CN113603644A - Two-photon fluorescent probe with response to formaldehyde positioned by endoplasmic reticulum as well as preparation method and application of two-photon fluorescent probe - Google Patents
Two-photon fluorescent probe with response to formaldehyde positioned by endoplasmic reticulum as well as preparation method and application of two-photon fluorescent probe Download PDFInfo
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Abstract
The invention discloses a two-photon fluorescent probe with an endoplasmic reticulum positioned formaldehyde response and a preparation method and application thereof, belonging to the technical field of analytical chemistry. The structural formula of the two-photon fluorescent probe responding to the endoplasmic reticulum positioning formaldehyde is as follows:
the preparation process is simple and the product yield is high. The two-photon fluorescent probe for endoplasmic reticulum positioning formaldehyde response can realize endoplasmic reticulum positioning and formaldehyde response, and further achieve the purpose of researching the stress of formaldehyde on endoplasmic reticulum. The present invention thus addresses the problem of poor targeting of particular organelles and biological samplesThe method has the problem of large damage, and can be applied to detecting the content of the endoplasmic reticulum formaldehyde.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and relates to a two-photon fluorescent probe with an endoplasmic reticulum positioning formaldehyde response, and a preparation method and application thereof.
Background
Leather products include genuine leather and artificial leather, and are now widely used in the square surfaces of life, including clothes, furniture, and partial protectors. However, residues in leather, such as Formaldehyde (FA) and the like, seriously affect the life health of human beings.
Research proves that the human body can transport the formaldehyde with strong volatility to the brain tissue with larger oxygen demand through respiration, and central nerve oxidative damage is generated. Oxidative damage, which is a major organ of intracellular protein synthesis, can lead to irregular changes in the endoplasmic reticulum morphology, which in turn leads to abnormal protein synthesis, or to interference with the correct folding of the protein, causing endoplasmic reticulum stress, and possibly causing neurodegenerative diseases such as alzheimer's disease and parkinson's disease. The in-situ detection of the formaldehyde level in the endoplasmic reticulum of nerve cells and the judgment of the endoplasmic reticulum stress caused by different leather manufacturing processes have very important functions on realizing the pathogenic mechanism and early diagnosis of neurodegenerative diseases.
However, the endoplasmic reticulum is located inside the cells, and how to realize real-time dynamic analysis of the content of the formaldehyde in the endoplasmic reticulum becomes a technical problem for technicians. The fluorescence imaging technology, particularly the two-photon confocal technology based on small molecules, has the advantages of in-situ real-time analysis, micro-wound, small biological damage, high signal-to-noise ratio, easy modification of molecules and the like, so that the fluorescence imaging technology is very colorful in evaluation of the formaldehyde content of a living body. Most importantly, the high spatiotemporal resolution of confocal imaging techniques makes this tool more likely to explore the relationship of formaldehyde to neural cell endoplasmic reticulum stress at the subcellular organelle level for research and to provide reliable help for early diagnosis of neurodegenerative diseases.
At the present stage, the formaldehyde probe lacks endoplasmic reticulum targeting property and two-photon characteristic, so that the formaldehyde probe has great damage to a biological sample and is not beneficial to living body imaging research. On the other hand, the endoplasmic reticulum stress caused by formaldehyde cannot be evaluated, and the research on the pathogenic mechanism and early diagnosis of the neurodegenerative diseases cannot be realized.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a two-photon fluorescent probe for endoplasmic reticulum positioning formaldehyde response, a preparation method and application thereof, solves the problems of poor targeting of a specific organelle and great damage of a biological sample, and provides a reasonable tool for researching the relationship between endoplasmic reticulum oxidative stress and neurodegenerative diseases.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a two-photon fluorescent probe for endoplasmic reticulum positioning formaldehyde response, which has the following structural formula:
the invention discloses a preparation method of a two-photon fluorescent probe with endoplasmic reticulum positioning formaldehyde response, which comprises the following steps: (1) uniformly dispersing 4-tosyl chloride and ethylenediamine in a solvent A for substitution reaction, and separating and purifying the obtained substitution product mixture after the substitution reaction is finished to obtain a compound 1; (2) uniformly dispersing the compound 1 and 4-bromo-1, 8-naphthalic anhydride in a solvent B, carrying out substitution reaction under the condition a, and separating and purifying an obtained product mixture after the substitution reaction is finished to obtain a compound 2; (3) and (3) uniformly dispersing the compound 2 and hydrazine hydrate in a solvent C for substitution reaction, and after the reaction is finished, separating and purifying the obtained product mixture to obtain the endoplasmic reticulum positioning formaldehyde response two-photon fluorescence probe SKD-1-HCHO.
Preferably, in step (1), the molar ratio of 4-toluenesulfonyl chloride to ethylenediamine is 1:5 to 20.
Preferably, in the step (2), the molar ratio of the compound 1 to the 4-bromo-1, 8-naphthalic anhydride is 1: 1-3.
Preferably, in the step (3), the molar ratio of the compound 2 to the hydrazine hydrate is 1: 1-3.
Preferably, in the step (1), the temperature of the substitution reaction is 30-50 ℃.
Preferably, in the step (2), the temperature of the substitution reaction is 70-90 ℃.
Preferably, in the step (3), the temperature of the substitution reaction is 70-90 ℃.
Preferably, in step (1), the solvent A is dichloromethane; in the step (2), the solvent B is ethanol or methanol; in the step (3), the solvent C is ethanol or methanol;
preferably, in the step (2), the condition a is an inert atmosphere protection reaction.
The invention discloses an application of the endoplasmic reticulum positioning formaldehyde response two-photon fluorescent probe or the endoplasmic reticulum positioning formaldehyde response two-photon fluorescent probe prepared by the preparation method in detecting the content of the endoplasmic reticulum formaldehyde.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a two-photon fluorescent probe for response of endoplasmic reticulum positioning formaldehyde. The naphthalimide fluorophore is used as a two-photon fluorophore of the probe, and the problem of great damage to biological samples is solved due to the unique two-photon property of the naphthalimide fluorophore; the p-toluenesulfonic acid is used as a positioning group of the endoplasmic reticulum, so that the accurate positioning of a specific organelle in a cell can be realized, and the problem of poor targeting property of the probe is solved; hydrazine is used as a recognition domain of formaldehyde molecules, hydrazone is generated through the substitution reaction of hydrazine groups and formaldehyde, and accurate recognition of the formaldehyde molecules can be achieved. Therefore, the two-photon fluorescent probe for response of the endoplasmic reticulum positioning formaldehyde can reduce sample damage and simultaneously realize accurate detection of the formaldehyde in the endoplasmic reticulum. Solves the problems that the prior fluorescent probe is limited by poor targeting of a specific organelle and great damage to a biological sample. Formaldehyde is a small molecule with physiological toxicity, which can cause damage to the nerve center after entering a living body. The formaldehyde probe at the present stage has the defects of large damage to a living body and is not beneficial to living body imaging due to the lack of targeting property and two-photon property; on the other hand, due to lack of targeting, the target can not be carried out aiming at a specific organelle in the cell, the endoplasmic reticulum stress caused by formaldehyde can not be evaluated, and the research on the pathogenic mechanism and the early diagnosis of the neurodegenerative disease can not be realized.
The invention discloses a preparation method of the endoplasmic reticulum positioning formaldehyde response two-photon fluorescence probe, which has the advantages of simple preparation process, high product yield and industrial expanded production and popularization value.
The invention discloses an application of the two-photon fluorescent probe for response of endoplasmic reticulum positioning formaldehyde in detecting the content of endoplasmic reticulum formaldehyde. The two-photon fluorescent probe for endoplasmic reticulum positioning formaldehyde response can realize endoplasmic reticulum positioning and formaldehyde response, and further achieve the purpose of researching the stress of formaldehyde on endoplasmic reticulum. The cytotoxicity experiment also shows that the probe has lower biological toxicity, and the two-photon confocal microscope imaging experiment shows that the probe has good permeability to PC12 cells, can effectively locate endoplasmic reticulum (the locating coefficient is 0.93) in the cells, is suitable for two-photon fluorescence imaging of the content of formaldehyde in the endoplasmic reticulum of the cells, and further realizes monitoring of oxidative stress of the endoplasmic reticulum.
Drawings
FIG. 1 is a graph showing the ultraviolet absorption spectrum and fluorescence emission spectrum of probe SKD-1-HCHO in organic solvents of different polarities; wherein (a) is an ultraviolet absorption spectrum of the probe molecule in Dioxane (Dioxane), Dichloromethane (dichromethane), Dimethylformamide (Dimethylformamide), Dimethyl sulfoxide (Dimethyl sulfoxide), Tetrahydrofuran (Tetrahydrofuran), Toluene (Toluene), Chloroform (Chloroform) and Ethanol (Ethanol), and (b) is a fluorescence emission spectrum of the probe molecule in Dioxane, Dichloromethane, Dimethylformamide, Dimethyl sulfoxide, Tetrahydrofuran, Toluene, Chloroform and Ethanol;
FIG. 2 is a graph of the linear relationship between the fluorescence intensity of probe SKD-1-HCHO and different formaldehyde concentrations;
FIG. 3 is a cross-sectional view showing the effective two-photon absorption of probe SKD-1-HCHO;
FIG. 4 is a graph showing the survival rate of PC12 cells in solutions of probe SKD-1-HCHO at various concentrations;
FIG. 5 is a confocal fluorescence imaging diagram of subcellular organelle co-localization experiment of probe SKD-1-HCHO; wherein (a) represents a case where the probe molecule is stained in PC12 cells for 30min, (b) represents a case where a commercially available ER-Tracker dye is stained in PC12 cells for 30min, (c) represents a superimposed image of (a) and (b), (d) represents intensity scattergrams of two channels corresponding to the endoplasmic reticulum, and (e) represents an ROI intensity distribution map of the endoplasmic reticulum. (f) The probe molecules were stained for 30min in PC12 cells, (g) the commercial Mito-Tracker dye was stained for 30min in PC12 cells, (h) the superimposed images of (f) and (g), (i) the intensity scattergrams of the two channels corresponding to mitochondria, and (j) the ROI intensity distribution map of mitochondria. (k) The probe molecules were stained for 30min in PC12 cells, (l) the commercial Lyso-Tracker dye was stained for 30min in PC12 cells, (m) the superimposed images of (k) and (l), (n) the intensity scattergrams of the two channels corresponding to the lysosome, and (o) the intensity profile of the ROI of the lysosome;
FIG. 6 is a plot of the fluorescence of the cells after 30min incubation of probe SKD-1-HCHO with different concentrations of formaldehyde (0, 1, 2, 4, 8, 10, 15, 20, 25, 30. mu.M); wherein a1), b1), c1), d1), e1), f1), g1), h1), i1), j1) are cell images in the bright field; furthermore, a2), b2), c2), d2), e2), f2), g2), h2), i2), j2) are images in the green fluorescence channel after cell incubation; furthermore, a3), b3), c3), d3), e3), f3), g3), h3), i3), j3) are images of cells after combining the bright field with the green fluorescence channel;
FIG. 7 is a graph showing the linear relationship between fluorescence intensities according to different formaldehyde concentrations in FIG. 6;
FIG. 8 is a two-photon fluorescence imaging of different depths in mouse brain tissue with probe SKD-1-HCHO; wherein (a) is fluorescence imaging of the penetration depth of the tissue in the control group at 260 μm, (b) is fluorescence imaging of the penetration depth of the tissue in the control group at 220 μm, (c) is fluorescence imaging of the penetration depth of the tissue in the control group at 180 μm, (d) is fluorescence imaging of the penetration depth of the tissue in the control group at 140 μm, (e) is fluorescence imaging of the penetration depth of the tissue in the control group at 100 μm, (f) is fluorescence imaging of the penetration depth of the tissue in the formaldehyde group at 260 μm, (g) is fluorescence imaging of the penetration depth of the tissue in the control group at 220 μm, (h) is fluorescence imaging of the penetration depth of the tissue in the formaldehyde group at 180 μm, (i) is fluorescence imaging of the penetration depth of the tissue in the formaldehyde group at 140 μm, (j) is fluorescence imaging of the penetration depth of the tissue in the formaldehyde group at 100 μm, (k) is fluorescence imaging of the penetration depth of the tissue in the anti-inflammatory group at 260 μm, (l) is fluorescence imaging of the penetration depth of the tissue in the anti-inflammatory group at 220 μm, (m) is fluorescence imaging of the penetration depth at 180 μm, (n) fluorescence imaging of tissue penetration depth of 140 μm in the anti-inflammatory group, (o) fluorescence imaging of tissue penetration depth of 100 μm in the anti-inflammatory group;
FIG. 9 is a graph showing the mean fluorescence intensity of brain tissue of rats in the control group, the formalin group and the anti-inflammatory group.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention discloses a two-photon fluorescent probe for endoplasmic reticulum positioning formaldehyde response, which can realize the detection of the content of the endoplasmic reticulum formaldehyde and has the following structural formula:
the invention discloses a preparation method of the endoplasmic reticulum positioning formaldehyde response two-photon fluorescence probe, which comprises the following steps:
step 1: under the condition of stirring, uniformly dispersing 4-tosyl chloride and ethylenediamine in a solvent dichloromethane, heating to react for 15 minutes at 30-50 ℃, and cooling to room temperature. The resulting substituted product mixture was washed twice with distilled water, dried over sodium sulfate and the solvent removed by rotary evaporation to give the product as a white solid, designated compound 1. Wherein the feeding molar ratio of the 4-tosyl chloride to the ethylenediamine is 1: 5-20.
Specifically, in the specific embodiment of the present invention, the feeding molar ratio of 4-tosyl chloride to ethylenediamine is 1: 10, the yield is 75-85%; is 1: 20, the yield is 55-60%; is 1: 8, the yield is 60-70%; is 1; 5, the yield is 50-55%.
Specifically, in the specific embodiment of the present invention, the yield is 25% to 30% at 30 ℃; at 40 ℃, the yield is 70-85%; the yield is 60-70% at 50 ℃.
Step 2: under the condition of stirring, uniformly dispersing the compound 1 and 4-bromo-1, 8-naphthalic anhydride in a solvent in an environment protected by nitrogen, carrying out reflux reaction at the temperature of 70-90 ℃ for 8 hours, and cooling to room temperature. The resulting substitution product mixture was washed twice with distilled water to give a solid powder, which was purified by distillation in a volume ratio of dichloromethane: methanol 20: 1 to obtain a grey solid product which is named as a compound 2. Wherein the feeding molar ratio of the compound 1, 4-bromine-1, 8-naphthalic anhydride is 1: 1-3. The solvent is methanol or ethanol.
Specifically, in a specific embodiment of the present invention, the compound 1, 4-bromo-1, 8-naphthalenic anhydride is fed in a molar ratio of 1:1, the yield is 80-85%; is 1: 2, the yield is 75-80%; is 1: 3, the yield is 65-70%.
Specifically, in the specific embodiment of the invention, the yield is 70-80% at 70 ℃; at 80 ℃, the yield is 80-85%; the yield is 70-75% at 90 ℃.
And step 3: under the condition of stirring, uniformly dispersing the compound 2 and hydrazine hydrate in a solvent, reacting at the temperature of 70-90 ℃ for 6 hours, and cooling to room temperature. And after suction filtration, carrying out vacuum drying on the filter cake to obtain white solid powder, namely the endoplasmic reticulum positioning formaldehyde response two-photon fluorescent probe, which is named as SKD-1-HCHO. Wherein the feeding molar ratio of the compound 2 to the hydrazine hydrate is 1: 1-3. The solvent is methanol or ethanol.
Specifically, in the specific embodiment of the present invention, the feeding molar ratio of the compound 2 and the hydrazine hydrate is 1:1, the yield is 80-85%; is 1: 2, the yield is 75-80%; is 1: 3, the yield is 70-75%.
Specifically, in the specific embodiment of the present invention, the yield is 70% to 75% at 70 ℃, 80% to 85% at 80 ℃, and 75% to 80% at 90 ℃.
The synthetic route of the two-photon fluorescent probe SKD-1-HCHO responding to endoplasmic reticulum positioning formaldehyde is as follows:
a) ethylenediamine, dichloromethane, 40 ℃, 15 minutes; b) 4-bromo-1, 8-naphthalic anhydride, ethanol or methanol, at 80 ℃ for 8 hours; c) hydrazine hydrate, ethanol or methanol, 80 ℃, 6 hours.
The application of the two-photon fluorescent probe for response of endoplasmic reticulum positioning formaldehyde is to realize the in-situ detection of the oxidative stress of the formaldehyde in a leather sample to the endoplasmic reticulum and the evaluation of induced neuroinflammation under the condition of two-photon excitation. The detection and evaluation method comprises the following steps:
the two-photon fluorescent probe responding to endoplasmic reticulum positioning formaldehyde is dissolved in DMSO solution to prepare 0.1mM mother liquor, 5 mu L of the mother liquor and 3mL of solvents with different polarities are respectively taken to obtain ultraviolet spectrograms of the probe SKD-1-HCHO in different solvents. Each set of solutions was then immediately tested for the uv-optimum excitation wavelength, the fluorescence optimum emission wavelength and the corresponding fluorescence quantum yield. To further characterize the response of the probe to formaldehyde, the fluorescence spectra of the probe SKD-1-HCHO were first measured in formaldehyde solutions of different concentrations, all excited at an excitation wavelength of 430 nm. Using two lights simultaneouslyThe two-photon fluorescence cross section of the probe SKD-1-HCHO is determined by a photon excitation fluorescence method. With increasing formaldehyde content. The fluorescence intensity of the probe solution at 550nm is gradually enhanced, and meanwhile, when the concentration of formaldehyde reaches 30 mu M, the fluorescence intensity of the probe gradually tends to be stable and does not change too much, which shows that the reaction of the probe and formaldehyde at the moment tends to be saturated, and a good linear relation exists between the concentration of formaldehyde and 0-20 mu M, wherein the linear equation is that y is 46.58+122.98x, R is20.96. The characterization experiment of the fluorescence intensity and the formaldehyde concentration shows that the probe has excellent response performance on formaldehyde detection.
The two-photon fluorescent probe for endoplasmic reticulum positioning formaldehyde response can realize endoplasmic reticulum positioning and formaldehyde response, and further achieve the purpose of researching the stress of formaldehyde on endoplasmic reticulum. The cytotoxicity experiment also shows that the probe has lower biological toxicity, and the two-photon confocal microscope imaging experiment shows that the probe has good permeability to PC12 cells, can effectively locate endoplasmic reticulum (the locating coefficient is 0.93) in the cells, is suitable for two-photon fluorescence imaging of the content of formaldehyde in the endoplasmic reticulum of the cells, and further realizes monitoring of oxidative stress of the endoplasmic reticulum.
The invention is further illustrated by the following examples and figures.
Example 1:
synthesis of two-photon fluorescent probe molecule SKD-1-HCHO with response of endoplasmic reticulum positioning formaldehyde
Step 1: 4-tosyl chloride (1.90g 10mmol) and ethylenediamine (6.0g 100mmol) were uniformly dispersed in methylene chloride (25mL) as a solvent with stirring, heated at 40 ℃ for 15 minutes and then cooled to room temperature. The resulting substituted product mixture was washed twice with distilled water (25mL), dried over sodium sulfate and the solvent removed by rotary evaporation to give the product as a white solid in 83% yield (1.78 g). Was designated as Compound 1.
Step 2: under stirring, compound 1(0.43g, 2.0mmol) and 4-bromo-1, 8-naphthalenic anhydride (0.56g, 2.0mmol) were uniformly dispersed in ethanol (20mL) under a nitrogen atmosphere, reacted at 80 ℃ for 8 hours, and then cooled to room temperature. The resulting substitution product mixture was washed twice with distilled water (40mL) to give a solid powder, which was purified with dichloromethane: methanol 20: 1 to give the product as a grey solid in 85% yield (0.80 g). Was designated as Compound 2.
And step 3: compound 2(141.9mg, 0.3mmol) and hydrazine hydrate (9.6mg, 0.3mmol) were mixed in absolute ethanol (3mL), the mixture was refluxed at 80 ℃ for 6 hours under nitrogen, then cooled, the solvent was removed by suction filtration, and the cake was dried under vacuum at 40 ℃ for 12 hours. Obtaining a white solid, namely the two-photon fluorescent probe SKD-1-HCHO responding to the endoplasmic reticulum positioning formaldehyde. The yield was 88% (112.06 mg).
1H NMR(600MHz,DMSO-d6)δ8.52(t,J＝7.3Hz,2H),8.31–8.15(m,2H),7.98(t,J＝7.8Hz,1H),7.77(t,J＝6.2Hz,1H),7.56(d,J＝8.0Hz,2H),7.19(d,J＝7.9Hz,2H),4.09(t,J＝6.7Hz,2H),3.10(q,J＝6.4Hz,2H),2.23(s,3H)。
Example 2:
synthesis of two-photon fluorescent probe molecule SKD-1-HCHO with response of endoplasmic reticulum positioning formaldehyde
Step 1: 4-tosyl chloride (1.90g 10mmol) and ethylenediamine (12.0g 200mmol) were uniformly dispersed in methylene chloride (25mL) as a solvent with stirring, heated at 30 ℃ for 15 minutes and then cooled to room temperature. The resulting substituted product mixture was washed twice with distilled water (25mL), dried over sodium sulfate and the solvent removed by rotary evaporation to give the product as a white solid, designated Compound 1. Wherein the feeding molar ratio of the 4-tosyl chloride to the ethylene diamine is 1: 20.
step 2: under stirring, compound 1(0.43g, 2.0mmol) and 4-bromo-1, 8-naphthalenic anhydride (1.12g, 4.0mmol) were uniformly dispersed in ethanol (20mL) under a nitrogen atmosphere, reacted at 70 ℃ for 8 hours, and then cooled to room temperature. The resulting substitution product mixture was washed twice with distilled water (40mL) to give a solid powder, which was purified with dichloromethane: methanol 20: 1 to obtain a grey solid product which is named as a compound 2.
And step 3: compound 2(141.9mg, 0.3mmol) and hydrazine hydrate (28.8mg, 0.9mmol) were mixed in absolute ethanol (3mL), the mixture was refluxed at 70 ℃ for 6 hours under nitrogen, then cooled, the solvent was removed by suction filtration, and the cake was dried under vacuum at 40 ℃ for 12 hours. Obtaining a white solid, namely the two-photon fluorescent probe SKD-1-HCHO responding to the endoplasmic reticulum positioning formaldehyde.
Example 3:
synthesis of two-photon fluorescent probe molecule SKD-1-HCHO with response of endoplasmic reticulum positioning formaldehyde
Step 1: 4-tosyl chloride (1.90g 10mmol) and ethylenediamine (4.8g 80mmol) were uniformly dispersed in dichloromethane (25mL) as a solvent with stirring, heated at 50 ℃ for 15 minutes and then cooled to room temperature. The resulting substituted product mixture was washed twice with distilled water (25mL), dried over sodium sulfate and the solvent removed by rotary evaporation to give the product as a white solid, designated Compound 1.
Step 2: under stirring, compound 1(0.43g, 2.0mmol) and 4-bromo-1, 8-naphthalenic anhydride (1.68g, 6.0mmol) were uniformly dispersed in ethanol (20mL) under a nitrogen atmosphere, reacted at 90 ℃ for 8 hours, and then cooled to room temperature. The resulting substitution product mixture was washed twice with distilled water (40mL) to give a solid powder, which was purified with dichloromethane: methanol 20: 1 to obtain a grey solid product which is named as a compound 2.
And step 3: compound 2(141.9mg, 0.3mmol) and hydrazine hydrate (19.2mg, 0.6mmol) were mixed in absolute ethanol (3mL), the mixture was refluxed at 90 ℃ for 6 hours under nitrogen, then cooled, the solvent was removed by suction filtration, and the cake was dried under vacuum at 40 ℃ for 12 hours. Obtaining a white solid, namely the two-photon fluorescent probe SKD-1-HCHO responding to the endoplasmic reticulum positioning formaldehyde.
Example 4:
synthesis of two-photon fluorescent probe molecule SKD-1-HCHO with response of endoplasmic reticulum positioning formaldehyde
Step 1: 4-tosyl chloride (1.90g 10mmol) and ethylenediamine (6.0g 100mmol) were uniformly dispersed in methylene chloride (25mL) as a solvent with stirring, heated at 30 ℃ for 15 minutes and then cooled to room temperature. The resulting substituted product mixture was washed twice with distilled water (25mL), dried over sodium sulfate and the solvent removed by rotary evaporation to give the product as a white solid, designated Compound 1.
Step 2: under stirring, compound 1(0.43g, 2.0mmol) and 4-bromo-1, 8-naphthalenic anhydride (0.56g, 2.0mmol) were uniformly dispersed in ethanol (20mL) under a nitrogen atmosphere, reacted at 90 ℃ for 8 hours, and then cooled to room temperature. The resulting substitution product mixture was washed twice with distilled water (40mL) to give a solid powder, which was purified with dichloromethane: methanol 20: 1 to obtain a grey solid product which is named as a compound 2.
And step 3: compound 2(141.9mg, 0.3mmol) and hydrazine hydrate (9.6mg, 0.3mmol) were mixed in absolute ethanol (3mL), the mixture was refluxed at 90 ℃ for 6 hours under nitrogen, then cooled, the solvent was removed by suction filtration, and the cake was dried under vacuum at 40 ℃ for 12 hours. Obtaining a white solid, namely the two-photon fluorescent probe SKD-1-HCHO responding to the endoplasmic reticulum positioning formaldehyde.
Example 5:
synthesis of two-photon fluorescent probe molecule SKD-1-HCHO with response of endoplasmic reticulum positioning formaldehyde
Step 1: 4-tosyl chloride (1.90g 10mmol) and ethylenediamine (3.0g 50mmol) were uniformly dispersed in dichloromethane (25mL) as a solvent with stirring, heated at 50 ℃ for 15 minutes and then cooled to room temperature. The resulting substituted product mixture was washed twice with distilled water (25mL), dried over sodium sulfate and the solvent removed by rotary evaporation to give the product as a white solid, designated Compound 1.
Step 2: under stirring, compound 1(0.43g, 2.0mmol) and 4-bromo-1, 8-naphthalenic anhydride (0.56g, 2.0mmol) were uniformly dispersed in ethanol (20mL) under a nitrogen atmosphere, reacted at 70 ℃ for 8 hours, and then cooled to room temperature. The resulting substitution product mixture was washed twice with distilled water (40mL) to give a solid powder, which was purified with dichloromethane: methanol 20: 1 to obtain a grey solid product which is named as a compound 2.
And step 3: compound 2(141.9mg, 0.3mmol) and hydrazine hydrate (9.6mg, 0.3mmol) were mixed in absolute ethanol (3mL), the mixture was refluxed at 70 ℃ for 6 hours under nitrogen, then cooled, the solvent was removed by suction filtration, and the cake was dried under vacuum at 40 ℃ for 12 hours. Obtaining a white solid, namely the two-photon fluorescent probe SKD-1-HCHO responding to the endoplasmic reticulum positioning formaldehyde.
Example 6:
spectrum test of two-photon fluorescent probe molecule with endoplasmic reticulum positioning formaldehyde response
The two-photon fluorescent probe with endoplasmic reticulum positioning formaldehyde response prepared in the embodiment 1 of the invention is dissolved in DMSO solution to prepare 0.1mM mother liquor, 5 mul of the mother liquor and 3mL of solvents with different polarities are respectively taken to obtain the ultraviolet absorption spectrum and the fluorescence emission spectrum of the probe SKD-1-HCHO in different solvents, and the ultraviolet absorption spectrum and the fluorescence emission spectrum are shown in figure 1. As shown in FIG. 1, under excitation of 430nm wavelength, the fluorescence intensity of the probe in PBS solution is the lowest, and the ultraviolet absorption wavelength of the probe is generally consistent among different solvents, so that the condition of PET occurrence can be best met by performing photophysical test on the probe in PBS solution.
Example 7:
to further verify the response ability of the probe to formaldehyde, the probes prepared in example 1 of the present invention were all tested in PBS solution (10mM, pH 7.4, containing 1% DMSO) to detect formaldehyde content in vitro. 2mg of probe SKD-1-HCHO was weighed and dissolved in 415. mu.L of DMSO and placed in an ep tube to obtain a 10-3mol/L probe stock solution. The probe stock was further diluted to 0.1mM with pure DMSO. Meanwhile, the purchased 37% formaldehyde solution is respectively diluted to 10-3mol/L and 10-4mol/L to be used as stock solutions. 300 mu L of the probe stock solution is measured and placed in a 2mL centrifuge tube, FA stock solutions (0, 5, 10, 15, 20, 30, 50, 60, 100, 110 mu M) with different concentrations are respectively and sequentially added into the centrifuge tube, and finally, the volume is increased to 3mL by PBS for detecting the fluorescence intensity. Unless otherwise specified, all fluorescence measurements were taken with excitation wavelength of 430nm and excitation and emission slit widths of 5nm to record changes in fluorescence spectra. As shown in figure 2, the probe SKD-1-HCHO has a good linear relation with the formaldehyde concentration of 0-20 mu M, and the linear equation is that y is 46.58+122.98x, R20.96. The characterization experiment of the fluorescence intensity and the formaldehyde concentration shows that the probe has excellent response performance on formaldehyde detection.
Example 8:
two-photon performance test of two-photon fluorescent probe molecule with response of formaldehyde positioned by endoplasmic reticulum
It was tested that the two-photon active cross-sections of the probe SKD-1-HCHO prepared in example 1 of the present invention in a pure PBS solution and in a solution added with formaldehyde are shown in the figure, and compared with the pure probe set, the two-photon active cross-section of the probe SKD-1-HCHO after the addition of formaldehyde appears at 550 nm. And as shown in FIG. 3, the two-photon cross section of the probe increased to 180GM after the addition of formaldehyde. The above demonstrates the ability of probe SKD-1-HCHO to be used for detecting formaldehyde and two-photon bioimaging.
Example 9:
cytotoxicity test
Cytotoxicity experiments were performed using the MTT (5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) method. When the probe SKD-1-HCHO (0, 5, 10, 20, 30. mu.M) prepared in example 1 was co-cultured with PC12 cells at various concentrations, as shown in FIG. 4, the toxicity of the probe SKD-1-HCHO to PC12 cells as a whole decreased the survival rate of the cells with increasing probe concentration, but the survival rate of PC12 cells was still 70% even at 30. mu.M. This indicates that the probe has low toxicity to cells and can be used for two-photon imaging experiments of biological samples.
Example 10:
organelle localization experiments
In order to further study the co-localization performance of subcellular organelles of the probe SKD-1-HCHO, the co-localization performance of the probe in endoplasmic reticulum, mitochondria and lysosome respectively is studied by co-staining the probe SKD-1-HCHO prepared in example 1 with commercial fluorescent dyes ER-Tracker Green, Mita-Tracker Green and Lyso-Tracker Green respectively. PC12 cells were incubated with FA (500. mu.M) for 30min, PC12 cells were washed three times with PBS, the washed cells were incubated with ER-Tracker Green (100nM), Mita-Tracker Green (100nM) and Lyso-Tracker Green (100nM) respectively with 10. mu.M probes SKD-1-HCHO for 30min, the PC12 cells were washed three times with PBS and finally subjected to fluorescence confocal imaging, which shows the imaging of the cells treated with ER-Tracker Green, Mita-Tracker Green and Ly-Tracker Green in the Green and red channels, respectively. As shown in FIG. 5, it can be seen that probe SKD-1-HCHO has the best co-localization in endoplasmic reticulum, the overlapping area of the green channel and the red channel is the largest, and the co-localization coefficient is 0.93. These data indicate that the probe can be well localized to the endoplasmic reticulum of the cell and image the FA of the endoplasmic reticulum.
Example 11:
in-situ detection of formaldehyde content in leather sample and evaluation of induced neuroinflammation
After exploring and proving that the two-photon fluorescent probe SKD-1-HCHO responding to the endoplasmic reticulum positioning formaldehyde prepared in the embodiment 1 of the invention has lower toxicity to cells, good light stability and good endoplasmic reticulum positioning capability, the two-photon fluorescent probe SKD-1-HCHO responding to the endoplasmic reticulum positioning formaldehyde is used for detecting the response of exogenous FA of the cells. PC12 cells were preincubated with N-ethylmaleimide (NEM, a widely used thiol blocking agent) at a concentration of 30 μ M for 30 minutes and then incubated with SKD-1-HCHO (5 μ M) for 30 minutes, as shown in FIG. 6, and hardly any fluorescence was observed in the green channel of the cells. In contrast, PC12 cells preincubated with NEM were incubated with SKD-1-HCHO (10. mu.M) for 30 minutes and then with different concentrations of formaldehyde (0, 1, 2, 4, 8, 10, 15, 20, 25, 30. mu.M) and PC12 cells incubated for 30 minutes showed fluorescence in the green channel (450-570 nm) with the intensity of fluorescence increasing with the increase in formaldehyde concentration, indicating that SKD-1-HCHO has good cell penetration ability and can image intracellular FA. FIG. 7 is a linear relationship graph of fluorescence intensity corresponding to different formaldehyde concentrations, wherein the linear equation is that y is 273.38225+64.13234x, R20.9685. The establishment of the linear equation provides an important clue for measuring the unknown content of formaldehyde in the leather sample.
Example 12:
detection of formaldehyde content in mouse brain
To explore the original imaging ability of the probe in the process of simulating and inducing Traumatic Brain Injury (TBI) of the mouse, the fluorescence intensity change of different depths of the brain tissue of the mouse is studied by a two-photon confocal Z-Stack technology (as shown in FIG. 8). By scanning the brain tissue layer by layer, it can be seen that there is essentially no fluorescence intensity at penetration depths below 100 μm, presumably due to PBS washing away probes that have not entered the tissue while washing the brain tissue cells. And when the penetration depth is 260 mu m, the fluorescence intensity is not high, which indicates that the penetration imaging depth of the mouse brain tissue experiment is 260 mu m. In the bar chart of FIG. 9, the fluorescence intensity of brain tissue with NS-398 added was the strongest compared to the fluorescence intensities of the other two groups, because the anti-inflammatory effect of NS-398 increased the free formaldehyde content in brain tissue, and the slightly increased fluorescence intensity compared to brain tissue with formaldehyde alone indicates that NS-398 has an inhibitory effect on formaldehyde-induced inflammation in mouse brain tissue.
In summary, the invention provides a two-photon fluorescent probe with an endoplasmic reticulum positioning formaldehyde response, and the structural formula is as follows:
the probe has good responsiveness and sensitivity to formaldehyde, and the role of formaldehyde in inducing endoplasmic reticulum stress and further causing inflammation of a living body is proved by PC12 cells. Promotes the understanding of the relationship between formaldehyde and endoplasmic reticulum stress. Meanwhile, the invention provides a synthesis method of the probe, the steps are simple, and the probe has endoplasmic reticulum targeting property. Has application prospect in further understanding and exploring endoplasmic reticulum stress, cell inflammation and other aspects.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
1. A two-photon fluorescent probe with response to endoplasmic reticulum positioning formaldehyde is characterized in that the structural formula is as follows:
2. the method for preparing the endoplasmic reticulum-localized formaldehyde-responsive two-photon fluorescent probe of claim 1, which comprises the following steps:
(1) uniformly dispersing 4-tosyl chloride and ethylenediamine in a solvent A for substitution reaction, and separating and purifying the obtained substitution product mixture after the substitution reaction is finished to obtain a compound 1;
(2) uniformly dispersing the compound 1 and 4-bromo-1, 8-naphthalic anhydride in a solvent B, carrying out substitution reaction under the condition a, and separating and purifying an obtained product mixture after the substitution reaction is finished to obtain a compound 2;
(3) and (3) uniformly dispersing the compound 2 and hydrazine hydrate in a solvent C for substitution reaction, and after the reaction is finished, separating and purifying the obtained product mixture to obtain the endoplasmic reticulum positioning formaldehyde response two-photon fluorescence probe SKD-1-HCHO.
3. The method according to claim 2, wherein in the step (1), the molar ratio of 4-toluenesulfonyl chloride to ethylenediamine is 1:5 to 20.
4. The preparation method according to claim 2, wherein in the step (2), the molar ratio of the compound 1 to the 4-bromo-1, 8-naphthalic anhydride is 1: 1-3.
5. The method according to claim 2, wherein in the step (3), the molar ratio of the compound 2 to the hydrazine hydrate is 1:1 to 3.
6. The method according to claim 2, wherein in the step (1), the temperature of the substitution reaction is 30 to 50 ℃;
in the step (2), the temperature of the substitution reaction is 70-90 ℃.
7. The method according to claim 2, wherein the temperature of the substitution reaction in the step (3) is 70 to 90 ℃.
8. The process according to claim 2, wherein in the step (1), the solvent A is dichloromethane; in the step (2), the solvent B is ethanol or methanol; in the step (3), the solvent C is ethanol or methanol.
9. The method according to claim 2, wherein in the step (2), the condition a is an inert atmosphere protection reaction.
10. The use of the two-photon fluorescent probe with response to formaldehyde for endoplasmic reticulum localization according to claim 1 or the two-photon fluorescent probe with response to formaldehyde for endoplasmic reticulum localization prepared by the preparation method according to any one of claims 2 to 9 in the detection of the content of formaldehyde in endoplasmic reticulum.
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|---|---|---|---|---|
| CN105542756A (en) * | 2016-02-03 | 2016-05-04 | 浙江理工大学 | Fluorescent probe for detecting formaldehyde as well as preparation method and application of fluorescent probe |
| CN106008342A (en) * | 2016-05-20 | 2016-10-12 | 济南大学 | Fluorescent probe for detecting formaldehyde in cytolysosome and preparation method thereof |
| CN106518762A (en) * | 2016-11-02 | 2017-03-22 | 济南大学 | Fluorescent probe for detecting formaldehyde in cell endoplasmic reticulums |
| CN110643355A (en) * | 2019-09-19 | 2020-01-03 | 济南大学 | Fluorescent probe for detecting polarity of endoplasmic reticulum as well as preparation method and application thereof |
-
2021
- 2021-08-31 CN CN202111015061.1A patent/CN113603644A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105542756A (en) * | 2016-02-03 | 2016-05-04 | 浙江理工大学 | Fluorescent probe for detecting formaldehyde as well as preparation method and application of fluorescent probe |
| CN106008342A (en) * | 2016-05-20 | 2016-10-12 | 济南大学 | Fluorescent probe for detecting formaldehyde in cytolysosome and preparation method thereof |
| CN106518762A (en) * | 2016-11-02 | 2017-03-22 | 济南大学 | Fluorescent probe for detecting formaldehyde in cell endoplasmic reticulums |
| CN110643355A (en) * | 2019-09-19 | 2020-01-03 | 济南大学 | Fluorescent probe for detecting polarity of endoplasmic reticulum as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SONG-JIAO LI等: "Efficient Two-Photon Fluorescent Probe for Imaging of Nitric Oxide during Endoplasmic Reticulum Stress", 《ACS SENS.》 * |
| 唐永和: "新型活性羰基(RCS)小分子荧光探针的设计、合成及其成像研究", 《济南大学博士学位论文》 * |
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