Fluorescent probe based on naphthalene derivative, preparation method of fluorescent probe and application of fluorescent probe in pH detection

Abstract

The invention belongs to the field of chemical detection, and relates to a fluorescent probe based on naphthalene derivatives, a preparation method of the fluorescent probe and application of the fluorescent probe in pH detection. The selected fluorophore naphthalene derivative has the advantages that the raw materials are simple and easy to obtain, the emission wavelength is in a visible light region, and the Stocks displacement is large (137 nm), so that the fluorescent probe pHP1 has the advantages of low background interference, small light damage to biological samples, strong sample penetrability, good light stability, high detection sensitivity and the like.

Description

Technical Field

[0001] The invention belongs to the field of chemical detection and relates to fluorescence detection of pH. Background Art

[0002] Hydrogen ions (H+) are essential and most reactive ions in living cells. Normal cellular function depends on pH homeostasis, and disruption of this homeostasis can trigger a variety of physiological abnormalities. For example, the normal extracellular pH ranges from 6.7 to 7.1, but most solid tumors exhibit abnormally low pH levels. Abnormal pH can also cause cardiopulmonary and neurological diseases. Given the crucial role of H+ in the body, developing detection tools that can rapidly, efficiently, and accurately detect pH changes is of great significance. Fluorescent probes are tools that convert intermolecular interactions into easily recognizable optical signals for external detection. Upon interaction with a specific target analyte, a fluorescent probe undergoes a significant change in the fluorescent signal, achieving the desired detection objective. Fluorescent probes offer advantages such as excellent selectivity, high sensitivity, simple and rapid operation, and minimal damage to the target. They have been widely used to detect metal cations, anions, and bioactive small molecules in environmental and biological systems. Application Publication No. CN108318462A discloses a fluorescent thin-film sensor using 2-(4-methoxyphenyl)quinolin-6-ol as a fluorescent probe molecule, utilizing its two fluorescence emission peaks for ratiometric measurement. Application Publication No. CN109776499A discloses a fluorescent probe, 4-morpholino-N-isonicotinylhydrazide, designed as a small molecule naphthalene imide compound, to address the challenge of detecting cellular pH under extremely acidic and alkaline conditions. Currently, a wide variety of probes are used to detect pH fluctuations. Fluorescent probes, with their advantages of high sensitivity, high-speed spatial analysis, and minimal biodamage, are widely used in pH detection in living cells, tissues, and organisms. However, many developed fluorescent probes for pH detection suffer from the following drawbacks: 1) Most require multiple synthesis steps and are costly to prepare; 2) most have a small Stokes shift, making them susceptible to interference from instrument background; and 3) most probes cannot detect pH with the naked eye, hindering real-time detection. Therefore, there is an urgent need to develop fluorescent pH probes that are easy to prepare, have a large Stokes shift, and can be detected with the naked eye. Summary of the Invention

[0003] To solve the above technical problems, the present invention proposes a fluorescent probe based on naphthalene derivatives, a preparation method thereof, and an application thereof in detecting pH.

[0004] The technical solution of the present invention is achieved as follows: A naphthalene derivative-based fluorescent probe for pH detection, the structural formula of the naphthalene derivative fluorescent probe is as follows: .

[0005] The synthetic route of the preparation method of the fluorescent probe for pH detection based on naphthalene derivatives is: The specific synthesis steps are: (1) 2,3,3-Trimethylindole and 2-bromoethanol were dissolved in anhydrous acetonitrile. After the reaction was completed under heating reflux, the reaction solution was cooled to room temperature, solids were precipitated, and intermediate 1 was obtained by filtration.

[0006] (2) The intermediate 1 and 6-methoxy-2-naphthaldehyde were dissolved in ethanol, pyridine was added, and the reaction was completed under heating reflux. The reaction solution was cooled to room temperature, filtered to obtain a crude product, and recrystallized from ethanol to obtain the probe pHP1.

[0007] In the above step (1), the molar ratio of 2,3,3-trimethylindole to 2-bromoethanol is 1:(1-6).

[0008] In the above step (1), the heating reflux reaction time is 12-48 hours.

[0009] In the above step (2), the molar ratio of the intermediate 1, 6-methoxy-2-naphthaldehyde and pyridine is 1:(1-6):(0.1-5).

[0010] In the above step (2), the heating reflux reaction time is 8-24 hours, and the yield of the probe is 60-85%.

[0011] The above fluorescent probe is used in detecting pH value for purposes other than disease diagnosis.

[0012] The above fluorescent probe is used to detect acidity in the environment or biological system for purposes other than disease diagnosis.

[0013] Furthermore, the detection concentration of the fluorescent probe is 1 mM, and the emission wavelength is 562 nm.

[0014] The recognition reaction mechanism of the fluorescent probe of this application is: .

[0015] The present invention has the following beneficial effects: (1) The fluorophore naphthalene derivative selected in the present invention has the advantages of simple probe synthesis steps, high yield, emission wavelength in the visible light region, and large Stokes shift (137 nm), which makes the fluorescent probe pHP1 have many advantages such as low background interference, small photodamage to biological samples, strong sample penetration, good photostability, and high detection sensitivity.

[0016] (2) The spirooxazolidine compound in the chemical structure of the fluorescent probe pHP1 of the present invention undergoes an intramolecular ring opening / closing reaction under the influence of the acidity of the environment. Under acidic conditions, the nitrogen on the indole is protonated, resulting in a decrease in electronegativity. The electrons mainly distributed on the benzene ring and the indole group increase, and the large conjugated system within the molecule is extended, thereby turning on fluorescence. This intramolecular reaction will cause a significant change in their absorption properties, thereby achieving efficient recognition of the pH of the environmental system. As the pH of the solution changes from acidic to alkaline, the color of the solution changes from yellow to colorless (see attached). Figure 9 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe pHP1 of the present invention.

[0019] Figure 2 This is the carbon nuclear magnetic resonance spectrum of the fluorescent probe pHP1 of the present invention.

[0020] Figure 3 This is a high-resolution mass spectrum of the fluorescent probe pHP1 of the present invention.

[0021] Figure 4 This is a fluorescence emission spectrum of the fluorescent probe pHP1 of the present invention in a DMSO-PBS (v / v=1 / 4, 10 mM) buffer solution with a pH of 4-9, with an excitation wavelength of 400 nm.

[0022] Figure 5 This is a study on the anti-interference ability of the fluorescent probe pHP1 of the present invention to common amino acids, with an excitation wavelength of 400 nm and an emission wavelength of 562 nm.

[0023] Figure 6 This is a study on the anti-interference ability of the fluorescent probe pHP1 of the present invention to common anions, with an excitation wavelength of 400 nm and an emission wavelength of 562 nm.

[0024] Figure 7 This study is a study on the anti-interference ability of the fluorescent probe pHP1 of the present invention to common metal ions, with an excitation wavelength of 400 nm and an emission wavelength of 562 nm.

[0025] Figure 8This is a photostability experiment of the fluorescent probe pHP1 of the present invention at different pH values, with an excitation wavelength of 400 nm and an emission wavelength of 562 nm.

[0026] Figure 9 This is a diagram showing the color change of the fluorescent probe pHP1 of the present invention in solutions at different pH values. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0029] Example 1 The preparation method of a fluorescent probe for pH detection based on naphthalene derivatives comprises the following steps: (1) The preparation method of intermediate 1 is as follows: compound 2,3,3-trimethylindole (159.2 mg, 1 mmol) and 2-bromoethanol (250 mg, 2 mmol) are dissolved in 60 mL of acetonitrile, refluxed at 85 degrees Celsius for 24 hours, cooled, solid precipitated, and intermediate 1 was filtered.

[0030] (2) The preparation method of probe pHP1 is as follows: intermediate 1 (284.2 mg, 1 mmol) and 6-methoxy-2-naphthaldehyde (186.2 mg, 1 mmol) are dissolved in 10 mL of ethanol, piperidine (85 mg, 1 mmol) is added, and the mixture is refluxed at 80°C for 12 hours. Filter and obtain 278.3 mg of white solid, which is the pH fluorescent probe pHP1, with a yield of 75%.

[0031] Fluorescent probe pHP1 H NMR spectroscopy determination ( Figure 1 ): 1 H NMR (CDCl3- d , 400 MHz) δ 1.30 (s,3 H), 1.57 (s, 3 H), 3.58 (m,1 H), 3.75 (m, 2 H), 3.90 (m, 1 H), 3.98 (s, 3H), 6.45 (d, J = 16.0 Hz, 1 H), 6.90 (d, J= 8.0 Hz, 1 H), 7.05 (m, 1 H), 7.11(d, J = 16.0 Hz, 1 H), 7.24 (m, 4 H), 7.71 (d, J = 2.0 Hz, 1 H), 7.78 (m, 3 H).

[0032] Fluorescent probe pHP1 nuclear magnetic resonance carbon spectrum determination ( Figure 2 ): 13 C NMR (CDCl3- d , 100 MHz) δ 20.5,28.6, 48.1, 50.2, 55.4, 63.7,105.9, 110.1, 112.1, 119.2, 121.8, 122.5, 124.4,125.2, 126.8, 127.3, 127.7, 129.0, 129.7, 131.9, 132.5, 134.4, 139.9, 150.7,157.9.

[0033] Fluorescent probe pHP1 high-resolution mass spectrometry determination ( Figure 3 ): HR-ESI-MS calculation for C 25 H 25 NO2:371.1185, found 372.1962 [M+H + ] + .

[0034] Example 2 The preparation method of a fluorescent probe for pH detection based on naphthalene derivatives comprises the following steps: (1) The preparation method of intermediate 1 is as follows: Compound 2,3,3-trimethylindole (159.2 mg, 1 mmol) and 2-bromoethanol (375 mg, 3 mmol) are dissolved in 60 mL of acetonitrile, refluxed at 85°C for 12 hours, cooled, solid precipitated, and intermediate 1 was filtered.

[0035] (2) The preparation method of probe pHP1 is as follows: intermediate 1 (284.2 mg, 1 mmol) and 6-methoxy-2-naphthaldehyde (372.4 mg, 2 mmol) were dissolved in 10 mL of ethanol, piperidine (170 mg, 2 mmol) was added, and the mixture was refluxed at 80 °C for 18 h. After filtration, 304.3 mg of white solid was obtained, which was the pH fluorescent probe pHP1, with a yield of 82%.

[0036] Fluorescent probe pHP1 H NMR spectrum determination: 1H NMR (CDCl3- d , 400 MHz) δ 1.30 (s, 3H), 1.57 (s, 3 H), 3.58 (m, 1 H), 3.75 (m, 2 H), 3.90 (m, 1 H), 3.98 (s, 3 H), 6.45 (d, J = 16.0 Hz, 1 H), 6.90 (d, J = 8.0 Hz, 1 H), 7.05 (m, 1 H), 7.11 (d, J =16.0 Hz, 1 H), 7.24 (m, 4 H), 7.71 (d, J = 2.0 Hz, 1 H), 7.78 (m, 3 H).

[0037] Fluorescent probe pHP1 nuclear magnetic resonance carbon spectrum determination: 13 C NMR (CDCl3- d , 100 MHz) δ 20.5, 28.6,48.1, 50.2, 55.4, 63.7, 105.9, 110.1, 112.1, 119.2, 121.8, 122.5,124.4,125.2, 126.8, 127.3, 127.7, 129.0, 129.7, 131.9, 132.5, 134.4, 139.9, 150.7,157.9.

[0038] High-resolution mass spectrometry of the fluorescent probe pHP1: HR-ESI-MS calculation for C 25 H 25 NO2: 371.1185, found 372.1962 [M+H + ] + .

[0039] Example 3 The preparation method of a fluorescent probe for pH detection based on naphthalene derivatives comprises the following steps: (1) The preparation method of intermediate 1 is as follows: compound 2,3,3-trimethylindole (159.2 mg, 1 mmol) and 2-bromoethanol (500 mg, 4 mmol) are dissolved in 60 mL of acetonitrile, refluxed at 85 degrees Celsius for 36 hours, cooled, solid precipitated, and intermediate 1 was filtered.

[0040] (2) The preparation method of probe pHP1 is as follows: intermediate 1 (284.2 mg, 1 mmol) and 6-methoxy-2-naphthaldehyde (186.2 mg, 2 mmol) were dissolved in 10 mL of ethanol, piperidine (8.5 mg, 0.1 mmol) was added, and the mixture was refluxed at 80 °C for 8 hours. After filtration, 222.7 mg of white solid was obtained, which is the pH fluorescent probe pHP1, with a yield of 60%.

[0041] Fluorescent probe pHP1 H NMR spectrum determination: 1 H NMR (CDCl3- d , 400 MHz) δ 1.30 (s, 3H), 1.57 (s, 3 H), 3.58 (m, 1 H), 3.75 (m, 2 H), 3.90 (m, 1 H), 3.98 (s, 3 H), 6.45 (d, J = 16.0 Hz, 1 H), 6.90 (d, J = 8.0 Hz, 1 H), 7.05 (m, 1 H), 7.11 (d, J =16.0 Hz, 1 H), 7.24 (m, 4 H), 7.71 (d, J = 2.0 Hz, 1 H), 7.78 (m, 3 H).

[0042] Fluorescent probe pHP1 nuclear magnetic resonance carbon spectrum determination: 13 C NMR (CDCl3- d , 100 MHz) δ 20.5, 28.6,48.1, 50.2, 55.4, 63.7, 105.9, 110.1, 112.1, 119.2, 121.8, 122.5,124.4,125.2, 126.8, 127.3, 127.7, 129.0, 129.7, 131.9, 132.5, 134.4, 139.9, 150.7,157.9.

[0043] High-resolution mass spectrometry of the fluorescent probe pHP1: HR-ESI-MS calculation for C 25 H 25 NO2: 371.1185,found 372.1962 [M+H + ] + .

[0044] Example 4 The preparation method of a fluorescent probe for pH detection based on naphthalene derivatives comprises the following steps: (1) The preparation method of intermediate 1 is as follows: compound 2,3,3-trimethylindole (159.2 mg, 1 mmol) and 2-bromoethanol (625 mg, 5 mmol) are dissolved in 60 mL of acetonitrile, refluxed at 85 degrees Celsius for 40 hours, cooled, solid precipitated, and intermediate 1 was filtered.

[0045] (2) The preparation method of probe pHP1 is as follows: intermediate 1 (284.2 mg, 1 mmol) and 6-methoxy-2-naphthaldehyde (1117.2 mg, 6 mmol) were dissolved in 10 mL of ethanol, piperidine (255 mg, 3 mmol) was added, and the mixture was refluxed at 80 °C for 16 h. After filtration, 296.9 mg of white solid was obtained, which was the pH fluorescent probe pHP1, with a yield of 80%.

[0046] Fluorescent probe pHP1 H NMR spectrum determination: 1 H NMR (CDCl3- d , 400 MHz) δ 1.30 (s, 3H), 1.57 (s, 3 H), 3.58 (m, 1 H), 3.75 (m, 2 H), 3.90 (m, 1 H), 3.98 (s, 3 H), 6.45 (d, J = 16.0 Hz, 1 H), 6.90 (d, J = 8.0 Hz, 1 H), 7.05 (m, 1 H), 7.11 (d, J =16.0 Hz, 1 H), 7.24 (m, 4 H), 7.71 (d, J = 2.0 Hz, 1 H), 7.78 (m, 3 H).

[0047] Fluorescent probe pHP1 nuclear magnetic resonance carbon spectrum determination: 13 C NMR (CDCl3- d , 100 MHz) δ 20.5, 28.6,48.1, 50.2, 55.4, 63.7, 105.9, 110.1, 112.1, 119.2, 121.8, 122.5,124.4,125.2, 126.8, 127.3, 127.7, 129.0, 129.7, 131.9, 132.5, 134.4, 139.9, 150.7,157.9.

[0048] High-resolution mass spectrometry of the fluorescent probe pHP1: HR-ESI-MS calculation for C 25 H 25 NO2: 371.1185,found 372.1962 [M+H + ] + .

[0049] Example 5 The preparation method of a fluorescent probe for pH detection based on naphthalene derivatives comprises the following steps: (1) The preparation method of intermediate 1 is as follows: compound 2,3,3-trimethylindole (159.2 mg, 1 mmol) and 2-bromoethanol (750 mg, 6 mmol) are dissolved in 60 mL of acetonitrile, refluxed at 85°C for 48 hours, cooled, solid precipitated, and intermediate 1 was filtered.

[0050] (2) The preparation method of probe pHP1 is as follows: intermediate 1 (284.2 mg, 1 mmol) and 6-methoxy-2-naphthaldehyde (1117.2 mg, 6 mmol) were dissolved in 10 mL of ethanol, piperidine (425 mg, 5 mmol) was added, and the mixture was refluxed at 80 °C for 24 h. 315.5 mg of white solid was obtained by filtration, which is the pH fluorescent probe pHP1, with a yield of 85%.

[0051] Fluorescent probe pHP1 H NMR spectrum determination: 1 H NMR (CDCl3- d , 400 MHz) δ 1.30 (s, 3H), 1.57 (s, 3 H), 3.58 (m, 1 H), 3.75 (m, 2 H), 3.90 (m, 1 H), 3.98 (s, 3 H), 6.45 (d, J = 16.0 Hz, 1 H), 6.90 (d, J = 8.0 Hz, 1 H), 7.05 (m, 1 H), 7.11 (d, J =16.0 Hz, 1 H), 7.24 (m, 4 H), 7.71 (d, J = 2.0 Hz, 1 H), 7.78 (m, 3 H).

[0052] Fluorescent probe pHP1 nuclear magnetic resonance carbon spectrum determination: 13 C NMR (CDCl3- d, 100 MHz) δ 20.5, 28.6,48.1, 50.2, 55.4, 63.7, 105.9, 110.1, 112.1, 119.2, 121.8, 122.5,124.4,125.2, 126.8, 127.3, 127.7, 129.0, 129.7, 131.9, 132.5, 134.4, 139.9,150.7,157.9.

[0053] High-resolution mass spectrometry of the fluorescent probe pHP1: HR-ESI-MS calculation for C 25 H 25 NO2: 371.1185, found 372.1962 [M+H + ] + .

[0054] Implementation effect example 1 PBS buffer solutions with a pH of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9 at a concentration of 10 mM were prepared. Mixed solutions A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, and A11 were prepared with these buffer solutions and DMSO at a volume ratio of 4:1. A 1 mM solution of the probe pHP1 was also prepared with DMSO. The fluorescence emission spectra of the probe pHP1 (10 µM) in the mixed solutions A1-A11 were observed using a fluorescence spectrometer. Figure 4 As shown in the figure, under the excitation condition of 400 nm, the fluorescence emission intensity of the probe pHP1 (10 µM) in the mixed solutions A1-A11 with pH values of 4-9 at 562 nm gradually decreases with the increase of the alkalinity of the mixed solution system, showing sensitive recognition ability to changes in solution pH.

[0055] Implementation effect example 2 PBS buffer solutions with a pH of 4 and 7.5 and a concentration of 10 mM were prepared respectively. Mixed solutions A and B with a volume ratio of 4:1 were prepared using the above buffer solutions and DMSO, and a probe pHP1 solution with a concentration of 1 mM was prepared using DMSO. The recognition effect of probe pHP1 for solutions with a pH of 4 and 7.5 in the presence of common amino acids, common anions, and common metal ions was investigated using a fluorescence spectrometer. 2940 μL of mixed solution A or B and 30 μL of pHP1 DMSO solution were added to a clean fluorescence cuvette, and then other analytes (common amino acids, common anions, common metal ions, etc.) were added. The fluorescence intensity at 562 nm corresponding to different analytes was detected on a fluorescence spectrometer, and a histogram of the fluorescence intensity at 562 nm was plotted to obtain a fluorescence emission histogram (see Appendix). Figure 5 , 6 and 7).

[0056] Experiments have shown that the probe pHP1's recognition of the pH of mixed solutions is not interfered with by common amino acids, common anions and common metal ions, and has good anti-interference ability.

[0057] Implementation effect example 3 PBS buffer solutions with pH values of 4, 7.5, and 9 and a concentration of 10 mM were prepared. Mixed solutions A, B, and C were prepared with the aforementioned buffer solutions and DMSO at a volume ratio of 4:1. A probe pHP1 solution was also prepared with DMSO at a concentration of 1 mM. Fluorescence spectrometry was used to investigate the changes in the fluorescence emission intensity of the probe pHP1 in mixed solutions A, B, and C over time. Figure 8 As shown in the figure, under continuous excitation conditions at an emission wavelength of 400 nm, the emission intensity of probe pHP1 (10 µM) at an emission wavelength of 562 nm in mixed solutions A, B and C with pH values of 4, 7.5 and 9, respectively, remained stable over time. The above experimental results indicate that the probe pHP1 has excellent optical stability.

[0058] Application Examples Given the excellent pH sensitivity of the pHP1 probe, we conducted quantitative pH measurements in various water samples (drinking water, tap water, irrigation water, and Dongfeng Canal water) to evaluate its potential value in environmental and physiological applications. These water samples were filtered through a microporous filter to remove impurities and prepare solutions at pH values of 4.50, 7.00, and 7.50. The fluorescence response of the pHP1 probe was measured at different pH values. By comparing the calibration results with the test results, the pH recovery rates for the different water samples were calculated (Table 1).

[0059] Table 1 Determination of pH values in various water samples by probe pHP1 As shown in Table 1, the recovery rate is between 98.00-104.40%. The probe pHP1 has the ability to monitor the pH of actual water samples, showing good practical application value.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fluorescent probe based on a naphthalene derivative having the following structural formula: 。 2. The method for preparing a fluorescent probe based on naphthalene derivatives according to claim 1, characterized in that: The steps are: (1) Dissolve 2,3,3-trimethylindole and 2-bromoethanol in anhydrous acetonitrile, heat under reflux until the reaction is complete, then cool the reaction solution to room temperature, precipitate a solid, and filter to obtain intermediate 1; (2) The intermediate 1 and 6-methoxy-2-naphthaldehyde were dissolved in ethanol, pyridine was added, and the mixture was heated under reflux until the reaction was complete. The reaction solution was then cooled to room temperature and filtered to obtain a crude product. The crude product was recrystallized from ethanol to obtain a fluorescent probe, namely pHP1.

3. The method for preparing a fluorescent probe based on naphthalene derivatives according to claim 2, wherein: In the step (1), the molar ratio of 2,3,3-trimethylindole to 2-bromoethanol is 1:1-6.

4. The method for preparing a fluorescent probe based on naphthalene derivatives according to claim 3, wherein: The heating reflux reaction time in step (1) is 12-48 hours.

5. The method for preparing a fluorescent probe based on naphthalene derivatives according to claim 2, wherein: The structural formula of the intermediate 1 is: .

6. The method for preparing a fluorescent probe based on naphthalene derivatives according to claim 2, wherein: In the step (2), the molar ratio of the intermediate 1, 6-methoxy-2-naphthaldehyde and pyridine is 1:1-6:0.1-5.

7. The method for preparing a fluorescent probe based on naphthalene derivatives according to claim 2, wherein: The heating reflux reaction time in step (2) is 8-24 hours, and the yield of the probe is 60-85%.

8. Use of the fluorescent probe according to claim 1 in detecting pH value for purposes other than disease diagnosis.

9. Use of the fluorescent probe according to claim 1 in detecting acidity in an environment or biological system for purposes other than disease diagnosis.

10. The use according to claim 8 or 9, characterized in that: The detection concentration of the fluorescent probe is 1 mM, and the emission wavelength is 562 nm.

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