CN114181171A - Fluorescent probe for early diagnosis of Alzheimer's disease - Google Patents
Fluorescent probe for early diagnosis of Alzheimer's disease Download PDFInfo
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- CN114181171A CN114181171A CN202111581474.6A CN202111581474A CN114181171A CN 114181171 A CN114181171 A CN 114181171A CN 202111581474 A CN202111581474 A CN 202111581474A CN 114181171 A CN114181171 A CN 114181171A
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- C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
- C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
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- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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Abstract
The invention discloses a fluorescent probe for early diagnosis of Alzheimer's disease, which has a structural formula as follows:
the preparation method comprises the following steps: (1) dissolving methylene blue in dichloromethane and water, adding sodium hydrosulfite and sodium bicarbonate, stirring, separating, extracting, combining and drying; (2) adding triethylamine, adding a dichloromethane solution containing triphosgene TPG, and stirring for reaction; (3) adding 4-aminomethyl phenol and triethylamine, stirring, adding dichloromethane, washing, drying, distilling, and purifying by silica gel chromatography; (4) dissolving in dichloromethane, adding dichloromethaneAdding triethylamine into the diluted cyclopropyl formyl chloride, stirring overnight, washing, and purifying by column chromatography to obtain the final product. The fluorescent probe can smoothly enter living cells or organisms to detect butyrylcholinesterase and active oxygen, has high sensitivity, and has important application prospect for early diagnosis of Alzheimer's disease.
Description
Technical Field
The invention relates to the technical field of detection of enzyme activity and active oxygen content, in particular to a dual-response fluorescent probe, a preparation method thereof and application thereof in detection of butyrylcholinesterase and active oxygen in patients with Alzheimer's disease.
Background
Alzheimer's Disease (AD) is a common neurodegenerative disease with cognitive and memory disorders that are a global concern today and a serious threat to the health of humans, especially the elderly. Unfortunately, there is no effective treatment to prevent or reverse the progression of the disease to date, and existing treatments only alleviate the current symptoms. In the asymptomatic long-term preclinical phase (10-20 years), the pathology becomes unknowingly irreversible, which leads to failure of drug therapy in later dosing and to severe disability and death.
Thus, early diagnosis of alzheimer's disease may provide the possibility for therapeutic intervention in devastating diseases. Several techniques have been used in the diagnosis of alzheimer's disease over several decades of endeavors, such as Computed Tomography (CT), Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), Single Photon Emission Computed Tomography (SPECT), and optical imaging. The methods commonly used in medicine, such as CT and PET, are mainly used to image plaques or tangles in the brain, that is, once found, the middle and late stages of alzheimer's disease.
Compared with the widely used imaging technology based on radioactive nuclides, the optical imaging tool has the advantages of being not negligible, including simple and convenient operation, low cost, no nuclear pollution and the like. The organic fluorescent receptor has been well developed for imaging by manipulating reaction sites, binding spaces and signal portions due to its synthetic properties to various objects, has a wide commercial value, and is an analysis method for biomarker analysis of alzheimer's disease, which is currently of application.
Therefore, how to provide a fluorescent probe with high sensitivity and high selectivity is a problem that needs to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of the above, the present invention aims to provide a fluorescent probe for early diagnosis of alzheimer's disease, a preparation method thereof and an application thereof, and in particular, to a dual-response fluorescent probe for detection of butyrylcholinesterase and active oxygen in alzheimer's disease patients, so as to solve the defects in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fluorescent probe for early diagnosis of Alzheimer's disease has a structural formula as follows:
the fluorescent probe takes butyrylcholine esterase and active oxygen as detection biomarkers, methylene blue as a fluorescent signal reporting group, a cyclopropyl formyl ester bond as an action site of the butyrylcholine esterase, and the other phenolic hydroxyl group for identifying the active oxygen is connected through the ester bond, so that the aim of double detection of the butyrylcholine esterase and the active oxygen is fulfilled.
A preparation method of a fluorescent probe for early diagnosis of Alzheimer's disease comprises the following steps:
(1) dissolving methylene blue in a mixed solution of dichloromethane and water at room temperature, adding mixed powder of sodium hydrosulfite and sodium bicarbonate, stirring until the water phase changes from blue to khaki, separating an organic layer, extracting the water layer with dichloromethane, combining the organic layers, and drying with anhydrous sodium sulfate to obtain reduced methylene blue R-MB;
(2) adding reduced methylene blue R-MB into triethylamine, cooling to 0 ℃, adding a dichloromethane solution containing triphosgene TPG, and stirring at room temperature to react to obtain an intermediate MB-Cl;
(3) adding 4-aminomethyl phenol and triethylamine into the intermediate MB-Cl, stirring overnight, adding dichloromethane, washing with water, drying an organic phase with anhydrous sodium sulfate, distilling off the solvent to obtain a crude product, and purifying by silica gel chromatography to obtain a compound P02 in the form of a white solid;
(4) dissolving the compound P02 in dichloromethane at 0 ℃, adding cyclopropyl formyl chloride diluted by dichloromethane, adding triethylamine, stirring overnight at room temperature, washing with saturated sodium bicarbonate solution, and purifying the crude product after removing the solvent by column chromatography to obtain the fluorescent probe P2 in the form of white powder.
The synthetic route of the preparation method of the fluorescent probe is as follows:
the synthesized double-response fluorescent probe is used for identifying and detecting butyrylcholine esterase and active oxygen, and the structure of the probe contains a cyclopropyl formyl ester bond of an identification site of the butyrylcholine esterase, and the other phenolic hydroxyl group for identifying the active oxygen is connected through the ester bond; the redox action of the active oxygen can be activated only after the butyrylcholinesterase catalyzed enzymatic reaction; methylene blue is used as a report unit and is connected with the logic reaction unit through an amido bond; only through the successive and reactive processes can the bond removal be driven to form CO2Generating emission methylene blue as near infrared fluorescence indicator.
Further, in the step (1), the molar ratio of methylene blue to sodium dithionite to sodium bicarbonate is 1: (1.5-2.5): (1.5-2.5); the stirring time was 20 min.
The beneficial effect of adopting the further technical scheme is that the oxidized methylene blue is reduced by the reducing agent sodium dithionite to form the reduced methylene blue under the alkaline environment provided by the sodium bicarbonate.
Further, in the step (2), the molar ratio of the reduced methylene blue R-MB, triethylamine and triphosgene TPG is 1: (1.2-1.5): (2.5-3.5); the reaction time was 0.5h with stirring.
The further technical scheme has the beneficial effects that the reduced methylene blue R-MB reacts with the triphosgene TPG to form an intermediate MB-Cl, and the organic base triethylamine is used as an acid-binding agent.
Further, in the step (3), the molar ratio of the intermediate MB-Cl, the 4-aminomethyl phenol and the triethylamine is 1: (1.5-2.5): (1.5-2.5).
The further technical scheme has the beneficial effects that the intermediate MB-Cl and 4-aminomethyl phenol are subjected to acylation reaction, and triethylamine is used as an acid-binding agent.
Further, in the step (4), the molar ratio of the compound P02, the cyclopropyl carbonyl chloride and the triethylamine is 1: (1-1.2): (2.5-3.5).
The further technical scheme has the beneficial effects that the compound P02 and cyclopropyl formyl chloride are subjected to esterification reaction to form cyclopropyl formic ether, and triethylamine is used as an acid-binding agent.
The invention also provides application of the fluorescent probe or the fluorescent probe prepared by the preparation method in detection of butyrylcholine esterase and active oxygen.
Further, in the above-mentioned detection process, the reaction concentration of the fluorescent probe was 10. mu. mol. L-1The reaction temperature was 37 ℃ and the reaction system was PBS buffer solution with pH 7.4, and the reaction time was 60 min.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. aiming at the reaction of synthesizing the fluorescent probe, the proportion of various raw materials is obtained through creative tests, and the proportion of triethylamine and triphosgene is particularly important, wherein the triethylamine influences the acid-base regulation of the reaction and is related to whether the reaction can be smoothly carried out; the content of triphosgene directly influences the degree of reaction, and is related to the reaction completion and the excess treatment step.
2. The inventor respectively characterizes by means of nuclear magnetic resonance hydrogen spectrum, carbon spectrum, mass spectrum and the like, and shows that the ratio type fluorescent probe is successfully synthesized.
3. The fluorescent probe can efficiently and selectively identify butyrylcholine esterase and active oxygen in a PBS buffer solution system, and has high sensitivity.
4. The fluorescent probe can smoothly enter living cells or organisms to detect butyrylcholinesterase and active oxygen, has high sensitivity, and has important application prospect for early diagnosis of Alzheimer's disease.
Drawings
FIG. 1 shows a fluorescent probe synthesized in example 1 of the present invention1H NMR spectrum;
FIG. 2 shows a fluorescent probe synthesized in example 1 of the present invention13A C NMR spectrum;
FIG. 3 is a mass spectrum of a fluorescent probe synthesized in example 1 of the present invention;
FIG. 4 is an absorption spectrum and a fluorescence emission spectrum of the fluorescent probe synthesized in example 1 of the present invention after reacting with butyrylcholinesterase and active oxygen;
FIG. 5 is a histogram of fluorescence intensity at the maximum emission wavelength of a fluorescent probe synthesized in example 1 of the present invention after treatment with active oxygen and addition of butyrylcholinesterase and various substances;
FIG. 6 is a fluorescence emission spectrum of the fluorescent probe synthesized in example 1 according to the present invention after being treated with active oxygen and added with butyrylcholinesterase at different concentrations;
FIG. 7 is a confocal fluorescence image of HEK293 cells synthesized by the fluorescent probe in example 1;
FIG. 8 is a fluorescence image of C57BL/6j mice and APP/PS1(B6) mice injected with the fluorescent probe synthesized in example 1 and with APP/PS1(B6) mice pre-treated with a drug for 0.5h and then injected with the fluorescent probe synthesized in example 1.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
Example 1
The preparation method of the fluorescent probe for early diagnosis of Alzheimer's disease comprises the following steps:
(1) dissolving methylene blue in a mixed solution of dichloromethane and water at room temperature, adding mixed powder of sodium hydrosulfite and sodium bicarbonate, stirring for 20min until the water phase turns from blue to khaki, separating an organic layer, extracting the water layer with dichloromethane, combining the organic layers, and drying with anhydrous sodium sulfate to obtain reduced methylene blue R-MB;
wherein the molar ratio of methylene blue to sodium dithionite to sodium bicarbonate is 1: 2: 2;
(2) adding reduced methylene blue R-MB into triethylamine, cooling to 0 ℃, adding a dichloromethane solution containing triphosgene TPG, and stirring at room temperature for reaction for 0.5h to obtain an intermediate MB-Cl;
wherein the molar ratio of the reduced methylene blue R-MB, the triethylamine and the triphosgene TPG is 1: 1.4: 3;
(3) adding 4-aminomethyl phenol and triethylamine into the intermediate MB-Cl, stirring overnight, adding dichloromethane, washing with water, drying an organic phase with anhydrous sodium sulfate, distilling off the solvent to obtain a crude product, and purifying by silica gel chromatography to obtain a compound P02 in the form of a white solid;
wherein the molar ratio of the intermediate MB-Cl, the 4-aminomethyl phenol and the triethylamine is 1: 2: 2;
(4) dissolving a compound P02 in dichloromethane at 0 ℃, adding cyclopropyl formyl chloride diluted by dichloromethane, adding triethylamine, stirring overnight at room temperature, washing with a saturated sodium bicarbonate solution, and purifying a crude product after removing the solvent by column chromatography to obtain a white powder-form fluorescent probe P2, wherein the structural formula is as follows:
wherein the mol ratio of the compound P02, the cyclopropyl formyl chloride and the triethylamine is 1: 1.1: 3.
example 2
The preparation method of the fluorescent probe for early diagnosis of Alzheimer's disease comprises the following steps:
(1) dissolving methylene blue in a mixed solution of dichloromethane and water at room temperature, adding mixed powder of sodium hydrosulfite and sodium bicarbonate, stirring for 20min until the water phase turns from blue to khaki, separating an organic layer, extracting the water layer with dichloromethane, combining the organic layers, and drying with anhydrous sodium sulfate to obtain reduced methylene blue R-MB;
wherein the molar ratio of methylene blue to sodium dithionite to sodium bicarbonate is 1: 1.5: 2.5;
(2) adding reduced methylene blue R-MB into triethylamine, cooling to 0 ℃, adding a dichloromethane solution containing triphosgene TPG, and stirring at room temperature for reaction for 0.5h to obtain an intermediate MB-Cl;
wherein the molar ratio of the reduced methylene blue R-MB, the triethylamine and the triphosgene TPG is 1: 1.2: 3.5;
(3) adding 4-aminomethyl phenol and triethylamine into the intermediate MB-Cl, stirring overnight, adding dichloromethane, washing with water, drying an organic phase with anhydrous sodium sulfate, distilling off the solvent to obtain a crude product, and purifying by silica gel chromatography to obtain a compound P02 in the form of a white solid;
wherein the molar ratio of the intermediate MB-Cl, the 4-aminomethyl phenol and the triethylamine is 1: 1.5: 2.5;
(4) dissolving a compound P02 in dichloromethane at 0 ℃, adding cyclopropyl formyl chloride diluted by dichloromethane, adding triethylamine, stirring overnight at room temperature, washing with a saturated sodium bicarbonate solution, and purifying a crude product after removing the solvent by column chromatography to obtain a white powder-form fluorescent probe P2, wherein the structural formula is as follows:
wherein the mol ratio of the compound P02, the cyclopropyl formyl chloride and the triethylamine is 1: 1: 3.5.
example 3
The preparation method of the fluorescent probe for early diagnosis of Alzheimer's disease comprises the following steps:
(1) dissolving methylene blue in a mixed solution of dichloromethane and water at room temperature, adding mixed powder of sodium hydrosulfite and sodium bicarbonate, stirring for 20min until the water phase turns from blue to khaki, separating an organic layer, extracting the water layer with dichloromethane, combining the organic layers, and drying with anhydrous sodium sulfate to obtain reduced methylene blue R-MB;
wherein the molar ratio of methylene blue to sodium dithionite to sodium bicarbonate is 1: 2.5: 1.5;
(2) adding reduced methylene blue R-MB into triethylamine, cooling to 0 ℃, adding a dichloromethane solution containing triphosgene TPG, and stirring at room temperature for reaction for 0.5h to obtain an intermediate MB-Cl;
wherein the molar ratio of the reduced methylene blue R-MB, the triethylamine and the triphosgene TPG is 1: 1.5: 2.5;
(3) adding 4-aminomethyl phenol and triethylamine into the intermediate MB-Cl, stirring overnight, adding dichloromethane, washing with water, drying an organic phase with anhydrous sodium sulfate, distilling off the solvent to obtain a crude product, and purifying by silica gel chromatography to obtain a compound P02 in the form of a white solid;
wherein the molar ratio of the intermediate MB-Cl, the 4-aminomethyl phenol and the triethylamine is 1: 2.5: 1.5;
(4) dissolving a compound P02 in dichloromethane at 0 ℃, adding cyclopropyl formyl chloride diluted by dichloromethane, adding triethylamine, stirring overnight at room temperature, washing with a saturated sodium bicarbonate solution, and purifying a crude product after removing the solvent by column chromatography to obtain a white powder-form fluorescent probe P2, wherein the structural formula is as follows:
wherein the mol ratio of the compound P02, the cyclopropyl formyl chloride and the triethylamine is 1: 1.2: 2.5.
performance testing
1. Test analysis
The fluorescent probe P2 synthesized in example 1 was used to perform the following procedures1H NMR spectrum,13C NMR spectrum and mass spectrum analysis. The results are shown in FIGS. 1 to 3.
FIG. 1 shows a fluorescent probe P2 synthesized in example 11H NMR spectrum. As can be seen from fig. 1, the specific spectral peaks are:1H NMR(500MHz,DMSO-d6) δ (ppm)7.28(d, J ═ 8.8Hz,2H),7.25(d, J ═ 8.4Hz,2H),7.03(d, J ═ 8.4Hz,2H),6.72(d, J ═ 2.7Hz,2H),6.66(dd, J ═ 8.8,2.6Hz,3H),4.21(d, J ═ 5.9Hz,2H),2.88(s,12H),1.87(dd, J ═ 12.3,8.0,4.7Hz,1H), 1.04-1.02 (m,2H),0.99(dd, J ═ 7.4,3.0Hz,2H), which corresponds to the probe group, which can prove successful probe synthesis.
FIG. 2 shows a fluorescent probe P2 synthesized in example 113C NMR spectrum. As can be seen from fig. 2, the specific spectral peaks are:13C NMR(214MHz,DMSO-d6) Delta (ppm)176.06,173.47,156.02,149.45,149.03,138.73,133.76,128.92,128.37,127.98,121.82,111.69,110.80,46.05,43.64,40.71,13.03,12.91,9.42,8.14, which corresponds to the probe group, further confirming the correct structure of the probe.
FIG. 3 is a mass spectrum of the fluorescent probe P2 synthesized in example 1. As can be seen from fig. 3, the specific information is: [ M + H ]]+：Chemical Formula:C28H31N4O3S+Exact masses: 503.2134, corresponding to the probe fragments, further confirmed that the probes were structurally correct.
2. Research on recognition performance of fluorescent probe on butyrylcholine esterase and active oxygen in buffer solution
The fluorescent probe synthesized in example 1 was adjusted to 5.0 mmol. multidot.L -12. mu.L of the aqueous solution of (4) was added to a buffer solution containing 1ml of LPBS (10 mmol. multidot.L)-1pH 7.4) in a centrifuge tube with 1 mM. L-1Active oxygen treatment, and addition of 250. mu.g/mL-1Butyrylcholinesterase was incubated at 37 ℃ for 60min and the changes in the absorption spectrum and fluorescence emission spectrum were detected. The results are shown in FIG. 4.
FIG. 4 is an absorption spectrum and a fluorescence emission spectrum before and after the reaction of the fluorescent probe synthesized in example 1 with butyrylcholinesterase and active oxygen. Wherein,the black curve shows fluorescent probe P2 (10. mu. mol. L)-1) The blue curve shows fluorescent probe P2 (10. mu. mol. L)-1) And reactive oxygen species ROS (1 mmol. L)-1) Green curve shows fluorescent probe P2 (10. mu. mol. L)-1) And butyrylcholinesterase BChE (250. mu.g.mL)-1) The red curve of the mixed solution (5) shows fluorescent probe P2 (10. mu. mol. L)-1) Reactive oxygen species ROS (1 mmol. L)-1) And butyrylcholinesterase BChE (250. mu.g.mL)-1) The mixed solution of (1).
As can be seen from FIG. 4, the fluorescent probe itself showed little reaction to butyrylcholinesterase and active oxygen, respectively, whereas when both butyrylcholinesterase and active oxygen were present, significant absorption occurred at 656nm, accompanied by a sharp increase in fluorescence centered around 690 nm.
Thus, the fluorescent probe of the present invention can react with butyrylcholinesterase and active oxygen in the presence of both. The determinant of this logical receptor is the coexistence of two reactive species, which improves the diagnostic accuracy of the target disease.
3. Adding active oxygen into a buffer solution for processing, adding different substances into the buffer solution, and testing the reaction capacity of butyrylcholinesterase
The fluorescent probe synthesized in example 1 was adjusted to 5.0 mmol. multidot.L -12. mu.L of the aqueous solution of (1) was taken out of 30 centrifugal tubes and added to a buffer solution (10 mmol. multidot.L) containing 1ml of LPBS-1pH 7.4) was added to the centrifuge tube in order from tube No. 2, and the change in fluorescence emission spectrum was measured before and after incubation at 37 ℃ for 60 min. The results are shown in FIG. 5.
FIG. 5 is a bar graph of fluorescence intensity at the maximum emission wavelength of the fluorescent probe synthesized in example 1 of the present invention after treatment with active oxygen and after addition of various substances. Wherein, butyrylcholinesterase shows obvious fluorescent response, and other substances have little influence on the fluorescence intensity.
Therefore, the fluorescent probe provided by the invention is not very responsive to most of the detected interfering substances and has good anti-interference capability.
4. Determination of butyrylcholine esterase minimum detection limit by fluorescent probe
At 37 deg.C, using fluorescence emission spectrum, in the presence of active oxygen, butyrylcholinesterase 0-1000 μ g/mL-1The concentrations were studied. The results are shown in FIG. 6.
As is clear from FIG. 6, the minimum detection limit of butyrylcholinesterase by the fluorescent probe synthesized in example 1 was 1.08. mu.g/mL by calculation-1。
Therefore, the fluorescent probe has high detection sensitivity on butyrylcholine esterase, and has potential application value in the aspect of high-efficiency detection.
5. Specific application of fluorescent probe in detection of butyrylcholine esterase and active oxygen in live cell and mouse imaging
(1) Specific application of fluorescent probe in detection of butyrylcholine esterase and active oxygen in living cells
HEK293 cells were incubated with 10. mu.M of the fluorescent probe synthesized in example 1 for 60min and fluorescence emission was collected at a wavelength range of 680-720nm upon excitation at 633 nm. The results are shown in FIG. 7.
FIG. 7 is a confocal fluorescence image of HEK293 cells synthesized by the fluorescent probe in example 1 of the present invention. In FIGS. 7(a) to 7(c), cells were incubated with a fluorescent probe (10. mu.M) for 60min and Lipopolysaccharide (LPS) (1.0. mu.g. mL) in that order-1) With propylene glycol methyl ether acetate (PMA) (0.5. mu.g.mL)-1) Pretreating for 30min, culturing with fluorescent probe for 60min, pretreating with N-acetylcysteine (NAC) (100mM) for 30min, and culturing with fluorescent probe for 60 min; FIGS. 7(d) -7 (f) are incubation of cells with fluorescent probe (10. mu.M) for 60min, or with tacrine (50 or 100. mu.M) pre-treatment for 30min, followed by incubation with fluorescent probe (10. mu.M) for 60min, respectively; FIGS. 7(g) to 7(h) are the mean fluorescence intensities of (a-c) and (d-f), respectively.
As can be seen from FIG. 7, after incubation with 10. mu.M fluorescent probe for 60min, weak fluorescence was exhibited, as shown in FIG. 7a, there was little change in cell morphology during imaging, indicating that cytotoxicity was low under such near infrared light irradiation. The responsive fluorescence is generated by methylene blue, which is revealed by the dual sensing reaction of the fluorescent probe with butyrylcholinesterase and active oxygen. This weak intensity may indicate a low expressed level of reactive oxygen species in the HEK293 cell line.
To test this hypothesis, lipopolysaccharide and propylene glycol methyl ether acetate were used to stimulate HEK293 cells to produce more reactive oxygen species, thereby mimicking the alzheimer's microenvironment, where an increase in intracellular emission intensity could be monitored, which means a significant increase in the release of the emitted methylene blue driven by the over-expressed reactive oxygen species. Another attempt to reduce the active oxygen content was also considered to further illustrate the active oxygen-triggered redox reaction. N-acetylcysteine has high sulfydryl reducibility and is widely used for eliminating active oxygen, so 100mM of N-acetylcysteine is applied to HEK293 cells.
After 30min pretreatment with N-acetylcysteine, HEK293 cells were incubated with fluorescent probes and cellular images of the same emission range were recorded in fig. 7c and 7g, with low intensity fluorescence indicating that the fluorescent probes produced little methylene blue, which was hindered by the lack of reactive oxygen species in the redox reaction, confirming the importance of the reactive oxygen species for producing significant fluorescence.
Further studies to validate butyrylcholinesterase function were proposed by experiments with increasing concentrations of tacrine inhibitors, as shown in fig. 7d and 7f, where significant fluorescence emission was observed in the absence of tacrine. After additional pretreatment with 50 μ M tacrine, a significant reduction in the emitted signal was obtained, and the inhibitory activity of butyrylcholinesterase was responsible for this loss of signal. When tacrine levels reached 100 μ M (FIG. 7h), the fluorescence emission was further reduced, confirming that butyrylcholinesterase was involved in the expected enzymatic reaction with P2 in cells.
In general, the logical and sensory strategy of butyrylcholinesterase and reactive oxygen species in cells is considered feasible and rational, which implies potential use in organisms.
(2) Specific application of fluorescent probe in detecting butyrylcholine esterase and active oxygen in organisms
APP/PS1(B6) mice are a classical living model for Alzheimer's disease studies. Three groups of APP/PS1(B6) mice with early Alzheimer's disease at 6 months of age were imaged in this protocol, and age-matched normal C57BL/6j mice were used as controls, taking into account the presence of a large number of potentially interfering species in the living organism. The results are shown in FIG. 8.
2h after injection of the probe P2, the C57BL/6j mice were exposed to a fixed near infrared excitation of 633nm and near infrared emission of 690nm laser. The fluorescent response of normal organisms was negligible, indicating that probe P2 responded very little to healthy individuals, providing excellent stability and showed no fluorescence in complex biological environments (fig. 8 a). When the fluorescent probe was applied to APP/PS1(B6) mice for 2h, it had overexpressed butyrylcholinesterase and active oxygen, with a fairly strong emission intensity, according to the bright fluorescence image shown in FIG. 8B. The responsive fluorescence was enhanced by more than 80-fold in comparison to the control sample.
Inhibition experiments were subsequently performed. After pretreatment with tacrine, APP/PS1(B6) mice applied probe P2 under exposure to the same imaging conditions. Apparently, a large decrease in fluorescence intensity was observed due to tacrine inhibition of butyrylcholinesterase activity (fig. 8 c). In addition, N-acetylcysteine was also used to eliminate reactive oxygen species in APP/PS1(B6) mice with alzheimer's disease, and no significant fluorescence emission was observed in fig. 8 d. This was quantified in FIG. 8e, and the weak fluorescence indicated that methylene blue was difficult to release due to the lack of active oxygen, which prevented the redox reaction of the fluorescent probe.
Therefore, the well-designed fluorescent probe shows excellent performance of identifying the Alzheimer's disease on a biological level, and the fluorescent probe is expected to be used as an index for early diagnosis of the Alzheimer's disease.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A fluorescent probe for early diagnosis of Alzheimer's disease, which has a structural formula as follows:
2. the method for preparing the fluorescent probe for the early diagnosis of Alzheimer's disease according to claim 1, which comprises the following steps:
(1) dissolving methylene blue in a mixed solution of dichloromethane and water at room temperature, adding mixed powder of sodium hydrosulfite and sodium bicarbonate, stirring until the water phase changes from blue to khaki, separating an organic layer, extracting the water layer with dichloromethane, combining the organic layers, and drying with anhydrous sodium sulfate to obtain reduced methylene blue R-MB;
(2) adding reduced methylene blue R-MB into triethylamine, cooling to 0 ℃, adding a dichloromethane solution containing triphosgene TPG, and stirring at room temperature to react to obtain an intermediate MB-Cl;
(3) adding 4-aminomethyl phenol and triethylamine into the intermediate MB-Cl, stirring overnight, adding dichloromethane, washing with water, drying an organic phase with anhydrous sodium sulfate, distilling off the solvent to obtain a crude product, and purifying by silica gel chromatography to obtain a compound P02 in the form of a white solid;
(4) dissolving the compound P02 in dichloromethane at 0 ℃, adding cyclopropyl formyl chloride diluted by dichloromethane, adding triethylamine, stirring overnight at room temperature, washing with a saturated sodium bicarbonate solution, and purifying a crude product after removing the solvent by column chromatography to obtain the white powder fluorescent probe P2.
3. The method for preparing a fluorescent probe for the early diagnosis of Alzheimer's disease according to claim 2, wherein in the step (1), the molar ratio of the methylene blue to the sodium dithionite to the sodium bicarbonate is 1: (1.5-2.5): (1.5-2.5).
4. The method for preparing a fluorescent probe for early diagnosis of Alzheimer's disease as claimed in claim 2, wherein the stirring time in step (1) is 20 min.
5. The method for preparing a fluorescent probe for the early diagnosis of Alzheimer's disease as claimed in claim 2, wherein in the step (2), the molar ratio of the reduced methylene blue R-MB, the triethylamine and the triphosgene TPG is 1: (1.2-1.5): (2.5-3.5).
6. The method for preparing a fluorescent probe for early diagnosis of Alzheimer's disease as claimed in claim 2, wherein the stirring reaction time in step (2) is 0.5 h.
7. The method for preparing a fluorescent probe for the early diagnosis of Alzheimer's disease according to claim 2, wherein in the step (3), the molar ratio of the intermediate MB-Cl, the 4-aminomethyl phenol and the triethylamine is 1: (1.5-2.5): (1.5-2.5).
8. The method for preparing a fluorescent probe for early diagnosis of Alzheimer's disease according to claim 2, wherein in the step (4), the molar ratio of the compound P02, the cyclopropyl carbonyl chloride and the triethylamine is 1: (1-1.2): (2.5-3.5).
9. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 8 for detecting butyrylcholinesterase and active oxygen.
10. A use as claimed in claim 9, wherein the fluorescent probe is present in a reaction concentration of 10 μmol-L-1Reaction temperatureThe reaction system was PBS buffer solution with pH 7.4 at 37 ℃ for 60 min.
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