CN108912084B

CN108912084B - Carbon monoxide fluorescent probe and preparation method and application thereof

Info

Publication number: CN108912084B
Application number: CN201810575253.XA
Authority: CN
Inventors: 林伟英; 高世滢; 唐永和
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2018-06-06
Filing date: 2018-06-06
Publication date: 2020-04-17
Anticipated expiration: 2038-06-06
Also published as: CN108912084A

Abstract

The invention provides a two-photon fluorescent probe for detecting CO, and a structure thereofThe formula is as follows:

. The probe provided by the invention can sensitively detect the existence of CO in a solution, particularly a cell; can react with CO in cells at low concentration, and can resist the interference of various active oxygen, amino acid and sulfhydryl-containing compounds. The CO-3 fluorescent probe has two-photon properties, and can avoid photobleaching and phototoxicity phenomena; has wide application prospect in the field of biomolecule detection.

Description

Carbon monoxide fluorescent probe and preparation method and application thereof

Technical Field

The invention relates to a sub-fluorescent probe for detecting CO in cells and application thereof, belonging to the field of organic small molecule fluorescent probes.

Background

Carbon monoxide (CO) has been studied for decades to demonstrate one of the important biological signaling molecules that can compete with oxygen in the body to bind hemoglobin, thereby preventing the normal transport of oxygen. If excessive intake of exogenous carbon monoxide can cause carbon monoxide poisoning in living organisms, carbon monoxide has long been recognized as merely a toxic and harmful substance, and whether endogenous carbon monoxide is actually present in the body and whether endogenous carbon monoxide has a physiological effect has been overlooked by people. Until 1949, Sjostrand found that endogenous carbon monoxide can be produced in humans; subsequently in 1987, Ullrich and Brune discovered that carbon monoxide can inhibit platelet aggregation by activating Guanylate Cyclase (GC). On the basis, Marks and the like speculate that endogenous carbon monoxide has a certain physiological function in organisms possibly; in 1993, Verma et al adopt an in situ hybridization research method to find that structural HO (HO-2) mRNA is widely distributed in the brain tissue of rats and has a high co-expression phenomenon with soluble GCm RNA, so that the research proves that endogenous carbon monoxide really has certain physiological effect in the body. Since then, carbon monoxide is considered to be both a gas molecule with toxic effects and a gas signaling molecule with specific biological activity. With the intensive research on the action of carbon monoxide in organisms, the carbon monoxide is found to be an important nerve transmission medium and participate in a plurality of important biological processes such as cardiovascular regulation, respiratory regulation, thermoregulation and the like in organisms. Thus, techniques and methods have been developed for the detection of the biologically active molecule carbon monoxide in biological samples with high sensitivity, rapidity and high selectivity.

Currently, many experimental methods have been developed for the analysis of the bioactive molecule carbon monoxide in environmental and biological samples, such as electrochemical methods, colorimetric methods, and infrared laser absorption spectroscopy. However, in view of the "superior" stability of the chemical properties of carbon monoxide molecules, the currently developed carbon monoxide fluorescence analysis methods based on bioactive molecules still yield several figures. Although some progress has been made in fluorescent probes based on carbon monoxide analysis, there is a certain distance from the requirements of practical application, and spectral properties such as sensitivity, long absorption and emission wavelength of the probe are still going to be further improved. Therefore, it is necessary to develop a fluorescent probe capable of recognizing carbon monoxide.

Disclosure of Invention

Aiming at the problems of long reaction time, short excitation wavelength and small Stokes displacement of the existing probe to carbon monoxide, the invention provides the fluorescent probe which has sensitive reaction, low detection limit, good specificity and two-photon property and can detect the carbon monoxide in cells.

Another object of the present invention is to provide a method for easily synthesizing the above fluorescent probe.

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

A fluorescent probe for detecting carbon monoxide has a chemical name of 7-diethylamino-3-allyl carbonate coumarin, CO-3 for short, and a structural formula shown in formula (I):

formula (I).

A method for synthesizing the fluorescent probe comprises the following steps:

(1) heating 7-diethylamino-3-hydroxycoumarin (1) in HCl solution for reaction to synthesize 7-diethylamino-3-hydroxycoumarin (2);

(2) stirring 7-diethylamino-3-hydroxycoumarin (2) and allyl chloride (3) in dichloromethane to react to obtain the final product, namely 7- (diethylamino) -2-oxo-2H-chromen-3-yl acrylate.

The molar ratio of the 7-diethylamino-3-hydroxycoumarin to the HCl is 1: 30.

The concentration of the HCl solution is 1-1.5 mol/L.

In the step (1), the reaction temperature is 100 ℃; the reaction time is 3-4 h.

The mol ratio of the 7-diethylamino-3-hydroxycoumarin to the allyl chloride is 1: 1-2.

In the step (2), the reaction temperature is 25 ℃, and the reaction time is 12-20 h.

The step (2) also comprises the step of separating and purifying the product: the reaction mixture was evaporated under reduced pressure to remove the solvent, and the dried solid was subjected to silica gel column chromatography with a eluent of dichloromethane/petroleum ether (V/V =5: 1).

The synthetic route is as follows:

；

。

the fluorescent probe is used for detecting the content of CO in solution and cells by single photon or two-photon fluorescence.

The mechanism of the fluorescent probe recognition in the invention is as follows:

according to the fluorescent probe for detecting CO, molecular fluorescence is quenched due to the strong electron pulling capacity of allyl acetate and coumarin structures, when the probe and CO molecules act, the compound CO-3 is reduced to 7-diethylamino-3-hydroxycoumarin, the ICT effect is enhanced due to the strong electron pushing and pulling effect of the hydroxyl groups and the coumarin structures, so that the fluorescence intensity is greatly improved, and the CO is identified in a fluorescence enhancement mode.

The recognition reaction is as follows:

。

the invention has the following advantages:

the CO-3 fluorescent probe can sensitively detect the existence of CO in a solution, particularly cells; can react with CO in cells at low concentration, and can resist the interference of various active oxygen, amino acid and sulfhydryl-containing compounds. The CO-3 fluorescent probe has two-photon properties, and can avoid photobleaching and phototoxicity phenomena; has wide application prospect in the field of biomolecule detection.

Drawings

FIG. 1 is a representation of CO-3¹H NMR spectrum;

FIG. 2 shows the selectivity of CO-3 fluorescent probes for different molecules or ions;

FIG. 3 is a graph showing the fluorescence intensity of CO-3 at different concentrations of CO;

FIG. 4 is an image of CO-3 single photon cell;

FIG. 5 is an image of CO-3 two-photon cell imaging.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.

EXAMPLE 1 Synthesis of CO-3 fluorescent Probe

(1) Adding 1 mmol of 7-diethylamino-3-aminocoumarin (1) into a 100 mL round-bottom flask, heating the flask to 100 ℃ in 20mL hydrochloric acid aqueous solution with the concentration of 1.5mol/L, reacting for 4 hours, and performing suction filtration to obtain a filter cake, namely 7-diethylamino-3-hydroxycoumarin (2), wherein the product is directly subjected to the next step without purification:

；

(2) stirring the compound 7-diethylamino-3-hydroxycoumarin (2) and 2mmol of allyl chloride (3) in 10mL of dichloromethane at 25 deg.C for 12h, distilling under reduced pressure, vacuum drying, and eluting with dichloromethane/petroleum ether (V/V =5: 1) to obtain siliconPerforming gel column chromatography to obtain a compound CO-3: 7-diethylamino-3-carbonato coumarin; yield: 77 percent. It is composed of¹The H NMR spectrum is shown in FIG. 1:

¹H NMR (400 MHz, DMSO-d6) δ 7.89 (s, 1H), 7.45 (d, 1H), 6.77 (dd, J =4.0 Hz, 1H), 6.61 (d, 1H),5.97(m,1H),5.42(dd, J = 4.0 Hz, 1H), 5.32(dd, J =4.0 Hz, 1H), 4.75(d, 2H), 3.44(m, 4H), 3.35(s, 1H),1.12(t, 5H)；

。

example 2 selectivity of CO-3 fluorescent probes for different molecules or ions

The CO-3 fluorescent probe of example 1 was prepared as a 1 mM stock solution.

Palladium chloride and ruthenium (II) tricarbonyl dichloride dimer (CORM-3) were dissolved in a DMSO solution to prepare 5mL of a mother liquor having a concentration of 10mM as a CO mother liquor.

The following were added: br^-, ClO^-, Cu²⁺, F^-, Fe²⁺, H₂O₂, HClO, Hg²⁺, HPO4^2-, Mg²⁺, Na⁺,Na₂S, NaHS, NO^-, NO^3-, OAC^-, SCN^-, SO₄ ^2-, Zn²⁺Hcy, CO was formulated in phosphate buffer (0.01 mM, pH = 7.4) into 5mL of 40 mM concentrated stock solution.

Taking 22 test tubes, respectively adding 25 mu L of probe mother liquor, 225 mu L of DMSO and mother liquor of each ion or molecule, and replacing interfering substances with equivalent water in a contrast; the volume was adjusted to 5mL with phosphate buffer (0.01 mM, pH = 7.4) to give a final concentration of interfering substance of 0.1 mM. Fluorescence detection was performed 60min after shaking each solution (λ ex = 400 nm, λ em = 504 nm). Drawing 2 by taking the fluorescence intensity as an ordinate and different molecules or ions as an abscissa; wherein 1-22 are respectively probe and Br^-, ClO^-, Cu²⁺,F^-, Fe²⁺, H₂O₂, HClO, Hg²⁺, HPO4^2-, Mg²⁺, Na⁺, Na₂S, NaHS, NO^-, NO^3-, OAC^-, SCN^-,SO₄ ^2-, Zn²⁺Hcy, CO. From fig. 2, it can be seen that the fluorescence intensity of the addition of other ions or molecules has little effect, while the addition of CO significantly enhances the fluorescence of compound CO-3.

EXAMPLE 3 fluorescence intensity of CO-3 at different concentrations of CO

Referring to the procedure of example 2, 10mL of a 100 mM CO stock solution was prepared and diluted with water to 1-9 mM for 17 equi-differential concentrations, with water as a control. The mother solution of CO-3 in example 2 was diluted to 5. mu.M, CO was added at different concentrations, and after 60min of reaction, fluorescence detection was performed (λ ex = 400 nm, λ em = 504 nm), and the fluorescence intensity in each system was measured, and a curve of fluorescence intensity versus CO concentration was plotted, as shown in FIG. 3. As can be seen from the figure, CO-3 has a fluorescence response when the CO concentration is the lowest test concentration (1 μ M); the fluorescence intensity of the reaction system is gradually enhanced along with the increase of the CO concentration, and the fluorescence intensity of the reaction system reaches a saturation state when the CO concentration reaches 9 mM.

EXAMPLE 4 CO-3 fluorescent Probe Single photon cell imaging

The fluorescent probe CO-3 is applied to HeLa cells for single photon fluorescence imaging to obtain a graph 4, wherein the 1 st to 3 rd line small graphs are respectively a bright field image, a single photon image and a superposed image of the bright field image, the single photon image and the superposed image, and the specific operation steps are as follows:

(1) 3 parts of the mixture with the density of 3 multiplied by 10⁵HeLa cells per mL at 37 ℃ with CO₂Culturing in 5% incubator for 12 hr;

(2) adding CO-3 into one cell to make its final concentration 5 μ M, incubating for 30min, washing the cell with PBS buffer solution for 3 times, preparing sample, bright field, and performing single photon fluorescence imaging (excitation wavelength 488nm, emission band 500-550 nm) to obtain graph A-A2;

(3) adding CO-3+ palladium chloride into another cell to make CO-3 final concentration 5 μ M and palladium chloride final concentration 10 μ M, incubating for 30min, and treating with the same method (2) to obtain diagram B-B2;

(4) another part of the cells was added with CO-3+ Palladium chloride + CORM-3 to give a final CO-3 concentration of 5. mu.M, Palladium chloride of 10. mu.M and CORM-3 of 10mM, and incubated for 30min to give the same result as in (2), as shown in FIG. C-C2.

As shown in FIG. 4, the fluorescent probe Co-3 can react with exogenous CO inside and outside the cell to make the cell emit strong fluorescence.

EXAMPLE 5 CO-3 fluorescent Probe two-photon cellular imaging

The specific operation steps of applying the fluorescent probe CO-3 of the invention to HeLa cells for two-photon fluorescence imaging are the same as in example 4, except that the excitation wavelength of fluorescence imaging is 760nm and the emission band is 500-550 nm, and FIG. 5 is obtained, wherein the panels in lines 1-2 are respectively a two-photon bright field and a two-photon fluorescence imaging graph.

As shown in FIG. 5, the fluorescent probe Co-3 can react with endogenous and exogenous CO in cells to make the cells emit strong fluorescence under two-photon excitation.

Claims

1. A fluorescent probe for detecting CO has a chemical name of 7-diethylamino-3-allyl carbonate coumarin, and a structural formula shown in formula (I):

formula (I).

2. A method for synthesizing the two-photon fluorescent probe according to claim 1, comprising the steps of:

(1) heating 7-diethylamino-3-aminocoumarin in HCl solution for reaction to synthesize 7-diethylamino-3-hydroxycoumarin;

(2) stirring 7-diethylamino-3-hydroxycoumarin and allyl chloroformate in dichloromethane to react to obtain the final product.

3. The method of claim 2, wherein the molar ratio of 7-diethylamino-3-aminocoumarin to HCl is 1: 30.

4. The synthesis method according to claim 2, wherein the HCl solution is in a concentration of 1-1.5 mol/L.

5. The synthesis method according to claim 2, wherein in the step (1), the reaction temperature is 100 ℃; the reaction time is 3-4 h.

6. The method of claim 2, wherein the molar ratio of 7-diethylamino-3-hydroxycoumarin to allyl chloroformate is from 1:1 to 2.

7. The synthesis method according to claim 2, wherein in the step (2), the reaction temperature is 25 ℃ and the reaction time is 12-20 h.

8. The synthesis method according to claim 2, wherein the step (2) further comprises the step of separating and purifying the product: the reaction mixture was evaporated under reduced pressure to remove the solvent, and the dried solid was subjected to silica gel column chromatography with a eluent of dichloromethane/petroleum ether (V/V =5: 1).

9. Use of a fluorescent probe according to claim 1 for the detection of CO in solutions and cells by single-photon or two-photon fluorescence.