CN113980010A

CN113980010A - Ratio type optical/photoacoustic dual-mode fluorescent probe and preparation method and application thereof

Info

Publication number: CN113980010A
Application number: CN202111389497.7A
Authority: CN
Inventors: 陈华; 柳丽; 许锦源; 沈星灿
Original assignee: Guangxi Normal University
Current assignee: Guangxi Normal University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-01-28

Abstract

The invention discloses a ratio type optical/photoacoustic dual-mode fluorescent probe DOP-CO and a preparation method and application thereof, and the fluorescent probe DOP-CO provided by the invention has the advantages of simple synthesis step, convenience in purification and excellent imaging; a double-ratio fluorescence and photoacoustic detection mode is adopted, so that the detection signal is accurate; the biocompatibility is good, and the damage to cells or animals is small; the method has the advantages of using non-ionizing radiation, non-invasive radiation and having no influence on normal tissues, can detect the generation of endogenous CO of the mouse in acute liver injury due to the complementary advantages of fluorescence and photoacoustic imaging, and is expected to be widely applied to the research of diseases and physiological processes related to liver injury.

Description

Ratio type optical/photoacoustic dual-mode fluorescent probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of preparation of organic small-molecule fluorescent probes, in particular to a ratio type optical/photoacoustic dual-mode fluorescent probe and a preparation method and application thereof.

Background

In recent years, the study of carbon monoxide (CO) has been of increasing interest, and such gas molecules have long been considered toxic, harmful, and even fatal. However, it is now considered to be an important gas signaling molecule which can be endogenously produced in human body and plays a very important physiological role in our lives, and CO is an important gas transmitter involved in many important physiological processes such as vasodilation, neurotransmission, anti-inflammation and anti-apoptosis, and there is increasing evidence that many diseases are associated with abnormalities in CO content in vivo such as oxidative stress, heart failure, etc., and although CO has many important physiological roles and pharmacological significance, many biological functions of CO are not clear, and for this reason, it is very valuable to develop an effective assay to sensitively and selectively detect dynamic changes in CO in complex living systems.

Although many fluorescent probes can detect intracellular CO at present, it is still a challenge to visualize the release behavior of endogenous CO, and the limited tissue penetration depth (<1mm) of fluorescence enables optical imaging to display only the surface, which can be overcome by Photoacoustic (PA) imaging, which has an imaging depth of several centimeters in tissue, but has low sensitivity and resolution, so it is of great importance to design a fluorescent probe with good imaging depth, high sensitivity and resolution.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a ratio type optical/photoacoustic dual-mode fluorescent probe, a preparation method and application thereof, and solves the technical problems of poor imaging depth and sensitivity and resolution of the conventional fluorescent probe.

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

a ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO, which has the following structural formula:

the invention provides a preparation method of the ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO, which comprises the following synthetic route:

preferably, the preparation method comprises the following steps:

(1) preparation of compound 2: under the ice-bath condition, phosphorus oxychloride is dropwise added into DMF and CH₂Cl₂Adding the commercially available raw material 1 dropwise into the mixed solution, stirring uniformly, removing the ice bath, heating for reaction, and after the reaction is finished, separating and purifying the reaction product to obtain a compound 2;

(2) preparation of compound 4: dissolving a commercially available raw material 3 in acetonitrile, adding iodoethane and potassium carbonate, uniformly mixing, reacting, and after the reaction is finished, separating and purifying a reaction product to obtain a compound 4;

(3) preparation of compound 5: dissolving the compound 2 obtained in the step (1) and the compound 4 obtained in the step (2) in a mixed solution of n-butyl alcohol and toluene, reacting, adding perchloric acid after the reaction is finished, and then separating and purifying a reaction product to obtain a compound 5;

(4) preparation of compound 6: dissolving the compound 5 obtained in the step (3) in DMF, adding m-mercaptophenol, uniformly stirring, then adding sodium hydride for reaction, and after the reaction is finished, separating and purifying a reaction product to obtain a compound 6;

(5) preparation of fluorescent probe DOP-CO: and (3) dissolving the compound 6 obtained in the step (4) in dichloromethane, adding triethylamine, stirring uniformly, adding allyl chloroformate under an ice bath condition for reaction, and after the reaction is finished, separating and purifying a reaction product to obtain the fluorescent probe DOP-CO.

Preferably, in the step (1), the molar ratio of the raw material 1 to the phosphorus oxychloride is 1:1.5, and DMF and CH₂Cl₂The volume ratio of (A) to (B) is 1:1, and the reaction conditions are as follows: reflux reaction is carried out under the protective atmosphere of nitrogen, the reaction temperature is 80-100 ℃, and the reaction time is 2-6h。

Preferably, in the step (2), the molar ratio of the raw material 3, the iodoethane and the potassium carbonate is 1-3:1-3:2-3, and the reaction conditions are as follows: carrying out reflux reaction under the protection of nitrogen, wherein the reaction temperature is 80-100 ℃, and the reaction time is 12-18 h.

Preferably, in the step (3), the molar ratio of the compound 2 to the compound 4 is 1:2, the volume ratio of n-butanol to toluene is 7:3, and the reaction conditions are as follows: reflux reaction is carried out under the atmosphere of nitrogen protection, the reaction temperature is 100-120 ℃, and the reaction time is 8-10 h.

Preferably, in the step (4), the molar ratio of the compound 5, the m-mercaptophenol and the sodium hydride is 1:3:2, and the reaction conditions are as follows: carrying out reflux reaction under the protection of nitrogen, wherein the reaction temperature is 45-60 ℃, and the reaction time is 10-16 h.

Preferably, in the step (5), the molar ratio of the compound 6, triethylamine and allyl chloroformate is 1-3:3-5:3-5, and the reaction conditions are as follows: carrying out reflux reaction under the protection of nitrogen, wherein the reaction temperature is 25-45 ℃, and the reaction time is 8-16 h.

The invention also provides application of the ratio type optical/photoacoustic dual-mode fluorescent probe in CO detection.

Preferably, the application specifically comprises the following steps:

(1) dissolving a fluorescent probe DOP-CO in dimethyl sulfoxide (DMSO) to prepare a probe mother solution;

(2) adding the probe mother solution into a solution to be detected and a biological sample;

(3) after adding CO, observing the change of ultraviolet and fluorescence spectrums of the solution to be detected containing the probe mother solution by using an ultraviolet spectrophotometer and a fluorescence spectrometer;

(4) diluting the probe mother liquor, injecting the diluted probe mother liquor into a mouse, and then performing near-infrared fluorescence and photoacoustic imaging to obtain CO near-infrared fluorescence and photoacoustic images in animal living bodies.

Compared with the prior art, the invention has the following beneficial effects:

(1) the probe DOP-CO provided by the invention has the advantages of simple synthesis steps, convenience in purification and excellent imaging;

(2) the probe DOP-CO provided by the invention adopts double-ratio type fluorescence and photoacoustic detection, and the detection signal is accurate;

(3) the probe DOP-CO provided by the invention has good biocompatibility and small harm to cells or animals;

(4) the probe DOP-CO provided by the invention has the advantages of using non-ionizing radiation, non-invasive radiation and having no influence on normal tissues.

Drawings

FIG. 1 shows that the probe DOP-CO was prepared¹H NMR spectrum;

FIG. 2 is a diagram of the prepared probe DOP-CO¹³A C NMR spectrum;

FIG. 3 is a result of a change test of titration ultraviolet and photoacoustic spectrograms of the prepared probe DOP-CO against CO;

FIG. 4 shows the change of the prepared probe DOP-CO in the titration fluorescence spectrum of CO and the linear relationship test result;

FIG. 5 shows the results of the test of selectivity of the prepared probe DOP-CO for CO;

FIG. 6 is the result of cytotoxicity test of the prepared probe DOP-CO;

FIG. 7 is the results of the prepared probe DOP-CO CO-localization test;

FIG. 8 shows the results of the test for detecting exogenous CO in cells using the prepared probe DOP-CO;

FIG. 9 shows the result of fluorescence imaging experiment of the prepared probe DOP-CO for detecting CO in the liver of a mouse;

FIG. 10 is the result of photoacoustic imaging experiment of the prepared probe DOP-CO for detecting CO in the liver of a mouse.

Detailed Description

The present invention will be described in more detail with reference to specific preferred embodiments, but the present invention is not limited to the following embodiments.

It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are commercially available.

Example 1

A preparation method of a ratio type optical/photoacoustic dual-mode fluorescent probe DOP-CO comprises the following steps:

(1) preparation of compound 2: 0.15mol of phosphorus oxychloride was added dropwise to 100mL of DMF and CH under ice bath conditions₂Cl₂In the mixed solution of (1), wherein DMF and CH₂Cl₂The volume ratio of the compound to the solvent is 1:1, then 0.1mol of a commercially available raw material 1 is dropwise added, the mixture is uniformly stirred, then the ice bath is removed, reflux reaction is carried out under the nitrogen protection atmosphere, the reaction temperature is 80 ℃, the reaction time is 3 hours, and after the reaction is finished, the reaction product is separated and purified to obtain a compound 2;

(2) preparation of compound 4: dissolving 0.1mol of a commercially available raw material 3 in 100mL of acetonitrile, then adding 0.1mol of iodoethane and 0.2mol of potassium carbonate, uniformly mixing, carrying out reflux reaction under the nitrogen protection atmosphere, wherein the reaction temperature is 80 ℃, the reaction time is 12 hours, and after the reaction is finished, separating and purifying a reaction product to obtain a compound 4;

(3) preparation of compound 5: 2.8g of Compound 2 and 10g of Compound 4 were dissolved in 100mL of a mixed solution of n-butanol and toluene in a volume ratio of 7:3, carrying out reflux reaction under the nitrogen protection atmosphere, wherein the reaction temperature is 100 ℃, the reaction time is 8h, adding 10mL perchloric acid after the reaction is finished, and then separating and purifying the reaction product to obtain a compound 5;

(4) preparation of compound 6: dissolving 100mg of compound 5 in 10mL of anhydrous DMF, adding 50 mu L of m-mercaptophenol, stirring for 5min, adding 10mg of sodium hydride, reacting at 50 ℃ for 10h under the nitrogen protection atmosphere, and after the reaction is finished, separating and purifying the reaction product to obtain a compound 6;

(5) preparation of fluorescent probe DOP-CO: dissolving 332mg of compound 6 in 5mL of dichloromethane, adding 310mg of triethylamine, stirring at room temperature for 10min, slowly adding 1.04g of allyl chloroformate under an ice bath condition for reaction, performing the reaction under the protection of nitrogen at 25 ℃ for 8h, distilling the reaction liquid under reduced pressure after the reaction is finished to evaporate the solvent, and then separating and purifying the reaction product by using a silica gel chromatographic column, wherein an eluent is dichloromethane/ethanol (V/V is 10:1), thus obtaining the fluorescent probe DOP-CO.

The fluorescent probe DOP-CO prepared in example 1 was subjected to nuclear magnetic and mass spectrometric characterization, and FIG. 1 shows that the probe DOP-CO is¹H NMR spectrum; FIG. 2 shows the probe DOP-CO¹³A C NMR spectrum;

¹H NMR(600MHz,DMSO-d₆)δ8.27(d,J＝14.8Hz,1H),7.86(d,J＝11.6Hz,1H),7.82(d,J＝8.0Hz,1H),7.74(d,J＝2.4Hz,1H),7.66(s,1H),7.61(d,J＝6.2Hz,1H),7.55(t,J＝7.4Hz,1H),7.33(t,J＝8.0Hz,1H),7.30(s,1H),6.90(d,J＝14.8Hz,1H),6.09–5.94(m,1H),5.43(d,J＝17.3Hz,1H),5.34(d,J＝10.5Hz,1H),4.78(dd,J＝19.3,8.5Hz,2H),4.54(d,J＝7.2Hz,2H),2.74(dt,J＝30.3,5.9Hz,4H),1.99(d,J＝6.0Hz,2H),1.76(s,6H),1.41(t,J＝7.2Hz,3H)。

¹³C NMR(151MHz,DMSO)δ178.87,174.77,152.82,151.26,149.65,145.74,142.98,140.64,134.72,134.05,132.82,132.08,130.14,128.15,127.64,127.20,123.54,121.68,119.60,118.00,114.58,109.24,69.60,51.58,35.58,31.76,27.92,27.02(2C),25.58,14.43.HRMS(ESI)m/z calcd for C₃₁H₃₂NO₃S⁺([M]⁺):498.2097；found 498.2123。

the fluorescent probe DOP-CO prepared in example 1 was subjected to experimental tests:

(1) ultraviolet absorption spectrum and photoacoustic spectrum experiment of reaction of fluorescent probe DOP-CO and CO

The fluorescent probe DOP-CO prepared in example 3 was dissolved in DMSO solvent to prepare a 5mM probe stock solution, and then 1998. mu.L of the liquid (PBS: DMSO ═ 7: 3) and 4. mu.L of the probe stock solution were added to a UV dish and an EP tube, respectively, and CO and PdCl were added₂The absorption spectrum and the photoacoustic spectrogram of the solution are tested by an ultraviolet spectrophotometer and a multispectral photoacoustic tomography imaging system, and the test result is shown in figure 3.

From FIG. 3a, it can be observed that the UV spectrum peak decreases at 610nm and increases at 740nm after CO addition, and the spectrum red-shifts from 670nm to 740nm, showing a change in ratio.

(2) Fluorescence emission of fluorescent probe DOP-CO and CO reaction and linear relation experiment thereof

Prepared by the method of example 1The fluorescent probe DOP-CO of (1) was dissolved in DMSO solvent to prepare a 5mM probe stock solution, 1998. mu.L of the solution (PBS: DMSO. about.7: 3) and 4. mu.L of the probe stock solution were added to a fluorescent dish, and then CO and PdCl were added to the dish₂The fluorescence emission spectrum of the solution is measured by a fluorescence spectrometer, and the measurement result is shown in figure 4.

From FIG. 4a, it can be observed that the fluorescence spectrum peak decreases at 715nm and increases at 785nm with the addition of CO, and from FIG. 4b, it can be observed that the fluorescence intensity ratio shows a good linear relationship with the CO concentration.

(3) Selective experiment of fluorescent probe DOP-CO on CO

The fluorescent probe DOP-CO prepared in example 1 was dissolved in DMSO solvent to prepare a probe stock solution of 5mM, and the probe stock solution and 10. mu.M Pd were examined at a concentration of 10. mu.M²⁺Adding GSH, Cys, Hcy, Ala, Leu, Trp, Gly, lle, Lys, and HS into the solution^-，HSO3^-，SO₃ ^2-，H₂O₂，ClO^-，·OH，ONOO^-，NO，NO^2-，NO^3-，N^3-，F^-，Cl^-，Br^-，.I^-，CN^-The fluorescence response of tBuOO and CO, the results of the test are shown in FIG. 5.

As shown in the figure, the fluorescence ratio is significantly enhanced only when CO is added, indicating that the probe DOP-CO has excellent selectivity for CO compared with other components, and can specifically detect CO in a complex biological environment.

(4) Cytotoxicity testing of fluorescent Probe DOP-CO

The digested HeLa, HepG2, HL7702 cell suspensions were added at 1X 10 per well, respectively⁵180. mu.L of each cell^-1The density of (2) was inoculated in a 96-well culture plate, the plate was placed in a cell incubator and cultured for 24 hours, when the cell density was 80% as observed in a microscope, 20. mu.L of each probe (0, 2, 4, 6, 8 and 10. mu.M) was added to each well, the plate was further cultured for 24 hours, then MTT reagent (10. mu.L, 5mg/mL MTT) was added thereto and incubated for 4 hours, the cell supernatant was removed, 100mL of DMSO was added to each well, the plate was placed on a shaker and shaken at a low speed for 10 minutes to sufficiently dissolve purple formazan crystals, and the plate was tested by a microplate readerFormazan absorbance at 570nm and cell survival calculated; cell viability was expressed as a percentage of the mean of the experimental group to the mean of the blank group, and the results are shown in FIG. 6.

As can be seen from the figure, the lower cytotoxicity of the probe DOP-CO in the cell indicates that the biocompatibility of the probe is better.

(5) Co-localization experiment of fluorescent probe DOP-CO

Culturing HepG2 cells in low-sugar culture solution containing 10% fetal calf serum by adherence at 37 deg.C under 5% CO₂Culturing in a saturated humidity incubator, replacing culture solution every 3 days, carrying out subculture, transferring cells into a confocal dish for culturing, taking out 1 confocal dish, replacing the culture solution in the confocal dish with 1mL of fresh culture solution, taking 2 mu L of probe mother solution, and incubating with a commercial probe LYDG (200nM) in the confocal dish for 15 minutes; then 1 confocal dish was removed and the culture medium was changed to 1mL fresh culture medium, 2. mu.L of 5mM probe stock solution was incubated with commercial probe MTDG (200nM) in the confocal dish for 15 min; carrying out confocal microscope imaging on the co-stained 4T1 cells, wherein the excitation wavelength of a green channel of the probe is 488nm, and the fluorescence collection wavelength is 505-560 nm; the excitation wavelength of the red channel is 638nm, and the fluorescence collection wavelength is 650-715 nm; the test results are shown in fig. 7.

As can be seen from the figure, the probe DOP-CO localized to lysosomes.

(6) Confocal fluorescence imaging experiment of exogenous response CO of fluorescent probe DOP-CO in cells

Inoculating HepG2 cells with the density of 80% to a confocal dish (35mm), after culturing for 24 hours, sucking the culture medium in the bottle by using a straw, adding 1mL of fresh culture medium, setting 1 blank group and 3 experimental groups for the exogenous response experiment of the fluorescent probe DOP-CO in the cells, adding 10 mu M of fluorescent probe into the blank group, incubating for 15min, removing the culture medium, rinsing for 2 times by using PBS (phosphate buffer solution), and imaging, wherein the experimental groups are firstly incubated for 15min by adding 10 mu M of probe, removing the culture medium, adding 1mL of fresh culture medium, respectively adding 5eq, 10eq and 15eq of CO for 30min, sucking the culture medium in the dish by using a straw, rinsing for 2 times by using PBS, and imaging, in the fluorescent imaging experiment process, an excitation source is selected to be 638nm, and the collection wave bands are respectively 660nm-715nm and 720nm-795nm, and the experimental result is shown in figure 8.

As can be seen from the figure, when no CO is added into the blank group, the channel 1 emits fluorescence, but the channel 2 does not emit fluorescence, when different equivalents of CO are added into the experimental group, the fluorescence of the channel 2 is continuously enhanced in a dose-dependent manner, and meanwhile, the fluorescence of the channel 1 is continuously weakened, which indicates that the probe DOP-CO has ratio fluorescence imaging capability and can well respond to CO in a cell environment.

(7) Fluorescent probe DOP-CO used for acute liver injury mouse living body fluorescence imaging experiment

APAP is injected into the abdominal cavity of an experimental group mouse, the same amount of normal saline is injected into the abdominal cavity of a control group mouse, after eight hours, fluorescence imaging is carried out by injecting a probe DOP-CO into the vein, and the experimental result is shown in figure 9.

As can be seen from the figure, in the experimental group, the image of channel 1(λ ex ═ 630nm filter 700nm) showed a decrease in fluorescence in the mouse liver region, while the image of channel 2(λ ex ═ 720nm filter 790nm) showed a significant increase in fluorescence in the mouse liver region; in contrast, in the control group, the image of channel 1(λ ex ═ 630nm filter 700nm) showed that fluorescence in the mouse liver region was enhanced, while the image of channel 2(λ ex ═ 720nm filter 790nm) showed that fluorescence in the mouse liver region was substantially unchanged, indicating that the probe DOP-CO was successfully used for acute liver injury model mouse live body imaging.

(8) Photoacoustic imaging experiment of fluorescent probe DOP-CO on mouse living body with acute liver injury

APAP is injected into the abdominal cavity of an experimental group mouse, the same amount of normal saline is injected into the abdominal cavity of a control group mouse, after eight hours, the photoacoustic imaging is carried out by injecting a probe DOP-CO into the vein, and the experimental result is shown in figure 10.

After injection of the probes DOP-CO and CO into the liver region of mice, in PA₆₇₀And PA₇₄₀A clear photoacoustic signal was observed in all channels and the experimental results are shown in fig. 10.

As can be seen from the figure, in the experimental group, the channel PA₆₇₀The image of (2) shows that the photoacoustic signal of the mouse liver region is falling, and the channel PA₇₄₀Image display of mouse liver region photoacoustic signalEnhancing the function; while in the control group, channel PA₆₇₀The image of (2) shows that the photoacoustic signal of mouse liver region is enhanced, and the channel PA₇₄₀The image of (a) shows that the photoacoustic signal of the mouse liver region is basically unchanged, indicating that the probe DOP-CO may be suitable as a PA probe for quantitatively detecting CO in the mouse liver.

Finally, it is to be noted that: the above examples do not limit the invention in any way. It will be apparent to those skilled in the art that various modifications and improvements can be made to the present invention. Accordingly, any modification or improvement made without departing from the spirit of the present invention is within the scope of the claimed invention.

Claims

1. A ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO is characterized in that the structural formula of the fluorescent probe is as follows:

2. the method for preparing the ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO according to claim 1, characterized in that the synthetic route thereof is as follows:

3. the method for preparing a ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO according to claim 2, characterized in that it comprises the following steps:

4. The method for preparing ratio-type optical/photoacoustic dual-mode fluorescence probe DOP-CO as claimed in claim 3, wherein in the step (1), the molar ratio of the raw material 1 to the phosphorus oxychloride is 1:1.5, and DMF and CH are added₂Cl₂The volume ratio of (A) to (B) is 1:1, and the reaction conditions are as follows: carrying out reflux reaction under the protection of nitrogen, wherein the reaction temperature is 80-100 ℃, and the reaction time is 2-6 h.

5. The method for preparing the ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO according to claim 3, wherein in the step (2), the molar ratio of the raw material 3, the iodoethane and the potassium carbonate is 1-3:1-3:2-3, and the reaction conditions are as follows: carrying out reflux reaction under the protection of nitrogen, wherein the reaction temperature is 80-100 ℃, and the reaction time is 12-18 h.

6. The method for preparing ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO according to claim 3, wherein in step (3), the molar ratio of compound 2 to compound 4 is 1:2, the volume ratio of n-butanol to toluene is 7:3, and the reaction conditions are as follows: reflux reaction is carried out under the atmosphere of nitrogen protection, the reaction temperature is 100-120 ℃, and the reaction time is 8-10 h.

7. The method for preparing the ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO according to claim 3, wherein in the step (4), the molar ratio of the compound 5, the m-mercaptophenol and the sodium hydride is 1:3:2, and the reaction conditions are as follows: carrying out reflux reaction under the protection of nitrogen, wherein the reaction temperature is 45-60 ℃, and the reaction time is 10-16 h.

8. The method for preparing a ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO according to claim 3, wherein in the step (5), the molar ratio of the compound 6, the triethylamine and the allyl chloroformate is 1-3:3-5:3-5, and the reaction conditions are as follows: carrying out reflux reaction under the protection of nitrogen, wherein the reaction temperature is 25-45 ℃, and the reaction time is 8-16 h.

9. Use of the ratiometric optical/photoacoustic dual-mode fluorescent probe DOP-CO according to claim 1 for the detection of CO.

10. The use according to claim 9, characterized in that it comprises in particular the following steps:

(3) after adding CO, observing the change of an ultraviolet absorption spectrum and a fluorescence emission spectrum of the liquid to be detected containing the probe mother liquor by using an ultraviolet spectrophotometer and a fluorescence spectrometer;