Coumarin-based micromolecular fluorescent probe M as well as preparation method and application thereof

Abstract

The invention discloses a coumarin-based micromolecular fluorescent probe M and a preparation method and application thereof, and relates to the technical field of fluorescent molecular probes, the preparation method of the probe M specifically comprises the following steps: S1, synthesizing a compound 2; s2, synthesizing a compound 3; s3, synthesizing a probe M: dissolving carboxylic coumarin and HBTU in an anhydrous DMF solution, and then sequentially adding a compound 3 and diisopropylamine to obtain a mixed solution; stirring and reacting the mixed solution at room temperature under the protection of inert gas; and after the reaction is finished, carrying out rotary evaporation to remove most of the solvent to obtain a crude product, and then purifying by using silica gel column chromatography to obtain a clean product probe M. The probe M synthesized in the invention has the advantages of strong selectivity, strong anti-interference performance, high sensitivity and strong reversibility, and can be used for pH detection.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent molecular probes, and in particular to a coumarin-based small molecule fluorescent probe M and a preparation method and application thereof. Background Art

[0002] pH is a key parameter that reflects the health of cells. It plays a critical role in many physiological and pathological processes, such as cell growth and apoptosis, enzyme activity, homeostasis of the body environment, and signal transmission between receptors.

[0003] Nowadays, there are many detection methods for detecting pH value, such as electrochemical measurement, acid-base indicator titration, potentiometric titration, absorption spectroscopy, microelectrode, etc. However, these methods have many disadvantages, requiring high instrument accuracy and processing procedures that require professionals to operate. In contrast, small molecule fluorescent probes have the advantages of simple operation and real-time monitoring.

[0004] Coumarin compounds have received extensive attention and application in the field of fluorescent probes due to their special structure and optical properties. The coumarin fluorescent probes reported so far have the following advantages: large Stokes shift, high quantum yield and photostability, good water solubility and biocompatibility, etc. There are many coumarin fluorescent probes reported so far, but most of them have problems such as low sensitivity, weak selectivity, and poor reversibility, which are technical problems that people in this field need to solve urgently. Summary of the invention

[0005] In view of the above problems, the present invention provides a coumarin-based small molecule fluorescent probe M and a preparation method and application thereof, which solves the problems of low sensitivity, weak selectivity, poor reversibility, etc. in the prior art.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a small molecule fluorescent probe M based on coumarin, wherein the probe M is 2-2-(2-(4-(7-hydroxy-2-oxo-2-hydro-benzopyran-3-carbonyl)piperazine)ethane)-6-morpholine-1-hydro-benzoisoquinoline-1,3(2-hydro)dione, and its structural formula is:

[0008]

[0009] The present invention also provides a method for preparing the above-mentioned coumarin-based small molecule fluorescent probe M, which specifically comprises the following steps:

[0010] S1: Synthesis of compound 2: Compound 1 (6-bromo-1,3-dihydro-benzoisoquinoline-1,3-dione) and morpholine were refluxed in ethylene glycol methyl ether under N2 protection, and the reaction solution was cooled to room temperature overnight. After crystals precipitated, they were filtered off under reduced pressure and dried to obtain compound 2 (6-morpholine-1,3-dihydro-benzoisoquinoline-1,3-dione). The synthesis route of compound 2 is as follows:

[0011]

[0012] S2: Synthesis of compound 3: Compound 2 and N-aminopiperidine were dissolved in ethanol and refluxed under the protection of inert gas; after the reaction was completed, the excess solvent was removed by rotary evaporator to obtain a crude product; the product was then dissolved in chloroform, washed with saturated NaCl solution for 3 times, the organic phase was collected, and then dried with anhydrous sodium sulfate solid, and the solvent was removed by rotary evaporation to obtain compound 3 (6-morpholine-2-(2-piperazineethyl)-1-hydrogen-benzoisoquinoline-1,3(2-hydrogen)-dione). The synthetic route of compound 3 is shown below:

[0013]

[0014] S3: Synthesis of probe M: Dissolve carboxylic acid coumarin and HBTU in anhydrous DMF solution, then add compound 3 and diisopropylamine in sequence to obtain a mixed solution; stir the mixed solution at room temperature under the protection of an inert gas to react; after the reaction is completed, remove most of the solvent by rotary evaporation to obtain a crude product, which is then purified by silica gel column chromatography to obtain a clean product probe M. The synthesis route of probe M is as follows:

[0015]

[0016] Furthermore, in step S1, the mass of compound 1 is 2.5 g, the mass of morpholine is 1.57 g, the volume of ethylene glycol methyl ether is 100 ml, and the reflux time is 24 h.

[0017] Furthermore, in step S2, the mass of compound 2 is 2.00 g, the mass of N-aminopiperidine is 1.28 g, the volume of ethanol is 30 mL, the inert gas is N2, and the reflux time is 16 h.

[0018] Furthermore, in step S3, the mass of carboxylic acid coumarin is 0.289 g, the mass of HBTU is 0.29 g, the volume of anhydrous DMF solution is 50 mL, the mass of compound 3 is 0.2 g, the volume of diisopropylamine is 0.11 mL, the inert gas is N2, and the stirring reaction time is 18 h.

[0019] An application of the above-mentioned coumarin-based small molecule fluorescent probe M in pH detection.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] In the present invention, probe M is based on the specific fluorescence of coumarin, whose carboxyl and hydroxyl groups can bind to hydrogen ions in the solution, causing the fluorescence intensity to change, thereby detecting pH. Probe M has a specific fluorescence for H in the pH range of 2.0-11.0. + There is good selectivity and sensitivity. In the range of pH value of 3.0-6.5, its fluorescence intensity has a good linear relationship with pH value, and the pH value can be accurately measured. Moreover, probe M still has good selectivity and anti-interference in the presence of common different metal ions and amino acids, and also possesses good reversibility and time stability. The synthesized probe M in the present invention has strong selectivity and anti-interference, high sensitivity, strong reversibility, and can be used for the detection of pH. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The UV-visible spectra of the probe M in solutions of different pH values ​​(2.0-11.0) in the present invention;

[0023] Figure 2 a) Fluorescence spectra of probe M in solutions of different pH values ​​(2.0-10.0) in the present invention, and b) fluorescence intensity ratio I 455 nm / I 558 Scatter plot of nm versus pH;

[0024] Figure 3 Schematic diagram of the response mechanism of probe M to pH in the present invention;

[0025] Figure 4 a) Fluorescence spectra of probe M in solutions with different pH values ​​(4.5-6.5) and b) fluorescence intensity ratio I 455 nm / I 558 Linear plot of nm versus pH;

[0026] Figure 5 is the fluorescence intensity ratio I of the probe M in different pH (2.0-10.0) solutions in the present invention. 558 nm / I 455 Scatter plot of nm versus pH;

[0027] Figure 6 a) Fluorescence spectra of probe M in solutions of different pH values ​​(3.0-5.0) in the present invention, and b) fluorescence intensity ratio I 558 nm / I 455 Linear plot of nm versus pH;

[0028] Figure 7 is the fluorescence intensity ratio of the probe M in the present invention (I 455nm / I 558 nm) after adding other ions at pH = 7.4;

[0029] Figure 8 a) Fluorescence spectrum of probe M at pH = 5.5 and pH = 7.4 in the present invention, and b) fluorescence intensity ratio I 558 nm / I 455 Reversibility results graph of nm;

[0030] Fig. 9 It is a scatter plot of the two-photon absorption cross-section values ​​of the probe M in the present invention in solutions with pH values ​​of 4.5 and 7.4 under different two-photon wavelength excitation. DETAILED DESCRIPTION

[0031] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0033] The reagents used in this example are all commercially available analytical grade chemicals (AR).

[0034] Instruments used in this example:

[0035] Nuclear magnetic resonance instrument (JEOLBCS 400M), mass spectrometer (Finnigan MAT LCQ), digital pH meter (pH-10C), UV-visible-near infrared spectrophotometer (Agilent cary 5000), fluorescence spectrometer (SpectrofiuorometerFS5).

[0036] Example 1

[0037] This embodiment provides a small molecule fluorescent probe M based on coumarin, and the probe M is 2-2-(2-(4-(7-hydroxy-2-oxo-2-hydro-benzopyran-3-carbonyl)piperazine)ethane)-6-morpholine-1-hydro-benzoisoquinoline-1,3(2-hydro)dione, and its structural formula is:

[0038]

[0039] This embodiment also provides a method for preparing the above-mentioned coumarin-based small molecule fluorescent probe M, which specifically comprises the following steps:

[0040] S1: Synthesis of compound 2: 2.5 g of compound 1 (6-bromo-1,3-dihydro-benzoisoquinoline-1,3-dione) and 1.57 g of morpholine were refluxed in 100 ml of ethylene glycol methyl ether under N2 protection for 24 h. The reaction solution was cooled to room temperature overnight. After crystals precipitated, they were filtered off under reduced pressure and dried to obtain compound 2 (6-morpholine-1,3-dihydro-benzoisoquinoline-1,3-dione). The synthesis route of compound 2 is as follows:

[0041]

[0042] S2: Synthesis of compound 3: 2.00g of compound 2 and 1.28g of N-aminopiperidine were dissolved in 30mL of ethanol and refluxed under N2 protection for 16h; after the reaction was completed, the excess solvent was removed by rotary evaporator to obtain a crude product; the product was then dissolved in chloroform, washed with saturated NaCl solution 3 times, the organic phase was collected, and then dried with anhydrous sodium sulfate solid, and the solvent was removed by rotary evaporation to obtain compound 3 (6-morpholine-2-(2-piperazineethyl)-1hydrogen-benzissoquinoline-1,3(2hydrogen)-dione). The synthetic route of compound 3 is as follows:

[0043]

[0044] S3: Synthesis of probe M: 0.289 g of carboxylic acid coumarin and 0.29 g of peptide coupling reagent HBTU were dissolved in 50 mL of anhydrous N, N-dimethylformamide (DMF) solution, and 0.2 g of compound 3 and 0.11 mL of diisopropylamine were added in sequence to obtain a mixed solution; the mixed solution was stirred at room temperature for 18 h under the protection of N2; after the reaction was completed, most of the solvent was removed by rotary evaporation to obtain a crude product, which was then purified by silica gel column chromatography to obtain a clean product probe M. The synthetic route of probe M is as follows:

[0045]

[0046] 1 HNMR(400MHz)δ8.45-8.42(1H,m),8.39(2H,d,J=3.5),8.22(2H,d,J=8.3),7.77(1H,t,J=8.0),7.52(1H,d,J=8.4), 6.81(1H,t,J=10.7),6.72(1H,s),3.22(4H,d,J=31.8),3.02(9H,q,J=7.2),2.45(5H,d,J=14.5).13CNMR(101MHz)δ

[0047] 164.05,163.88,163.24,158.55,156.21,148.51,139.89,135.01,132.73,131.21,130.83,128.01,126.62,125.78,12 3.04,119.67,116.30,115.59,114.35,111.04,102.61,66.72,55.54,53.55,46.05,41.94.ESI-MSm / z[(M+H)+]:605.4.

[0048] This embodiment also provides an application of the above-mentioned coumarin-based small molecule fluorescent probe M in pH detection.

[0049] The probe M prepared in the above embodiment was tested as follows:

[0050] Preparation of test solution:

[0051] Probe storage solution (1 mmoL / L): Probe M can be prepared by dissolving it in DMSO (dimethyl sulfoxide).

[0052] Other cationic analyte solutions (10mmoL / L): CaCl2, KCl, ZnCl2, NaNO3, Na2SO3, NaClO and FeSO4 were dissolved in pure water. Small molecule analyte solution (10mmoL / L): GSH (glutathione), Cys (cysteine), Hcy (homocysteine), Na2S and H2O2 were dissolved in PBS buffer solution (pH = 7.4). The above solution ions were used in the subsequent probe selectivity test.

[0053] Tris buffer solution: Weigh tris (hydroxymethyl)aminomethane (Tris) and dissolve it in pure water, add appropriate amount of HCl or NaOH, adjust the pH with a pH meter, and prepare 20 mL of each solution with a pH of 2-11.

[0054] 1. Ultraviolet spectrum test

[0055] The UV-visible spectra of 5 μM probe M were measured in different pH solutions (pH=2.0-11.0). The UV-visible spectra were obtained by testing with an Agilentcary5000 UV-visible-near infrared spectrophotometer.

[0056] like Figure 1As shown in the figure, as the solution pH increases from 2.0 to 11.0, the absorption peak of probe M at 342nm decreases, while a new absorption peak is generated at 390nm and gradually increases, with its isosbestic point at 356nm. Since the typical absorption peak of coumarin is at 342nm, and the absorption peak of naphthaleneimide is at 390nm, the experimental results also show that as the pH increases, the luminescence shifts from coumarin to naphthaleneimide.

[0057] 2. Fluorescence spectral response

[0058] The spectrum test experiment of probe M was carried out. Different pH solutions were prepared by adjusting Tris buffer solution and dilute hydrochloric acid solution or sodium hydroxide solution. The fluorescence spectrometer was used for measurement, and the excitation wavelength was set to 390 nm and the slit width was set to 5.0 / 2.5 nm.

[0059] like Figure 2 As shown, with the increase of pH (2.0-10.0), the emission peak of probe M at 455nm is significantly enhanced, which is the characteristic emission peak of coumarin; and the emission peak at 558nm is slightly enhanced, which is the characteristic emission peak of naphthaleneimide. This result shows that under 390nm excitation, the energy resonance transfer effect between coumarin and naphthaleneimide is not ideal.

[0060] Based on the above studies, the response mechanism of probe M to pH can be obtained, such as Figure 3 As shown. According to the FRET principle, after the probe M is excited, the energy is transferred from the donor coumarin part to the acceptor naphthalimide part, that is, the fluorescence emission of the donor coumarin will weaken as the FRET effect occurs. However, according to Figure 5 Studies have shown that under 390nm excitation, probe M exhibits strong blue fluorescence emission provided by the coumarin part and weak green fluorescence emission provided by the naphthaleneimide part, and as the pH increases, the fluorescence intensity of the coumarin part increases rapidly, which is contrary to the FRET mechanism. A reasonable explanation for this phenomenon is that the ICT effect has a dominant influence, which enhances the fluorescence intensity of coumarin. Therefore, as the pH increases, the fluorescence emission of both the energy donor (coumarin) and the energy acceptor (naphthaleneimide) is enhanced.

[0061] 3. Probe M for quantitative detection of pH

[0062] The properties of probe M for quantitative detection of pH were studied. Figure 2 -b) shows that the fluorescence intensity ratio R(I 455 nm / I 558 nm) increased from 3.14 to 33.94 with pH change (4.0-8.0), an increase of 11 times. Within this pH range, the linear relationship between the two was studied, such as Figure 4As shown, R value (I 455 nm / I 558 nm) has a good linear correlation in the pH range of 4.5-6.5. 2 =0.9928.

[0063] In addition, if Figure 5 As shown in Figure 2, when the pH range decreases from 10.0 to 2.0, the fluorescence intensity of probe M is inversely proportional to R'(I 558 / I 455 ) increases. And the R' value shows a good linear relationship between pH 3.0 and 5.0. 2 =0.9912( Figure 6 ). Therefore, by these two ratiometric data analysis methods, probe M can have a good linear response in a wide pH range (pH 3.0-6.5); including the pH of lysosomes (4.5-5.5).

[0064] 4. Study on the selectivity of probe M

[0065] At pH = 7.4, 25 μM of the above test solution ions (H + , Ca 2+ , ClO - 、Cys、Fe 3+ , GSH, H2O2, Hcy, K + 、Na2S、NO3 - 、SO3 2- 、Zn 2+ ), the selectivity of probe M was studied.

[0066] like Figure 7 As shown in Figure 2, the fluorescence intensity ratio of probe M was not affected by the addition of other biologically relevant analytes (including Ca 2+ , ClO - 、Cys、Fe 3+ , GSH, H2O2, Hcy, K + 、Na2S、NO3 - 、SO3 2- 、Zn 2+ ) and has obvious changes. This result shows that the fluorescence value of the fluorescent probe will not be interfered by other metal ions and amino acids, but will only change with H + The selectivity of probe M is higher.

[0067] 5. Reversibility study of probe M

[0068] Reversibility experiments were performed in different pH solutions (pH=5.5 and 7.4).

[0069] like Figure 8As shown in the figure, probe M has at least 5 good reversible cycles under pH 5.5 and pH 7.4. On this basis, probe M can be used to reversibly detect the pH of cell lysosomes without interference.

[0070] 6. Two-photon absorption cross section measurement

[0071] The two-photon cross-section values ​​of probe M in solutions with pH values ​​of 4.5 and 7.4 were studied using two-photon excitation fluorescence spectroscopy.

[0072] like Fig. 9 As shown in the figure, at pH 7.4, the maximum two-photon cross-section value of probe M is 74.2GM under 790nm wavelength excitation, and at pH 4.5, the maximum two-photon cross-section value of probe M is 35.5GM under 770nm wavelength excitation, and the quantum yield of the probe increases from 18.1 to 4.36. This result shows that probe M can be used as a two-photon fluorescent probe for further bioluminescence imaging under 790nm excitation.

[0073] Based on the above, it can be seen that the probe M of this embodiment is based on the specific fluorescence of coumarin, and its carboxyl and hydroxyl groups can combine with hydrogen ions in the solution to change the fluorescence intensity, thereby detecting pH. Probe M has a specific fluorescence for H in the pH range of 2.0-11.0. + It has good selectivity and sensitivity. In the pH range of 3.0-6.5, its fluorescence intensity has a good linear relationship with the pH value, and the pH value can be accurately measured. Moreover, probe M still has good selectivity and anti-interference in the presence of common different metal ions and amino acids, and also has good reversibility and time stability.

[0074] In summary, the probe M synthesized in this example is a small molecule fluorescent probe with strong selectivity, strong anti-interference, high sensitivity, strong reversibility, and can be used to detect pH.

[0075] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A small molecule fluorescent probe M based on coumarin, characterized in that: The structural formula of the probe M is:

2. A method for preparing a coumarin-based small molecule fluorescent probe M as claimed in claim 1, characterized in that: The specific steps include: S1: Synthesis of Compound 2: Compound 1 and morpholine were refluxed in ethylene glycol methyl ether under N2 protection, and the reaction solution was cooled to room temperature overnight. After crystals precipitated, they were filtered under reduced pressure and dried to obtain Compound 2. The synthesis route of Compound 2 is as follows: S2: Synthesis of compound 3: Compound 2 and N-aminopiperidine were dissolved in ethanol and refluxed under the protection of inert gas; after the reaction was completed, the excess solvent was removed by rotary evaporator to obtain a crude product; the product was then dissolved in chloroform, washed with saturated NaCl solution for 3 times, the organic phase was collected, and then dried with anhydrous sodium sulfate solid, and the solvent was removed by rotary evaporation to obtain compound 3. The synthetic route of compound 3 is shown below: S3: Synthesis of probe M: Dissolve carboxylic acid coumarin and HBTU in anhydrous DMF solution, then add compound 3 and diisopropylamine in sequence to obtain a mixed solution; stir the mixed solution at room temperature under the protection of an inert gas to react; after the reaction is completed, remove most of the solvent by rotary evaporation to obtain a crude product, which is then purified by silica gel column chromatography to obtain a clean product probe M. The synthesis route of probe M is as follows:

3. The method for preparing a coumarin-based small molecule fluorescent probe M according to claim 2, characterized in that: In the step S1, the mass of compound 1 is 2.5 g, the mass of morpholine is 1.57 g, the volume of ethylene glycol methyl ether is 100 ml, and the reflux time is 24 h.

4. The method for preparing a coumarin-based small molecule fluorescent probe M according to claim 2, characterized in that: In step S2, the mass of compound 2 is 2.00 g, the mass of N-aminopiperidine is 1.28 g, the volume of ethanol is 30 mL, the inert gas is N2, and the reflux time is 16 h.

5. The method for preparing a coumarin-based small molecule fluorescent probe M according to claim 2, characterized in that: In the step S3, the mass of carboxylic acid coumarin is 0.289 g, the mass of HBTU is 0.29 g, the volume of anhydrous DMF solution is 50 mL, the mass of compound 3 is 0.2 g, the volume of diisopropylamine is 0.11 mL, the inert gas is N2, and the stirring reaction time is 18 h.

6. Use of the coumarin-based small molecule fluorescent probe M as claimed in claim 1 in pH detection.

Images