble coumarin Fe with structural formula 3B. 3+ Fluorescent probe.

[0060] Example 3

[0061] This invention prepares water-soluble coumarin Fe with the structural formula 3C. 3+ The preparation method of the fluorescent probe is as follows:

[0062] Step 1: Accurately weigh 0.60 g (4.3 mmol) of 2,4-dihydroxybenzaldehyde and 0.98 g (6.5 mmol) of p-tolueneacetic acid. Use 12 mL of acetic anhydride as solvent, heat to 100 °C, and after the mixture dissolves, add 906 μL (6.5 mmol) of triethylamine. Heat to 150 °C and react for 6 h. After the reaction is completed by TLC, cool at room temperature. The acetylated coumarin core recrystallizes and precipitates. Filter to obtain pale yellow crystals. Add the above product to 10 mL of methanol, slowly add 90 μL of concentrated hydrochloric acid (the concentration of concentrated hydrochloric acid is 12 mol / L according to national standards), heat to 65 °C and react for 4 h. The pale yellow crystals gradually turn into a white transparent solid. After the reaction is completed, filter to obtain benzopyranone coumarin intermediate (1).

[0063] Step 2: Add the above-mentioned benzopyranone coumarin intermediate (1) (0.3 g, 1.2 mmol) and potassium carbonate (0.66 g, 4.8 mmol) to 15 mL of acetonitrile, add bromopropyne (206 μL, 2.4 mmol) dropwise, heat to 60 °C, and react for 4 h. After the reaction is complete, filter, concentrate, dissolve in ethyl acetate, wash with saturated ammonium chloride and saturated brine in sequence, retain the organic layer, dry with anhydrous sodium sulfate, concentrate under reduced pressure to obtain the alkynyl-containing intermediate (2);

[0064] Step 3: Dissolve sodium azide (0.6 g, 9.2 mmol) in a small amount of water (1.5 mL) and add DCM (15 mL) as solvent. Add trifluoromethanesulfonic anhydride (3 mL, 18.4 mmol) dropwise at 0 °C. After 4 h, add potassium carbonate (1 g, 7.2 mmol), γ-aminobutyric acid (9.2 mmol), and anhydrous copper sulfate (0.92 mmol). React for 4 h. Remove the organic solvent and adjust the pH to 2. Extract with ethyl acetate. Dry the organic layer with anhydrous sodium sulfate and evaporate to dryness to obtain azidoaminobutyric acid. 1 mmol of azidoaminobutyric acid was dissolved in tert-butanol, and then an alkynyl-containing intermediate (2) (0.3 g, 1 mmol), copper sulfate pentahydrate (0.112 g, 0.4 mmol), sodium ascorbate (0.168 g, 0.85 mmol), and 2 mL of pure water were added. The mixture was heated to 60 °C and reacted for 5 h. After the reaction was monitored by TLC, the mixture was washed directly with saturated brine, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethanol = 40:1; 20:1; 10:1 gradient elution) to obtain water-soluble coumarin Fe with the structural formula 3C. 3+ Fluorescent probe.

[0065] Example 4

[0066] This invention prepares water-soluble coumarin Fe with a 3D structural formula. 3+ The preparation method of the fluorescent probe is as follows:

[0067] Step 1: Accurately weigh 0.60 g (4.3 mmol) of 2,4-dihydroxybenzaldehyde and 0.98 g (6.5 mmol) of p-tolueneacetic acid. Use 12 mL of acetic anhydride as solvent, heat to 100 °C, and after the mixture dissolves, add 906 μL (6.5 mmol) of triethylamine. Heat to 150 °C and react for 6 h. After the reaction is completed by TLC, cool at room temperature. The acetylated coumarin core recrystallizes and precipitates. Filter to obtain pale yellow crystals. Add the above product to 10 mL of methanol, slowly add 90 μL of concentrated hydrochloric acid (the concentration of concentrated hydrochloric acid is 12 mol / L according to national standards), heat to 65 °C and react for 4 h. The pale yellow crystals gradually turn into a white transparent solid. After the reaction is completed, filter to obtain benzopyranone coumarin intermediate (1).

[0068] Step 2: Add the above-mentioned benzopyranone coumarin intermediate (1) (0.3 g, 1.2 mmol) and potassium carbonate (0.66 g, 4.8 mmol) to 15 mL of acetonitrile, add bromopropyne (206 μL, 2.4 mmol) dropwise, heat to 60 °C, and react for 4 h. After the reaction is complete, filter, concentrate, dissolve in ethyl acetate, wash with saturated ammonium chloride and saturated brine in sequence, retain the organic layer, dry with anhydrous sodium sulfate, concentrate under reduced pressure to obtain the alkynyl-containing intermediate (2);

[0069] Step 3: Dissolve sodium azide (0.6 g, 9.2 mmol) in a small amount of water (1.5 mL) and add DCM (15 mL) as solvent. Add trifluoromethanesulfonic anhydride (3 mL, 18.4 mmol) dropwise at 0 °C. After 4 h, add potassium carbonate (1 g, 7.2 mmol), L-threonine (9.2 mmol), and anhydrous copper sulfate (0.92 mmol) and react for 4 h. Remove the organic solvent and adjust the pH to 2. Extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, and evaporate to dryness to obtain azide-threonine. 1 mmol of azidothreoamino acid was dissolved in tert-butanol, and then an alkynyl-containing intermediate (2) (0.3 g, 1 mmol), copper sulfate pentahydrate (0.112 g, 0.4 mmol), sodium ascorbate (0.168 g, 0.85 mmol), and 2 mL of pure water were added. The mixture was heated to 60 °C and reacted for 5 h. After the reaction was monitored by TLC, the mixture was washed directly with saturated brine, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethanol = 40:1; 20:1; 10:1 gradient elution) to obtain water-soluble coumarin Fe with a 3D structural formula. 3+ Fluorescent probe.

[0070] See Figure 8-13 MS, 1H-NMR, and 13C-NMR spectra of each compound.

[0071] Among them, the benzopyranone coumarin intermediate (1) has the following structural formula:

[0072]

[0073] Physicochemical properties (1): White solid, yield 72%, mp 188.0-189.4℃ ESI-MS m / z: 253.0862 ([M+H] + ),275.0681([M+Na] + ), 1H NMR(600MHz,DMSO-d6)δ8.10(s,1H,H-4),7.60–7.58(m,2H,H-2',6'),7.58(s,1H,H-5),7.24(d ,J=7.9Hz,2H,H-3',5'),6.81(dd,J=8.5,2.3Hz,1H,H-6),6.75(s,1H,H-8),2.34(s,3H,H-7'). 13 C NMR (151MHz, DMSO-d6) δ161.09,160.11,154.81,140.43,137.44,132.18,129.86,128.74,128.09,122.12,113.35,112.05,101.69,20.78.

[0074] The alkynyl-containing intermediate (2) has the following structural formula:

[0075]

[0076] Physicochemical properties (2): White solid, yield 80%; mp 156.3-158.1℃, ESI-MS m / z: 291.1014 ([M+H] + ), 1 H NMR(500MHz,DMSO-d6)δ8.17(s,1H,H-4),7.70(d,J=8.7Hz,1H,H-5),7.63–7.59(m,2H,H-2',6'),7.25(d,J=7.8Hz,2H,H-3',H-5'),7. 09(d,J=2.4Hz,1H,H-8),7.02(dd,J=8.6,2.4Hz,1H,H-6),4.95(d,J=2.4Hz,2H,H-2”),3.68(t,J=2.4Hz,1H,H-3”),2.34(s,3H,H-7′). 13 C NMR(126MHz,DMSO-d6)δ159.99,159.94,154.42,140.06,137.76,131.97,129.6 5,128.82,128.20,123.59,113.68,113.12,101.31,78.98,78.56,56.16,20.85.

[0077] Water-soluble coumarin Fe 3+ The fluorescent probe (3A) has the following structural formula:

[0078]

[0079] Physicochemical properties (3A): Pale yellow solid, yield 74%, mp 182.0-183.9℃, ESI-MS m / z: 448.1873 ([M+H]). + ); 470.1685([M+Na] + ), 1 H NMR (500MHz, DMSO-d6) δ8.45(s,1H,H-4),8.18(s,1H,H-7”),7.70(d,J=8.6Hz,1H,H-5),7.61( d,J=8.1Hz,2H,H-2',6'),7.25(d,J=7.9Hz,2H,H-3',5'),7.21(d,J=2.4Hz,1H,H-8),7.06(dd ,J=8.7,2.3Hz,1H,H-6),5.48(dd,J=11.4,4.5Hz,1H,H-8”),5.29(s,2H,H-2”),2.34(s,3H,H- 7'),2.32–2.12(m,1H,H-10”),2.01–1.93(m,1H,H-11),0.85(dd,J=22.6,6.6Hz,6H,H-12,13). 13 C NMR(126MHz,DMSO-d6)δ170.99,161.28,160.37,154.95,142.18,140.51,138.06,132.36,1 30.53,129.17,128.54,125.43,123.66,101.46,62.11,60.99,39.36,24.75,22.93,21.18.

[0080] Water-soluble coumarin Fe 3+ The fluorescent probe (3B) has the following structural formula:

[0081]

[0082] Physicochemical properties (3B): Pale yellow solid, yield 73%; mp 174.9-175.8℃, EIS-MS m / z: 434.1718 ([M+H] + ); 456.1538([M+Na] + ), 1H NMR (600MHz, DMSO-d6) δ8.39(s,1H,H-4),8.16(s,1H,H-7”),7.69(d,J=8.6Hz,1H,H-5),7. 61(d,J=8.0Hz,2H,H-2',6'),7.25(d,J=7.9Hz,2H,H-3',5'),7.20(d,J=2.4Hz,1H,H-8),7 .06(dd,J=8.6,2.4Hz,1H,H-6),5.29(s,2H,H-2”),5.20(d,J=8.0Hz,1H,H-8”),2.34(s,3H ,H-7'),1.25–1.14(m,1H,H-10”),0.95(d,J=6.7Hz,3H,H-11),0.81(d,J=6.7Hz,3H,H-12). 13 C NMR(151MHz,DMSO-d6)δ169.58,160.938,159.99,154.58,141.85,140.09,137.70,131.99,129.62, 128.79,128.16,125.12,123.35,113.38,112.67,101.14,68.10,61.68,30.44,20.80,18.97,18.28.

[0083] Water-soluble coumarin Fe 3+ The fluorescent probe (3C) has the following structural formula:

[0084]

[0085] Physicochemical properties (3C): Pale yellow solid, yield 71%; mp 172.6-173.1℃, EIS-MS m / z: 420.1561 ([M+H]). + ), 1 H NMR(500MHz,DMSO-d6)δ8.31(s,1H,H-4),8.17(s,1H,H-7”),7.69(d,J=8.7Hz,1H ,H-5),7.61(d,J=7.8Hz,2H,H-2',6'),7.25(d,J=7.8Hz,2H,H-3',5'),7.19(s,1 H,H-8),7.04(dd,J=8.7,2.5Hz,1H,H-6),5.27(s,2H,H-2”),4.41(t,J=7.1Hz,2H ,H-8”),2.34(s,3H,H-7’),2.23(t,J=7.3Hz,2H,H-9”),2.07–2.01(m,2H,H-10”). 13CNMR(126MHz,DMSO-d6)δ174.30,160.95,160.05,154.63,142.03,140.19,137.73,132.05,129. 68,128.84,128.22,124.93,123.33,113.40,113.17,101.09,62.27,49.20,30.38,25.29,20.87.

[0086] Water-soluble coumarin Fe 3+ Fluorescent probes (3D) have the following structural formula:

[0087]

[0088] Physicochemical properties (3D): Pale yellow solid, yield 72%; mp 183.1-184.4℃, EIS-MS m / z: 436.1509 ([M+H]). + ); 458.1328([M+Na] + ), 1 H NMR(500MHz,DMSO-d6)δ8.31(s,1H,H-4),8.17(s,1H,H-7”),7.69(d,J=8.7Hz,1H ,H-5),7.61(d,J=7.8Hz,2H,H-2',6'),7.25(d,J=7.8Hz,2H,H-3',5'),7.19(s,1 H,H-8),7.04(dd,J=8.7,2.5Hz,1H,H-6),5.27(s,2H,H-2”),4.41(t,J=7.1Hz,2H ,H-8”),2.34(s,3H,H-7’),2.23(t,J=7.3Hz,2H,H-9”),2.07–2.01(m,2H,H-10”). 13 C NMR(126MHz,DMSO-d6)δ174.30,160.95,160.05,154.63,142.03,140.19,137.73,132.05,129.6 8,128.84,128.22,124.93,123.33,113.40,113.17,101.09,62.27,49.20,30.38,25.29,20.87.

[0089] The following experiment further illustrates the water-soluble coumarin Fe 3+ Application of fluorescent probes in the fluorescence detection of Fe3+ ions.

[0090] Example 5

[0091] Water-soluble coumarin Fe 3+ Water solubility test of fluorescent probes (3A-3D)

[0092] Weigh 1 mg of each of the four coumarin fluorescent probes 3A-3D and dissolve all samples in ultrapure water. Select the completely dissolved samples and then dissolve the samples that are insoluble in ultrapure water in different proportions of acetonitrile-water solvent to determine the minimum amount of acetonitrile required to dissolve the sample at the same concentration.

[0093] like Figure 1 As shown, fluorescent probes 3A-3D all have good water solubility, with water content ranging from 40% to 75%, with 3D > 3C > 3B > 3A. It is worth noting that the water solubility of the designed and synthesized fluorescent probes is higher than that of the parent nucleus.

[0094] Example 6

[0095] Water-soluble coumarin Fe 3+ Fluorescent probes (3A-3D) for Fe 3+ Selective detection of ions

[0096] A water-soluble benzopyranone coumarin amino acid Fe was prepared with an acetonitrile:water ratio of 0.5:9.5 (v / v). 3+ Ion fluorescent probes, respectively added with Mn 2+ Mg 2+ Ag + Na + Ni 2+ Ca 2+ Cd 2+ Co 2+ Cu 2+ Fe 3 + Fe 2+ Zn 2+ Pb 2+ A 0.01 mol / L aqueous solution of ionic perchlorate was mixed in 10 μL and allowed to react for 5 min. The fluorescence emission spectrum was measured using 331 nm as the excitation wavelength.

[0097] like Figure 2 As shown, the fluorescent probe has an emission peak at 400-500 nm. When Fe is added... 3+ Subsequently, the emission peak of the fluorescent probe solution at 400-500 nm was significantly weakened. The addition of other cations, such as Mn, further reduced this effect. 2+ Mg 2+ Ag + Na + Ni 2+ Ca 2+ Cd2+ Co 2+ Cu 2 + Fe 2+ Zn 2+ Pb 2+ After ionization, the fluorescent probe solution showed no significant change at 420-450 nm. Therefore, the experimental results indicate that only solutions containing Fe... 3+ Only then can a significant decrease in fluorescence intensity be observed in the fluorescent probe solution at 420-450 nm. Experiments show that compounds 3A-3D all affect Fe... 3+ Identification: 3D has high water solubility, but is susceptible to Fe. 3+ The sensitivity of 3C is not as good as that of 3A and 3B. The sensitivity of 3A and 3B is not much different. 3B is more water-soluble than 3A, and 3C is more water-soluble than 3B. However, 3C has no specificity. The following uses compound 3B as an example to show other studies on fluorescence performance.

[0098] Example 7

[0099] Water-soluble coumarin Fe 3+ Fluorescent probe (3B) for Fe 3+ Ion recognition competition experiment

[0100] Prepare a 0.05 mM probe 3B solution using acetonitrile:water = 0.5:9.5 (v / v). Transfer 2 mL of probe 3B solution to a series of analytical vials, and add 10 μL of Fe... 3+ After reacting the stock solution for 5 minutes, add 10 μL of Mn. 2+ Mg 2+ Ag + Na + Ni 2+ Ca 2+ Cd 2+ Co 2+ Cu 2+ Fe 2+ Zn 2+ Pb 2+ Ion stock solution (0.01 mol / L). Using acetonitrile:water = 0.5:9.5 (v / v) as the reference solvent, the probe 3B solution and Fe were measured sequentially. 3+ The fluorescence spectrum of the mixed solution with probe 3B solution was analyzed, and finally the coexisting ion pairs of Fe were determined sequentially. 3+ For the fluorescence spectrum of the solution when the probe interferes, see [link to relevant documentation]. Figure 3 As shown in the left figure; Figure 3 The right-hand image shows the fluorescence spectrum of the probe 3B interacting with a single metal ion. Figure 3 To date, the fluorescence intensity did not change significantly after the addition of other metal ion interfering substances, and the other coexisting cations had an effect on Fe.3+ Fluorescent recognition of ions is largely unaffected; probe 3B is effective for Fe. 3+ Ion recognition is not affected by the presence of other metal ions.

[0101] Example 8

[0102] Water-soluble coumarin Fe 3+ Fluorescent probe (3B) for Fe 3+ Fluorescent titration of ions

[0103] In an acetonitrile:water = 0.5:9.5 (v / v) solution system, Fe was gradually added to the probe 3B solution (0.05 mmol / L). 3+ ionic (Fe) 3+ The fluorescence emission spectrum was measured using ion concentrations of 0-0.065 mmol / L and excitation wavelength of 331 nm.

[0104] Depend on Figure 4 As is known, in the acetonitrile:water = 0.5:9.5 (v / v) system, with Fe... 3+ As the ion concentration gradually increased, the fluorescence intensity of the probe 3B solution at 400-500 nm gradually decreased, further verifying the effectiveness of probe 3B in detecting Fe. 3+ The fluorescence quenching performance of ions belongs to the "turn-off" mode.

[0105] Example 9

[0106] Water-soluble coumarin Fe 3+ Fluorescent probe (3B) for Fe 3+ Calculation of the detection limit of ions

[0107] Based on the titration results, the curves of probe 3B with Fe were plotted. 3+ The working curve of concentration change. The limit of detection (LOD) is calculated using the formula: LOD = 3σ / κ (LOD represents the detection limit, σ symbolizes the standard deviation of the blank test, and κ represents the slope of the calibration curve).

[0108] Depend on Figure 4 It can be seen that the probe's effect on Fe was calculated. 3+ The limit of detection for the ions is 1.1 μM (0.06 mg / L).

[0109] Example 10

[0110] Water-soluble coumarin Fe 3+ Fluorescence changes of fluorescent probe (3B) at different pH values

[0111] To detect the pH value of the solution, probe 3B and Fe were used. 3+The effect of ion interaction was investigated by preparing solutions with different pH values ​​(2.0-13.0) and adding Fe to probe 3B solutions with different pH values. 3+ The fluorescence spectrum of the ions was plotted at 436 nm to measure the changes in their fluorescence spectrum.

[0112] Depend on Figure 5 It can be seen that within the pH range of 2-8, the fluorescence spectrum of probe 3B alone does not change significantly, but the fluorescence weakens significantly when the pH increases to 8-13; probe 3B reacts with Fe... 3+ After ion interaction, the fluorescence intensity decreased significantly at pH 2-8, but without much fluctuation. When the pH increased to 8-13, the fluorescence intensity increased markedly. This indicates that in a buffer system with pH < 8, pH significantly affects the detection of Fe by probe 3B. 3+ The impact is small, and the detection effect is the most obvious.

[0113] Example 11

[0114] EDTA on water-soluble coumarin Fe 3+ Effects of fluorescent probe (3B)

[0115] Transfer 2 ml of probe 3B (0.05 mM, acetonitrile-water = 0.5:9.5, pH = 7.0) into a quartz tube, and continuously increase the concentration of EDTA (0-0.12 mM). Simultaneously, accurately transfer another 2 ml of 3B (0.05 mM, acetonitrile-water = 0.5:9.5, pH = 7.0), and continuously increase the concentration of Fe. 3+ The changes in fluorescence intensity were measured by determining the amounts of (0-0.12mM) and EDTA (0-0.12mM) at an excitation wavelength of 331nm.

[0116] Depend on Figure 6 It can be seen that EDTA has no effect on the fluorescence intensity of the probe. However, as the concentration of EDTA increases, the fluorescence intensity of the probe 3B+Fe increases. 3+ The fluorescence intensity did not change significantly; therefore, we believe that probe 3B and Fe... 3+ It has strong chelating ability, especially unaffected by chelating agents (such as EDTA).

[0117] Example 12: Water-soluble coumarin Fe 3+ Application of fluorescent probe (3B) in zebrafish imaging

[0118] As a control group, three-day-old zebrafish were pretreated with probe 3B (15.0 μM) in E3 embryonic culture medium (34.8 g NaCl, 1.6 g KCl, 5.8 g CaCl2-2H2O, 9.78 g MgCl2-6H2O) for 0.5 hours. In the experimental group, zebrafish were first cultured with 3B for 0.5 hours, and then treated with different concentrations of Fe...3+ Treatment with (10.0, 50.0 μM) for 0.5 hours. After incubation at 28°C for 4 days, fluorescence images of probe 3B were acquired at 400-460 nm wavelength (blue channel). Figure 7 It was observed that probe 3B (10 μM) quickly entered the zebrafish, demonstrating good biocompatibility and producing a blue fluorescent signal. With Fe... 3+ With the gradual addition of (0-50 μM), the blue fluorescence significantly weakened, indicating that probe 3B can specifically recognize Fe in zebrafish. 3+ These findings indicate that probe 3B has the capability to observe Fe in zebrafish. 3+ The ability to detect Fe in vivo. 3+ Its potential.

[0119] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0120] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Water-soluble coumarin Fe 3+ Fluorescent probe, characterized in that, Its structural formula is as follows: Where R is or or .

2. Water-soluble coumarin Fe 3+ A method for preparing a fluorescent probe, characterized in that, Preparation of water-soluble coumarin Fe as described in claim 1 3+ The fluorescent probe involves the following steps: Step 1: Using p-tolueneacetic acid and 2,4-dihydroxybenzaldehyde as starting materials, dissolve them in acetic anhydride, add triethylamine as a catalyst to react and obtain acetylated coumarin nuclei; add the acetylated coumarin nuclei to methanol, slowly add concentrated hydrochloric acid, and remove the acetyl group in the acidic environment to obtain benzopyranone coumarin intermediate 1. Step 2: Using benzopyranone coumarin intermediate 1, bromopropyne and potassium carbonate as raw materials, and acetonitrile as solvent, intermediate 2 containing an alkyne group is obtained through nucleophilic substitution. Step 3: After dissolving the azide amino acid in tert-butanol, add intermediate 2 containing an alkyne group, then add copper sulfate pentahydrate, sodium ascorbate, and pure water. Use click chemistry to synthesize water-soluble coumarin Fe. 3+ Fluorescent probe; The acetylated coumarin nucleus structure is as follows: ; Benzopyranone coumarin intermediate 1 has the following structural formula: The acetylene-containing intermediate 2 has the following structural formula: .

3. The water-soluble coumarin Fe as described in claim 2 3+ A method for preparing a fluorescent probe, characterized in that, Step one specifically involves: dissolving p-tolueneacetic acid and 2,4-dihydroxybenzaldehyde in acetic anhydride as a solvent, adding triethylamine as a catalyst, reacting at 130-150℃ for 6-8 hours, cooling after the reaction, and recrystallizing to obtain acetylated coumarin nuclei; adding the acetylated coumarin nuclei to methanol, slowly adding concentrated hydrochloric acid, heating to 60-65℃, and reacting for 3-4 hours; filtering the white transparent solid after the reaction to obtain benzopyranone coumarin intermediate 1.

4. The water-soluble coumarin Fe as described in claim 3 3+ A method for preparing a fluorescent probe, characterized in that, The reaction temperature in step two is 55-60℃, and the reaction time is 3-4 hours. After the reaction is completed, the mixture is filtered, concentrated, dissolved in ethyl acetate, and washed with saturated ammonium chloride and saturated brine in sequence. The organic layer is retained, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate 2 containing alkynyl groups.

5. The water-soluble coumarin Fe as described in claim 4 3+ A method for preparing a fluorescent probe, characterized in that, The azide amino acid in step three is azide-leucine, azide-valine, or azide-threonine, corresponding to the water-soluble coumarin Fe synthesized accordingly. 3+ The fluorescent probes have the following structural formulas: ; ； 。 6. The water-soluble coumarin Fe as described in claim 5 3+ A method for preparing a fluorescent probe, characterized in that, The azide amino acid was prepared by the following steps: sodium azide was dissolved in water and DCM was added as a solvent. Trifluoromethanesulfonic anhydride was added dropwise at 0°C. After 3-4 hours, potassium carbonate, amino acid, and anhydrous copper sulfate were added and reacted for 3-4 hours. The organic solvent was removed, the pH was adjusted to 2, and the mixture was extracted with ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and then evaporated to dryness to obtain the azide amino acid. The molar volume ratio of sodium azide:DCM:trifluoromethanesulfonic anhydride:potassium carbonate:amino acid:anhydrous copper sulfate was 9.2 mmol:15 mL:18.4 mmol:7.2 mmol:9.2 mmol:0.92 mmol. Wherein, the amino acid is L-leucine, L-valine or L-threonine, and the corresponding azide amino acids are azide-leucine, azide-valine or azide-threonine, respectively.

7. The water-soluble coumarin Fe as described in claim 6 3+ A method for preparing a fluorescent probe, characterized in that, The click chemistry method in step three is as follows: Azide amino acids are dissolved in tert-butanol, then intermediate 2 containing an alkyne group, copper sulfate pentahydrate, sodium ascorbate, and pure water are added. The mixture is heated to 55-60℃ and reacted for 4-5 hours. After the reaction is completed under TLC monitoring, saturated brine is added for washing, followed by extraction with ethyl acetate, drying with anhydrous sodium sulfate, concentration under reduced pressure, and purification by silica gel column chromatography to obtain water-soluble coumarin Fe. 3+ Fluorescent probe; wherein the column chromatography silica gel column purification uses a gradient elution of dichloromethanol = 40:1; 20:1; 10:

1.

8. The water-soluble coumarin Fe as described in any one of claims 2-7 3+ A method for preparing a fluorescent probe, characterized in that, In step one, the molar ratio of p-tolueneacetic acid: 2,4-dihydroxybenzaldehyde: triethylamine is (1.5-2): 1: (1.5-2); the volume molar ratio of acetic anhydride: methanol: concentrated hydrochloric acid: 2,4-dihydroxybenzaldehyde is 2.5-3 mL: 2-2.5 mL: 90-100 μL: 1 mmol. In step two, the molar volume ratio of benzopyranone coumarin intermediate 1: potassium carbonate: bromopropyne: acetonitrile is 1-1.2 mmol: 4-4.8 mmol: 2.4 mmol: 15 mL; In step three, the molar ratio of azide amino acid: alkyne-containing intermediate: copper sulfate pentahydrate: sodium ascorbate is 1:1:0.4:0.85, and the molar volume ratio of alkyne-containing intermediate 2 to pure water is 1 mmol:2 mL.

9. The water-soluble coumarin Fe as described in claim 1 3+ Fluorescent probes in the preparation of Fe in zebrafish 3+ Applications in fluorescence imaging detection reagents.

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