CN112480052B

CN112480052B - Ratio type near-infrared fluorescent probe for detecting pH, preparation method and application

Info

Publication number: CN112480052B
Application number: CN202011444382.9A
Authority: CN
Inventors: 秦涛; 冯丽恒
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2022-05-31
Anticipated expiration: 2040-12-08
Also published as: CN112480052A

Abstract

The invention relates to a ratio-type near-infrared fluorescent probe for detecting pH, in particular to a ratio-type near-infrared fluorescent probe for detecting pH, a preparation method and application thereof. The invention provides a ratio type near-infrared fluorescent probe containing a benzopyran structure and used for detecting pH. Specifically, 7-hydroxy-3-carboxyl coumarin is used as a raw material, and the 7-hydroxy-3-formyl chloride coumarin is obtained through an acyl chlorination reaction; and carrying out amidation reaction on the 7-hydroxy-3-formyl chloride coumarin and (E) -2- (2- (4-aminostyryl) -4H-chromene-4-methylene) malononitrile to obtain the near-infrared fluorescent probe PCHC containing chromene and coumarin. The near-infrared fluorescent Probe (PCHC) obtained by the invention can emit different lights under different pH conditions, realizes ratio detection on pH according to the change of fluorescence, effectively avoids background interference, and has good application value in the aspect of pH detection of an actual sample. The adopted synthetic route is short, the preparation method is simple to operate, and the reaction conditions are mild.

Description

Ratio type near-infrared fluorescent probe for detecting pH, preparation method and application

Technical Field

The invention relates to a ratio-type near-infrared fluorescent probe for detecting pH, in particular to a ratio-type near-infrared fluorescent probe containing a benzopyran structure and used for detecting pH, a preparation method and application thereof.

Background

pH is an important parameter in many fields. The pH of the living environment is a very important physiological parameter, which plays a crucial role in a series of tissue activities, such as enzymatic activity, cell proliferation and apoptosis, drug resistance, ion transport, endocytosis and muscle contraction. Abnormal pH is an indicator of the occurrence of many common diseases, such as cancer, stroke, and alzheimer's disease, among others. In addition, the determination of the pH of industrial waste water, soil, pharmaceuticals, and food is of great importance to the fields of industrial, agricultural, pharmaceutical, and food safety inspection. Therefore, it is important to accurately detect the pH change in the sample.

The fluorescence spectrum method is used for measuring the pH value by utilizing the change of fluorescence parameters such as fluorescence intensity, fluorescence lifetime and the like, has the advantages of high sensitivity, good selectivity, high response speed, high signal-to-noise ratio and the like, and more importantly, can monitor the change of the pH value of a sample in situ in real time, so that the fluorescence probe for detecting the pH value is developed rapidly. However, many pH fluorescent probes have reported a small pH range so far, and few fluorescent probes capable of detecting pH in an alkaline environment have been reported. However, in some samples in actual tests, a wider range of pH values is required for the test. The limited range of pH detection makes many fluorescent probes unable to meet practical requirements, and other detection methods are required to detect pH. In addition, many test samples emit fluorescence by themselves under excitation by visible light, which severely interferes with fluorescence detection and visualization of the sample. The maximum emission wavelength of the near-infrared fluorescent probe is 650-900nm, so that background interference can be avoided. However, benzopyran-based Near Infrared (NIR) fluorescence probes (absorption and emission in the range of 650-900 nm) have been reported only rarely. Therefore, the benzopyran near-infrared fluorescent probe for detecting the wide-range pH is designed and synthesized, and has important significance in pH detection.

Disclosure of Invention

In view of the above problems, the present invention provides a ratiometric near-infrared fluorescent probe for detecting pH, which can detect the pH of a sample by the change of emission wavelength under different pH conditions. The preparation method is simple to operate and mild in reaction conditions.

In order to achieve the purpose, the invention adopts the following technical scheme:

a ratiometric near infrared fluorescent Probe (PCHC) for detecting pH, having the formula:

a method for preparing a ratiometric near infrared fluorescent Probe (PCHC) for detecting pH comprises the following steps:

step 1, in N₂Under protection, 7-hydroxy-3-carboxycoumarin and thionyl chloride are mixed and subjected to heating reflux reaction to obtain 7-hydroxy-3-formyl chloride coumarin; the reaction formula is as follows:

step 2, in N₂Under protection, (E) -2- (2- (4-aminostyryl) -4H-chromene-4-methylene) malononitrile and potassium carbonate are mixed, then an anhydrous toluene solution in which the 7-hydroxy-3-formyl chloride coumarin obtained in the step 1 is dissolved is added, heating reflux reaction is carried out, after the reaction is finished, the solvent and the column are removed by rotation, the product is washed by water, suction filtration is carried out until no blue fluorescence exists in the water, the solid in the filter cake is collected, vacuum drying is carried out, and the ratio type near infrared fluorescence probe containing chromene and coumarin for detecting pH is obtained, namely (E) -N- (4- (2- (4-propanedicyanomethylene) -4H-chromen-2-yl) vinyl) phenyl) -7-hydroxy-3-carboxamidocoumarin; the reaction formula is as follows:

further, the dosage of the 7-hydroxy-3-carboxycoumarin in the step 1 is 0.50-5.00mmol, and the dosage of the thionyl chloride is 5-20 mL.

Further, the heating reflux reaction temperature in the step 1 is 80-100 ℃, and the heating reflux reaction time is 4-8 h.

Further, in the step 2, the dosage of the (E) -2- (2- (4-aminostyryl) -4H-chromene-4-methylene) malononitrile is 1.00 to 3.00mmol, the dosage of the potassium carbonate is 2.00 to 6.00mmol, and the dosage of the anhydrous toluene is 5 to 30 mL.

Further, the temperature of the heating reflux reaction in the step 2 is 110-.

An application of a ratio type near infrared fluorescent probe for detecting pH in the aspect of pH detection.

The pH detection range of the ratio type near infrared fluorescent probe for detecting the pH is 4.0-9.0.

Compared with the prior art, the invention has the following advantages:

the near-infrared fluorescent Probe (PCHC) obtained by the invention takes benzopyran as a skeleton structure, takes 7-hydroxycoumarin as an electron donating group, takes styryl as a conjugated connecting group, and takes dicyanomethylene-4H-chromene as an electron withdrawing group to form a D-pi-A type large conjugated structure. The coumarin has excellent optical and biological properties such as high fluorescence quantum yield, good light stability, high color development intensity, wide chromatographic coverage range, good biocompatibility and the like, and the near infrared fluorescent Probe (PCHC) obtained by connecting the coumarin and the near infrared chromophore dicyanomethylene-4H-chromene has thermal, optical and chemical stability, large molar extinction coefficient and wide-range spectral absorption. The near-infrared fluorescent Probe (PCHC) obtained by the invention can emit different lights under different pH conditions, realizes the ratio detection of the pH according to the change of fluorescence, effectively avoids the interference of the background, and has better application value in the aspect of the pH detection of an actual sample. The adopted synthetic route is short, the preparation method is simple to operate, and the reaction conditions are mild.

Drawings

FIG. 1 is a UV absorption spectrum of the fluorescent probe PCHC of the present invention with pH change.

FIG. 2 is a fluorescence emission spectrum of the fluorescent probe PCHC of the present invention with pH.

FIG. 3 is a graph showing the anti-interference ability of the fluorescent probe PCHC of the present invention to various ions and bioactive small molecules in BR buffer solution with pH values of 5.0, 7.0 and 8.0. Wherein the abscissa 0 to 19 represents: blank group, Mg²⁺、Cd²⁺、Ca²⁺、Co² ⁺、K⁺、Cu²⁺、Ag⁺、Zn²⁺、Fe³⁺、Fe²⁺Sodium nitrite, Vc, L-cysteine, glutathione, glucose, sodium hypochlorite and Cl^-L-glutamic acid, L-arginine.

FIG. 4 is a graph showing the color change of the fluorescent probe PCHC of the present invention in solutions of different pH values.

FIG. 5 is a reversibility test chart of the fluorescent probe PCHC of the present invention at a pH value between 4.0 and 7.0.

FIG. 6 is a reversibility test chart of the fluorescent probe PCHC of the present invention at a pH value between 7.0 and 9.0.

Detailed Description

The technical solution of the present invention will be specifically and specifically described below with reference to the embodiments of the present invention and the accompanying drawings.

Example 1

Preparation of a ratiometric near infrared fluorescent Probe (PCHC) for detecting pH:

1. in a 50mL three-necked round-bottomed flask equipped with magnetons, 392mg (1.90mmol) of 7-hydroxy-3-carboxycoumarin was added, and then nitrogen gas was introduced three times under vacuum, 10mL of thionyl chloride was added, followed by heating and reflux reaction at 90 ℃ for 6 hours. And (3) after the reaction is finished, reducing pressure and removing the solvent to obtain a crude product of the 7-hydroxy-3-formyl chloride coumarin, and directly carrying out the next reaction without further purification.

2. Into a 50mL three-necked round-bottomed flask equipped with magnetons were charged 590mg (1.90mmol) of (E) -2- (2- (4-aminostyryl) -4H-chromen-4-methylene) malononitrile and 525mg (3.80mmol) of K₂CO₃Vacuumizing and introducing nitrogen for three times, adding 10mL of anhydrous toluene into the crude acyl 7-hydroxy-3-formyl chloride coumarin which is spun out of the solvent to dissolve, quickly adding into a three-neck round-bottom flask, and carrying out reflux reaction for 16 hours at 120 ℃. And (3) after the reaction is finished, removing the solvent in a rotating way, enabling pure dichloromethane to pass through a column, enabling pure ethyl acetate to pass through the column, removing the solvent in a rotating way, washing the product with water, performing suction filtration until blue fluorescence does not exist in water, collecting the solid in the filter cake, and performing vacuum drying to obtain a dark red solid, namely 93mg of the near infrared fluorescent probe (E) -N- (4- (2- (4-propylene dicyanomethylene) -4H-chromen-2-yl) vinyl) phenyl) -7-hydroxy-3-formamido coumarin (PCHC), wherein the yield is 9.8%.¹H NMR(400MHz,DMSO)δ11.19(s,1H),10.84(s,1H),8.87(s,1H),8.72(d,J＝8.4Hz,1H),7.93(t,J＝8.4Hz,1H),7.86(d,J＝8.7Hz,1H),7.80(t,J＝9.6Hz,4H),7.74(d,J＝11.0Hz,1H),7.69(s,1H),7.61(t,J＝7.8Hz,1H),7.44(d,J＝16.0Hz,1H),7.00(s,1H),6.90(d,J＝8.6Hz,1H),6.83(s,1H)；HRMS-ESI for C₃₀H₁₇N₃O₅(m/z)500.1235[M+H]⁺。

Example 2

Test experiment for near infrared fluorescent Probe (PCHC) detection of pH:

the fluorescent Probe (PCHC) prepared in example 1 was dissolved in DMSO to prepare a stock solution of the fluorescent probe at 1 mmol/L. Acetic acid, phosphoric acid, boric acid and sodium hydroxide are respectively used for preparing pH: 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 pH buffer solution (BR buffer solution). Adding fluorescent probe stock solution into buffer solutions with different pH values to make the final concentration of the fluorescent probe stock solution be 10.0 μ M, and obtaining ultraviolet absorption spectrum and fluorescence emission spectrum after 30min (the excitation is 415.0nm, and the widths of the excitation slit and the emission slit are both 10.0 nm). As shown in FIG. 1, the near infrared fluorescent Probe (PCHC) absorption spectrum is gradually red-shifted with increasing pH. As shown in FIG. 2, the fluorescence intensity of the near infrared fluorescent Probe (PCHC) was gradually increased at a wavelength of 453.0nm with an increase in pH. As the pH increased, the fluorescence intensity of the near-infrared fluorescent Probe (PCHC) gradually decreased at a wavelength of 662.0 nm.

Example 3

Near infrared fluorescent Probe (PCHC) for anti-ion interference performance test of pH detection:

2mL of BR buffer solutions with pH values of 5.0, 7.0 and 8.0 were put into a four-way cuvette and added to the stock solutions of the fluorescent probes prepared in example 2. Then adding various ions and biologically related active small molecules (Mg) respectively²⁺、Cd²⁺、Ca²⁺、Co²⁺、K⁺、Cu²⁺、Ag⁺、Zn²⁺、Fe³⁺、Fe²⁺Sodium nitrite, Vc, L-cysteine, glutathione, glucose, sodium hypochlorite and Cl^-L-glutamic acid, L-arginine) so that the final concentrations of the fluorescent probe and the interfering molecule were 10. mu. mol/L and 50. mu. mol/L, respectively, and the fluorescence values were measured after 0.5 hour reaction with the fluorescent probe set alone as a blank (excitation: 415.0nm, and widths of both excitation slit and emission slit: 10.0 nm). As shown in FIG. 3, the probe of the present invention has good anti-interference ability.

Example 4

Fluorescence photograph of near infrared fluorescent Probe (PCHC) as a function of pH:

BR buffer solutions with different pH values of 4.0, 5.5, 6, 6.5, 7.0, 8.0, and 9.0 were adjusted, and the fluorescent probe stock solutions prepared in example 2 were added, respectively, as shown in fig. 4, the red light was gradually reduced and the blue light was gradually increased with the increase of pH.

Example 5

Reversibility test for pH detection of near infrared fluorescent Probe (PCHC):

the stock solutions of the fluorescent probes prepared in example 2 were added to a four-way cuvette, the pH was adjusted to vary between 4.0 and 7.0, and between 7.0 and 9.0, and the change in fluorescence intensity was recorded separately (excitation at 415.0nm, excitation slit and emission slit width each 10.0 nm). As shown in fig. 5, 6, indicating that the process can be performed reversibly for at least five cycles. The fluorescence probe of the invention is shown to have good reversibility to the response of pH.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and it will be apparent to those skilled in the art that several modifications and improvements can be made without departing from the principle of the present invention, and all of them are included in the protection scope of the present invention.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A ratio type near-infrared fluorescent probe for detecting pH is characterized in that the structural formula of the near-infrared fluorescent probe is as follows:

。

2. the method for preparing a ratiometric near infrared fluorescent probe for detecting pH according to claim 1, comprising the steps of:

step 1, in N₂Under protection, 7-hydroxy-3-carboxycoumarin and thionyl chloride are mixed and subjected to heating reflux reaction to obtain 7-hydroxy-3-chloroformyl coumarin;

step 2, in N₂Under protection, (E) -2- (2- (4-aminostyryl) -4H-chromene-4-methylene) malononitrile and potassium carbonate are mixed, then an anhydrous toluene solution in which the 7-hydroxy-3-chloroformyl coumarin obtained in the step 1 is dissolved is added, heating reflux reaction is carried out, after the reaction is finished, the solvent is removed by rotation, the solvent is passed through a column, the solvent is removed by rotation, the product is washed by water and is filtered until blue fluorescence does not exist in the water, solids in a filter cake are collected, and vacuum drying is carried out to obtain the pH detection ratio type near infrared fluorescence probe containing chromene and coumarin according to claim 1, namely, (E) -N- (4- (2- (4-malononitrile) -4H-chromene-2-yl) vinyl) phenyl) -7-hydroxy-3-carbamyl coumarin And (4) element.

3. The method for preparing a ratiometric near-infrared fluorescent probe for detecting pH according to claim 2, wherein: in the step 1, the dosage of the 7-hydroxy-3-carboxycoumarin is 0.50-5.00mmol, and the dosage of the thionyl chloride is 5-20 mL.

4. The method for preparing a ratiometric near-infrared fluorescent probe for detecting pH of claim 2, which comprises: the temperature of the heating reflux reaction in the step 1 is 80-100 ℃, and the time of the heating reflux reaction is 4-8 h.

5. The method for preparing a ratiometric near-infrared fluorescent probe for detecting pH according to claim 2, wherein: in the step 2, the dosage of the (E) -2- (2- (4-aminostyryl) -4H-chromene-4-methylene) malononitrile is 1.00 to 3.00mmol, the dosage of the potassium carbonate is 2.00 to 6.00mmol, and the dosage of the anhydrous toluene is 5 to 30 mL.

6. The method for preparing a ratiometric near-infrared fluorescent probe for detecting pH according to claim 2, wherein: the temperature of the heating reflux reaction in the step 2 is 110-130 ℃, and the time of the heating reflux reaction is 12-20 h.

7. The method for preparing a ratiometric near-infrared fluorescent probe for detecting pH according to claim 2, wherein: and in the step 2, pure dichloromethane is firstly used for column chromatography, and then pure ethyl acetate is used for column chromatography.

8. The use of the ratiometric near-infrared fluorescent probe for detecting pH of claim 1 for pH detection.

9. The use of the ratiometric near-infrared fluorescent probe for detecting pH of claim 8, wherein: the pH detection range is 4.0-9.0.