CN115403573B - Foam cell specific recognition probe and synthesis method thereof - Google Patents
Foam cell specific recognition probe and synthesis method thereof Download PDFInfo
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 14
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- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
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- 231100000240 steatosis hepatitis Toxicity 0.000 description 2
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
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- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention relates to a foam cell specific recognition probe and a synthesis method thereof, wherein the method comprises the following steps: adding 4-fluoro-2-hydroxybenzaldehyde and alkaline organic liquid into a first solvent, stirring uniformly, then adding benzothiazole-2-acetonitrile, and separating an intermediate product CN-F after the first full reaction; adding CN-F and piperidine into a second solvent, and separating out a target product CN-PD after the second full reaction. The specific recognition probe prepared by the method has excellent solvent effect, hardly emits light in a water phase, emits green band strong light in an organic phase, emits strong fluorescence only in the presence of liposome, has good selectivity for unresponsiveness to other biomacromolecules such as nucleic acid, protein, saccharides and the like, has rapid response to liposome, and can specifically display the formation of foam cells through response to lipid droplets.
Description
Technical Field
The invention relates to the technical field of specific recognition probes, in particular to a foam cell specific recognition probe and a synthesis method thereof.
Background
Foam cells constitute a core component of atherosclerotic plaques. Fluorescent probes designed for foam cell specific recognition can be used for fluorescent imaging of atherosclerotic plaques. Currently, the surface of the nanoprobe is modified mainly by an antibody capable of recognizing a foam cell surface biomarker in the design of the specific probe. For example, the osteopontin antibody and near infrared fluorescent probe ICG are simultaneously loaded on Ti by the university of Beijing Zheng Lemin teaching team 3 C 2 On the nanosheets, construct antibody/Ti 3 C 2 ICG nanoprobe for in vivo fluorescence imaging of vulnerable plaque.
Although nanoprobe design strategies based on antibody recognition have proven useful for detection and in vivo imaging of foam cells within vulnerable plaques. However, there are still several disadvantages to this strategy: 1. the efficiency of distinguishing foam cells by the probes with antibody recognition function is low, and great background interference can be caused at the cell level; 2. complex nanostructures can lead to poor biocompatibility, making clinical transformations difficult; 3. the nano probe containing the antibody has fragile structure, is easy to deteriorate and is not beneficial to long-term storage and transportation; 4. the nanoscale structural tissue has poor penetration, and it is difficult to pass through the endothelial layer to the foam cell layer at the living level, so that it is not suitable for screening of stable plaques.
Disclosure of Invention
In order to solve the objective drawbacks existing in the current foam cell specific probe design and function. The invention provides a foam cell specific probe which has the characteristics of high recognition efficiency, good biocompatibility, stable structure, strong penetrability in cortex and the like.
In order to achieve the purpose, the invention uses lipid droplets which are abnormally aggregated in foam cells as a biological marker to design and synthesize the stimulus-responsive activatable small molecule fluorescent probe. According to the solvent color-changing effect, coumarin fluorophor is selected for structural modification, and a novel green fluorescent probe with extremely high response times is designed, synthesized and screened for specific fluorescent imaging of foam cells. The scheme of the invention is specifically as follows.
A foam cell specific recognition probe has a structural formula of
Wherein R1, R2 and R3 are all hydrogen or alkyl.
As a further improvement of the foam cell specific recognition probe, R1, R2 and R3 are all hydrogen, and the structural formula of the foam cell specific recognition probe is as follows
A method of synthesizing a foam cell-specific recognition probe as described above, comprising the steps of: adding 4-fluoro-2-hydroxybenzaldehyde and alkaline organic solution into a first solvent, stirring uniformly, then adding benzothiazole-2-acetonitrile, and separating an intermediate product CN-F (code number) after the first full reaction; adding CN-F and piperidine into a second solvent, and separating out a target product CN-PD (code number) after the second full reaction.
Wherein the structural formula of the CN-F is
The structural formula of the CN-PD is
As a further improvement of the method for synthesizing the foam cell-specific recognition probe of the present invention, the molar ratio of 4-fluoro-2-hydroxybenzaldehyde to benzothiazole-2-acetonitrile was added in (1 to 10): 1 to 10.
As a further improvement of the method for synthesizing a foam cell-specific recognition probe of the present invention, the basic organic liquid is triethylamine (Et) 3 N); the addition amount of the triethylamine is 50-150% of the addition amount of the 4-fluoro-2-hydroxybenzaldehyde by mass.
As a further improvement of the synthesis method of the foam cell specific recognition probe, the first solvent is a mixed solution of EtOH/DCM, and the volume ratio of EtOH (ethanol) to DCM (dichloromethane) is 1:1; the second solvent is DMF (dimethylformamide).
As a further improvement of the method for synthesizing a foam cell-specific recognition probe of the present invention, the condition of the first sufficient reaction is a reaction at a room temperature of 10 to 45℃for 1 to 50 hours, and the condition of the second sufficient reaction is a reaction at a temperature of 40 to 60℃for 1 to 50 hours.
As a further improvement of the method for synthesizing the foam cell-specific recognition probe of the present invention, the intermediate product CN-F is isolated in the following manner: the first solvent was removed under reduced pressure, and the remaining product was suspended with ethyl acetate to wash the remaining product, followed by suction filtration under reduced pressure to give compound CN-F.
As a further improvement of the method for synthesizing the foam cell-specific recognition probe of the present invention, the molar ratio of addition of CN-F and piperidine was 1: (5-30).
As a further improvement of the synthesis method of the foam cell specific recognition probe, the target product CN-PD is separated in the following way: extracting the reaction liquid after the second reaction by using EA (ethyl acetate) and water, retaining an EA phase, then decompressing to remove EA in the EA phase, and purifying the residue by a normal phase silica gel column to obtain a target product CN-PD; wherein, when purifying by using normal phase silica gel column, the flushing liquid uses PE/EA mixed liquid, and the PE/EA volume ratio is=20:1. PE is petroleum ether and EA is ethyl acetate.
Compared with the prior art, the invention has the beneficial effects that: the specific recognition probe has excellent solvent effect, hardly emits light in a water phase, emits green band strong light in an organic phase, emits strong fluorescence only in the presence of liposome, has good selectivity for nonresponsion of other biomacromolecules such as nucleic acid, protein, saccharides and the like, has rapid response to liposome, and can specifically display the formation of foam cells through response to lipid droplets.
The specific recognition probe has high recognition efficiency, better biocompatibility and stable structure. Compared with a specific probe adopting an antibody macromolecule to carry out surface modification, the specific recognition probe is a small molecular organic compound and has stronger penetrability in the cortex of a human body.
Drawings
In order to more clearly illustrate the technical solution of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described. It is to be understood that the following drawings illustrate only certain embodiments of the invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally relevant drawings without inventive effort by those of ordinary skill in the art.
FIG. 1 is a graph showing the results of the hydrogen spectrum characterization of CN-PD provided in test example 1.
FIG. 2 is a graph showing the results of the carbon spectrum characterization of CN-PD provided in test example 1.
Fig. 3 is a graph of high resolution mass spectrum characterization results of CN-PD provided in test example 1.
FIG. 4 is a diagram of the spectroscopy analysis of CN-PD provided in test example 2.
FIG. 5 is a spectrum of CN-PD fluorescence emission provided in test example 3, outlining the three-dimensional distribution of intracellular lipid droplets.
FIG. 6 is a Person co-localization coefficient test pattern of CN-PD and a commercial lipid droplet probe as provided in test example 3.
FIG. 7 is a co-localization coefficient test pattern of CN-PD and other organelle probes provided in test example 3.
FIG. 8 is a graph showing the lipid droplets distribution of CN-PD in fatty liver tissue as provided in test example 3.
FIG. 9 is a photograph showing fluorescence of the whole fat cells in the fluorescence of the fat tissue by CN-PD provided in test example 3.
FIG. 10 is a comparative image of the staining analysis of monocytes and macrophages with CN-PD, respectively, as provided in test example 3.
FIG. 11 is a graph showing the contrast of images obtained by staining macrophages and foam cells with CN-PD, respectively, as provided in test example 3.
FIG. 12 is a graph showing the comparison of the size and fluorescence intensity of lipid droplets in macrophages and foam cells obtained by staining the macrophages and foam cells with CN-PD, respectively, as provided in test example 3.
FIG. 13 is a graph showing the comparison of fluorescence intensity of macrophages and foam cells, respectively, by staining the macrophages and foam cells with CN-PD, as provided in test example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
1. The compound 4-fluoro-2-hydroxybenzaldehyde (100.0 mg,0.714 mmol) and 0.1mL Et 3 N adding EtOH/DCM mixingTo the reaction mixture (10 mL:10 mL) was stirred at room temperature for 5min, benzothiazole-2-acetonitrile (124.3 mg, 0.514 mmol) was then added and reacted at room temperature of 20 to 30℃for 16h overnight, the solvent was removed under reduced pressure, the remaining product was suspended with 20mL EA, and then suction filtration under reduced pressure was performed to obtain compound CN-F.
2. Compound CN-F (100.0 mg,0.337 mmol) and 0.5mL piperidine were added to 10mL DMF and reacted at 50℃for 16h overnight. The reaction solution was extracted with EA and water, the EA phase was retained, then EA in the EA phase was removed under reduced pressure, and the remaining product was purified by a normal phase silica gel column (PE/ea=20:1) to give the objective product CN-PD.
Wherein the structural formula of the CN-F is
The structural formula of the CN-PD is
Example 2
1. The compound 4-fluoro-2-hydroxybenzaldehyde (100.0 mg,0.714 mmol) and 0.2mL Et 3 N was added to EtOH/DCM mixture (10 mL:10 mL), stirred at room temperature for 5min, then benzothiazole-2-acetonitrile (12.43 mg,0.071 mmol) was added to the reaction solution, reacted at room temperature of 10-18℃for 24h overnight, the solvent was removed under reduced pressure, the remaining product was suspended with 20mL EA, and then suction filtered under reduced pressure to give compound CN-F.
2. Compound CN-F (10.0 mg,0.034 mmol) and 0.1mL of piperidine were added to 10mL of DMF and reacted at 40℃for 6h. The reaction solution was extracted with EA and water, the EA phase was retained, then EA in the EA phase was removed under reduced pressure, and the remaining product was purified by a normal phase silica gel column (PE/ea=20:1) to give the objective product CN-PD.
Example 3
1. The compound 4-fluoro-2-hydroxybenzaldehyde (100.0 mg,0.714 mmol) and 0.1mL Et 3 N was added to EtOH/DCM mixture (10 mL:10 mL), stirred at room temperature for 5min, and benzothiazole-2-one was then added to the reaction solutionAcetonitrile (1243 mg,7.14 mmol), at room temperature of 30-40℃for 4h, the solvent was removed under reduced pressure, the remaining product was suspended with 20mL of EA, and then suction filtration under reduced pressure was performed to obtain the compound CN-F.
2. Compound CN-F (100.0 mg,0.337 mmol) and 0.2mL of piperidine were added to 10mL of DMF and reacted at 60℃for 46h. The reaction solution was extracted with EA and water, the EA phase was retained, then EA in the EA phase was removed under reduced pressure, and the remaining product was purified by a normal phase silica gel column (PE/ea=20:1) to give the objective product CN-PD.
Test example 1
The results of structural characterization of the target product obtained in example 1 by using hydrogen spectrum, carbon spectrum and high-resolution mass spectrum are shown in fig. 1-3, and the results show that the structure of the target product obtained in example 1 is
Test example 2
Panel A in FIG. 4 shows the fluorescence intensity of the target product CN-PD obtained in example 1 dissolved in various solvents including various organic solvents (toluene, chloroform, methylene chloride, acetone, ethyl acetate, DMF, DMSO, meCN, meOH) and water. The test example firstly proves that CN-PD has excellent solvent effect, hardly emits light in the water phase, emits strong light in the green wave band in the organic phase, and has more than 75 times of fluorescence intensity difference of the CN-PD in the organic phase/the water phase (figure 4A).
Panel B in FIG. 4 shows that the target product CN-PD obtained in example 1 is dissolved in a solvent system composed of methanol and water, and the fluorescence intensity of CN-PD is enhanced with the increase of the methanol content and has good linear correlation (FIG. 4B).
Panel C in FIG. 4 is an in vitro model of intracellular lipid droplets using liposomes, and the response ability of the objective product CN-PD obtained in example 1 to liposomes was observed. Experiments have found that CN-PD emits strong fluorescence only in the presence of liposomes, exhibiting good selectivity for other biomacromolecules such as nucleic acids, proteins, carbohydrates, etc. without response (fig. 4C).
Panel D in FIG. 4 shows the change in fluorescence intensity of the objective product CN-PD obtained in example 1 with respect to liposomes over time. CN-PD responded extremely rapidly to liposomes, approaching the intensity maximum almost within 5 minutes (fig. 4D). With existing antibodies/Ti 3 C 2 The ICG nano probe has stronger response capability.
Test example 3
The cells were rendered and fluorous developed using CN-PD, the results are shown in figure 5. The left panel in FIG. 5 shows the effect of CN-PD on fluorescence in cells; left two is an enlarged view of the square area of left one; third left is the emission spectrum of fluorescent points of ROI1, ROI2, ROI3 and ROI4 in second left; the left four is a three-dimensional profile indicating intracellular lipid droplets by CN-PD fluorescence.
At the cellular level, the maximum emission wavelength of CN-PD is around 511nm (as shown in the left-hand dichotomy in fig. 5), so 488nm is chosen as the excitation channel and the reception range is set to 500-560nm. Under this test condition, CN-PD clearly delineates the shape, size, and three-dimensional distribution of lipid droplets within the cell (as shown in the left four graphs of fig. 5). The pearson co-localization coefficient of CN-PD with commercial lipid drop probes reached 0.95 (fig. 6), while co-localization with other organelle probes was very poor (fig. 7), indicating lipid drop selectivity of CN-PD at the cellular level. At the tissue level, CN-PD staining can show lipid droplet morphology, size and distribution in fatty liver (fig. 8). Whereas in adipose tissue, the whole adipocytes were almost fluorescent (fig. 9), showing lipid selectivity of CN-PD.
The mononuclear cells, macrophages and foam cells were stained with CN-PD. No significant differences in lipid droplets between macrophages and monocytes were found (fig. 10); however, the lipid droplets in the foam cells were drastically changed, and the diameter of the lipid droplets was significantly increased (fig. 11). The average diameter of the foam intracellular lipid droplets is 2.28 μm, 5.6 times larger than that of macrophages; meanwhile, the volume of lipid droplets in foam cells was 6.20. Mu.m 3 174.5 times larger than the volume of macrophages; the fluorescence intensity of the foam cell cross section was 13.4 times that of macrophages (fig. 12). The overall comparison of fluorescence intensity by flow cytometry showed lipid pairing with CN-PDAfter staining the droplets, the foam cells were more fluorescent than the macrophages (fig. 13). These experiments demonstrate that CN-PD can specifically show foam cell formation through response to lipid droplets.
The above description is only of the preferred embodiments of the present invention, but the protection scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalent substitutions or alterations to the technical solution and the inventive concept thereof within the scope of the present invention.
Claims (10)
1. A foam cell-specific recognition probe, characterized in that: the structure is as follows
Wherein R1, R2 and R3 are all hydrogen.
2. The foam cell-specific recognition probe of claim 1, wherein: the structural formula of the foam cell specific recognition probe is
3. A method of synthesizing the foam cell-specific recognition probe according to claim 2, comprising the steps of:
adding 4-fluoro-2-hydroxybenzaldehyde and alkaline organic liquid into a first solvent, stirring uniformly, then adding benzothiazole-2-acetonitrile, and separating an intermediate product CN-F after the first full reaction; adding CN-F and piperidine into a second solvent, and separating out a target product CN-PD after the second full reaction;
wherein the structural formula of the CN-F is
The structural formula of the CN-PD is
4. The method for synthesizing a foam cell-specific recognition probe according to claim 3, wherein the molar ratio of 4-fluoro-2-hydroxybenzaldehyde to benzothiazole-2-acetonitrile added is (1 to 10): 1 to 10.
5. The method for synthesizing a foam cell-specific recognition probe according to claim 3, wherein the basic organic liquid is triethylamine; the addition amount of the triethylamine is 50-150% of the addition amount of the 4-fluoro-2-hydroxybenzaldehyde by mass.
6. The method for synthesizing a foam cell-specific recognition probe according to claim 3, wherein the first solvent is a mixed solution of EtOH and DCM, and the volume ratio of EtOH to DCM is 1:1; the second solvent is DMF.
7. The method for synthesizing a foam cell-specific recognition probe according to claim 3, wherein the first sufficient reaction condition is a reaction at a room temperature of 10 to 45℃for 1 to 50 hours, and the second sufficient reaction condition is a reaction at a temperature of 40 to 60℃for 1 to 50 hours.
8. The method of claim 3, wherein the isolation of intermediate CN-F is performed by:
the first solvent was removed under reduced pressure, and the remaining product was suspended with ethyl acetate to wash the remaining product, followed by suction filtration under reduced pressure to give compound CN-F.
9. The method for synthesizing a foam cell-specific recognition probe according to claim 3, wherein the molar ratio of CN-F to piperidine added is 1: (5-30).
10. The method for synthesizing a foam cell-specific recognition probe according to claim 3, wherein the target product CN-PD is isolated by:
extracting the reaction liquid after the second reaction by using EA and water, retaining an EA phase, then decompressing to remove EA in the EA phase, and purifying the remainder by a normal phase silica gel column to obtain a target product CN-PD; wherein, when purifying by using normal phase silica gel column, the flushing liquid uses PE/EA mixed liquid, and the PE/EA volume ratio is=20:1.
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