CN113336701B - Nitric oxide two-photon lipid droplet locking fluorescent probe, preparation method thereof and application thereof in detecting neuroinflammation - Google Patents
Nitric oxide two-photon lipid droplet locking fluorescent probe, preparation method thereof and application thereof in detecting neuroinflammation Download PDFInfo
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- CN113336701B CN113336701B CN202110414471.7A CN202110414471A CN113336701B CN 113336701 B CN113336701 B CN 113336701B CN 202110414471 A CN202110414471 A CN 202110414471A CN 113336701 B CN113336701 B CN 113336701B
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- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 title claims abstract description 179
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 44
- 150000002632 lipids Chemical class 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 208000036110 Neuroinflammatory disease Diseases 0.000 title claims abstract description 24
- 230000003959 neuroinflammation Effects 0.000 title claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 79
- 150000001875 compounds Chemical class 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 36
- 229940126214 compound 3 Drugs 0.000 claims description 22
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 20
- 229940125904 compound 1 Drugs 0.000 claims description 17
- 229940125782 compound 2 Drugs 0.000 claims description 16
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 239000003054 catalyst Substances 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 11
- 230000035484 reaction time Effects 0.000 claims description 8
- WFQDTOYDVUWQMS-UHFFFAOYSA-N 1-fluoro-4-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C=C1 WFQDTOYDVUWQMS-UHFFFAOYSA-N 0.000 claims description 7
- 229940126062 Compound A Drugs 0.000 claims description 7
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- PAMIQIKDUOTOBW-UHFFFAOYSA-N N-methylcyclohexylamine Natural products CN1CCCCC1 PAMIQIKDUOTOBW-UHFFFAOYSA-N 0.000 claims description 7
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- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 6
- OISVCGZHLKNMSJ-UHFFFAOYSA-N 2,6-dimethylpyridine Chemical compound CC1=CC=CC(C)=N1 OISVCGZHLKNMSJ-UHFFFAOYSA-N 0.000 claims description 6
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- VZAWCLCJGSBATP-UHFFFAOYSA-N 1-cycloundecyl-1,2-diazacycloundecane Chemical compound C1CCCCCCCCCC1N1NCCCCCCCCC1 VZAWCLCJGSBATP-UHFFFAOYSA-N 0.000 claims description 4
- BWZVCCNYKMEVEX-UHFFFAOYSA-N 2,4,6-Trimethylpyridine Chemical compound CC1=CC(C)=NC(C)=C1 BWZVCCNYKMEVEX-UHFFFAOYSA-N 0.000 claims description 4
- ITQTTZVARXURQS-UHFFFAOYSA-N 3-methylpyridine Chemical compound CC1=CC=CN=C1 ITQTTZVARXURQS-UHFFFAOYSA-N 0.000 claims description 4
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 4
- FKNQCJSGGFJEIZ-UHFFFAOYSA-N 4-methylpyridine Chemical compound CC1=CC=NC=C1 FKNQCJSGGFJEIZ-UHFFFAOYSA-N 0.000 claims description 4
- NTSLROIKFLNUIJ-UHFFFAOYSA-N 5-Ethyl-2-methylpyridine Chemical compound CCC1=CC=C(C)N=C1 NTSLROIKFLNUIJ-UHFFFAOYSA-N 0.000 claims description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 4
- JLTDJTHDQAWBAV-UHFFFAOYSA-N N,N-dimethylaniline Chemical compound CN(C)C1=CC=CC=C1 JLTDJTHDQAWBAV-UHFFFAOYSA-N 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 claims description 4
- XJHCXCQVJFPJIK-UHFFFAOYSA-M caesium fluoride Chemical compound [F-].[Cs+] XJHCXCQVJFPJIK-UHFFFAOYSA-M 0.000 claims description 4
- 239000012973 diazabicyclooctane Substances 0.000 claims description 4
- ZCSHNCUQKCANBX-UHFFFAOYSA-N lithium diisopropylamide Chemical compound [Li+].CC(C)[N-]C(C)C ZCSHNCUQKCANBX-UHFFFAOYSA-N 0.000 claims description 4
- 230000004807 localization Effects 0.000 claims description 4
- 150000007530 organic bases Chemical class 0.000 claims description 4
- YJVFFLUZDVXJQI-UHFFFAOYSA-L palladium(ii) acetate Chemical group [Pd+2].CC([O-])=O.CC([O-])=O YJVFFLUZDVXJQI-UHFFFAOYSA-L 0.000 claims description 4
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- SMUQFGGVLNAIOZ-UHFFFAOYSA-N quinaldine Chemical compound C1=CC=CC2=NC(C)=CC=C21 SMUQFGGVLNAIOZ-UHFFFAOYSA-N 0.000 claims description 4
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 claims description 4
- GJFNRSDCSTVPCJ-UHFFFAOYSA-N 1,8-bis(dimethylamino)naphthalene Chemical compound C1=CC(N(C)C)=C2C(N(C)C)=CC=CC2=C1 GJFNRSDCSTVPCJ-UHFFFAOYSA-N 0.000 claims description 3
- AVFZOVWCLRSYKC-UHFFFAOYSA-N 1-methylpyrrolidine Chemical compound CN1CCCC1 AVFZOVWCLRSYKC-UHFFFAOYSA-N 0.000 claims description 3
- AHVYPIQETPWLSZ-UHFFFAOYSA-N N-methyl-pyrrolidine Natural products CN1CC=CC1 AHVYPIQETPWLSZ-UHFFFAOYSA-N 0.000 claims description 3
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- DIAIBWNEUYXDNL-UHFFFAOYSA-N n,n-dihexylhexan-1-amine Chemical compound CCCCCCN(CCCCCC)CCCCCC DIAIBWNEUYXDNL-UHFFFAOYSA-N 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
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- 229910019142 PO4 Inorganic materials 0.000 claims description 2
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Abstract
The invention discloses a nitric oxide two-photon lipid droplet locking fluorescent probe, a preparation method thereof and application thereof in detecting neuroinflammation. The two-photon fluorescent probe (SUN-TAN) is a compound with a structure shown in a formula I. In the two-photon probe, the aromatic secondary amine is used as the NO recognition domain, so that the interference of other active oxygen substances when the o-phenylenediamine is used as the NO recognition domain can be avoided, and the selectivity of the probe can be improved; the modified polyethylene glycol chain structure (amino triethylene glycol monomethyl ether) can increase the solubility of the probe and improve the biocompatibility of the probe in vivo, and is favorable for crossing a blood brain barrier to enter deep brain tissue; meanwhile, the triphenylamine structure is used as a lipid drop positioning group, so that targeted positioning to a neuroinflammation part is facilitated, and the detection accuracy is improved; and the naphthalimide structural unit is used as a fluorescent group, so that the naphthalimide structural unit has the advantages of high stability and high quantum yield, and is favorable for obtaining clearer imaging results.
Description
Technical Field
The invention belongs to the technical field of chemical and biological analysis detection imaging, and particularly relates to a nitric oxide two-photon lipid droplet locking fluorescent probe, a preparation method thereof and application thereof in detecting neuroinflammation.
Background
Neuroinflammation, an immune inflammatory response generated by activation of the central nervous system, commonly occurs in neurodegenerative diseases, including Alzheimer's Disease (AD), Parkinson's Disease (PD), and Amyotrophic Lateral Sclerosis (ALS). Detection of neuroinflammatory responses is considered to be of crucial importance for the early diagnosis and treatment of neurodegenerative diseases. Current methods of detecting neuroinflammatory responses include Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and Single Photon Emission Computed Tomography (SPECT), among others. However, the above method has certain limitations: for example, MRI has long image acquisition time and low sensitivity; PET can generate radiation to human bodies; SPECT has a problem of high detection cost. Therefore, it is highly desirable to develop a rapid, low-cost, and highly sensitive method for detecting neuroinflammatory responses.
In recent years, molecular fluorescent probe technology has been widely used in biochemical and medical cross fields because of its advantages of low cost, high sensitivity, etc., and the realization of rapid, noninvasive, and in-situ imaging of cells or living animals. Compared with a single photon fluorescence probe, the two-photon fluorescence probe combines a focus excitation technology and a point scanning technology, and can carry out clearer imaging. Meanwhile, two-photon fluorescent probes based on reactive oxygen species response are increasingly used for neuroinflammatory response detection. Nitric Oxide (NO) is a reactive oxygen species present in the body and is also an important proinflammatory factor in the body. When the neuroinflammation reaction occurs, a large amount of NO can be released by the activated microglia, and the construction of the NO two-photon fluorescent probe becomes an effective means for detecting the neuroinflammation reaction. However, the detection of neuroinflammatory reaction based on the two-photon fluorescent probe with NO response has the following technical problems: (1) lack of targeting, easily cause false positive; (2) poor ability to cross the blood brain barrier and difficult access to brain tissue. Therefore, how to construct a two-photon fluorescent probe with high targeting property, strong capability of crossing blood brain barrier, high fluorescence maximum response multiple, high sensitivity to NO response and good selectivity for NO response also becomes a technical problem to be solved in the field.
Disclosure of Invention
In order to improve the technical problem, the invention provides a compound with a structure shown in a formula I:
the invention also provides a preparation method of the compound with the structure shown in the formula I, which comprises the following steps: reacting the compound 3 with a compound A to obtain a compound of a formula I;
the compound 3 has the following structure:
the compound A has the following structure:
according to an embodiment of the present invention, during the above reaction, it is preferable to add a base and a catalyst.
According to an embodiment of the invention, the catalyst may be selected from: tris (o-methylphenyl) phosphonium, 1' -bisdiphenylphosphinoferrocene, palladium dichloride, palladium acetate (Pd (OAc))2) At least one of tetrakis (triphenylphosphine) palladium and tris (dibenzylideneacetone) dipalladium; preferably palladium acetate (Pd (OAc)2) And tris (o-methylphenyl) phosphorus.
According to an embodiment of the present invention, the base may be an organic base or an inorganic base.
For example, the inorganic base may be selected from at least one of the following compounds: hydrides, hydroxides, acetates, fluorides, phosphates, carbonates and bicarbonates of alkali metals or alkaline earth metals; preferably, the inorganic base is selected from sodium amide, sodium hydride, sodium hydroxide, potassium hydroxide, sodium acetate, sodium phosphate, potassium fluoride, cesium fluoride, sodium carbonate, potassium bicarbonate, sodium bicarbonate or cesium carbonate.
For example, the organic base may be selected from at least one of the following compounds: alkoxides, tertiary amines, substituted or unsubstituted pyridines and substituted or unsubstituted triethylamine, trimethylamine, N, N-diisopropylethylamine, tri-N-propylamine, tri-N-butylamine, tri-N-hexylamine, tricyclohexylamine, N-methylcyclohexylamine, N-methylpyrrolidine, N-methylpiperidine, N-ethylpiperidine, N, N-dimethylaniline, N-methylmorpholine, pyridine, 2, 3-or 4-methylpyridine, 2-methyl-5-ethylpyridine, 2, 6-dimethylpyridine, 2,4, 6-trimethylpyridine, 4-dimethylaminopyridine, quinoline, methylquinoline, N, N, N-tetramethylethylenediamine, N, N-dimethyl-1, 4-diazacyclohexane, N, N-diethyl-1, 4-diazabicyclocyclohexane, 1, 8-bis (dimethylamino) naphthalene, Diazabicyclooctane (DABCO), Diazabicyclononane (DBN), Diazabicycloundecane (DBU), butylimidazole and methylimidazole, lithium diisopropylamide, sodium methoxide, potassium tert-butoxide; triethylamine is preferred.
According to an embodiment of the present invention, the preparation method may be performed in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from N, N-Dimethylformamide (DMF).
According to an embodiment of the invention, the molar ratio of compound 3 to compound a is 1 (1-2), exemplary 1:1, 1:1.44, 1: 2.
According to an embodiment of the invention, the catalyst is used in an amount of 0.5 to 4% by mass, illustratively 0.5%, 1%, 2%, 3% by mass, based on the mass of compound 3.
According to an embodiment of the invention, the molar ratio of compound 3 to base is 1 (5-10), exemplary 1:5, 1:7, 1: 9.2.
According to an embodiment of the invention, the temperature of the reaction is 60-100 ℃, exemplary 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃; the reaction time is 12-36 h, and 12h, 24h and 36h are exemplified.
According to an embodiment of the present invention, the preparation method further comprises a step of precipitating a solid product from the reacted mixture after the reaction is completed. For example, the precipitation process may be to pour the mixture into ice water and collect the resulting solid product. Further, the preparation method further comprises the step of purifying the solid product. For example, the purification may be performed using a column chromatography column. Preferably, the eluent for the column chromatography column separation is petroleum ether, ethyl acetate 1- (1-5) (v/v), exemplarily 1:1, 1:2, 1: 5.
Preferably, the synthetic route for the compounds of formula I is as follows:
according to an embodiment of the present invention, said compound 3 is prepared by a process comprising: reacting the compound 2 with 4-bromo-1, 8-naphthalic anhydride to obtain the compound 3;
the compound 2 has the following structure:
according to an embodiment of the present invention, the above reaction may be carried out in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from ethanol.
According to an embodiment of the invention, the molar ratio of compound 2 and 4-bromo-1, 8-naphthalic anhydride is 1 (0.5 to 1.0), exemplary 1:0.5, 1:0.6, 1:0.8, 1:1.
According to the embodiment of the invention, the reaction temperature is 60-100, and 60 examples, 70 examples, 80 examples and 100 examples are illustrated; the reaction time is 1-6 h, and 1h, 4h and 6h are exemplified.
According to an embodiment of the present invention, the preparation method further comprises a step of separating a crude product after the reaction is completed. For example, the mixture after the reaction is extracted with an extractant, the filtrate is collected, and the filtrate is distilled under reduced pressure to obtain the crude product. Preferably, the extractant may be dichloromethane and saturated brine. Further, the preparation method also comprises the step of purifying the crude product to obtain the compound 3. For example, the purification may be performed using column chromatography to provide compound 3.
Preferably, the synthetic route of the compound 3 is as follows:
according to an embodiment of the present invention, said compound 2 is prepared by a process comprising: reacting the compound 1 with hydrazine hydrate to obtain a compound 2;
the compound 1 has the following structure:
according to an embodiment of the present invention, the above reaction may be carried out in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from ethanol.
According to an embodiment of the present invention, during the above reaction, a catalyst is preferably added. Preferably, the catalyst may be selected from: Pd/C powder, 1' -bis-diphenylphosphino ferrocene, palladium dichloride, palladium acetate (Pd (OAc))2) At least one of tetrakis (triphenylphosphine) palladium and tris (dibenzylideneacetone) dipalladium. Preferably Pd/C powder.
According to the embodiment of the invention, the mass ratio of the catalyst to the compound 1 is 1 (20-40), and the mass ratio is 1:20, 1:25 and 1: 28.
According to an embodiment of the invention, the ratio of the amount of hydrazine hydrate to compound 1 is 1mL (0.1-0.3) g, exemplary 1mL:0.1g, 1mL:0.14g, 1mL:0.2 g.
According to the embodiment of the invention, the reaction temperature is 60-100, and 60 examples, 70 examples, 80 examples and 100 examples are illustrated; the reaction time is 1-4 h, and 1h, 2h and 4h are exemplified.
According to an embodiment of the present invention, the preparation method further comprises a step of separating a crude product after the reaction is completed. For example, the mixture after the reaction is extracted with an extractant, the filtrate is collected, and the filtrate is distilled under reduced pressure to obtain the crude product. Preferably, the extractant may be dichloromethane and saturated brine. Further, the preparation method further comprises the step of purifying the crude product. For example, the purification may be performed using a column chromatography column.
Preferably, the synthetic route of the compound 2 is as follows:
according to an embodiment of the present invention, the compound 1 is prepared by a method comprising: reacting 1-fluoro-4-nitrobenzene with aminotriglycol monomethyl ether to obtain a compound 1.
According to an embodiment of the present invention, the above reaction may be carried out in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from DMSO.
According to an embodiment of the invention, the molar volume ratio of the 1-fluoro-4-nitrobenzene to the solvent is 1mmol (1-2) mL, for example 1mmol:1.35 mL.
According to an embodiment of the invention, the molar ratio of the 1-fluoro-4-nitrobenzene to the aminotriglycol monomethyl ether is 1 (0.5-1), exemplary 1:0.5, 1:0.83, 1:1.
According to an embodiment of the invention, the temperature of the reaction is 60 to 100 ℃, exemplary 60 ℃, 70 ℃, 80 ℃, 100 ℃; the reaction time is 4-8 h, and 4h, 6h and 8h are exemplified.
According to an embodiment of the present invention, the preparation method further comprises a step of separating a crude product after the reaction is completed. For example, the mixture after the reaction is extracted with an extractant, and the lower organic phase is collected and distilled under reduced pressure to obtain a crude product. Preferably, the extractant may be dichloromethane and water. Further, the preparation method further comprises the step of purifying the crude product. For example, the purification may be performed using a column chromatography column.
Preferably, the synthetic route of the compound 1 is as follows:
according to an embodiment of the present invention, the preparation method of the compound having the structure shown in formula I comprises the following steps:
s1, dissolving 1-fluoro-4-nitrobenzene and aminotriglycol monomethyl ether in DMSO (dimethyl sulfoxide), reacting, cooling the mixture to room temperature, extracting with dichloromethane and water, collecting a lower organic phase, distilling under reduced pressure to obtain a crude product, preparing a sample of the crude product, and separating the crude product by using a column chromatography column to obtain a compound 1, wherein the structural formula of the compound 1 is as follows:
s2, mixing the compound 1 obtained in the step S1, Pd/C powder, hydrazine hydrate and ethanol, reacting under the protection of nitrogen, cooling the mixture to room temperature after the reaction is finished, and removing the Pd/C powder in the system by suction filtration under reduced pressure; then extracting the filtrate with dichloromethane and saturated saline solution, collecting an organic phase, distilling under reduced pressure to obtain a crude product, preparing a sample of the crude product, and separating by using a column chromatography chromatographic column to obtain a compound 2, wherein the structural formula of the compound 2 is as follows:
s3, dissolving the compound 2 obtained in the step S2 and 4-bromo-1, 8-naphthalic anhydride in ethanol, reacting, extracting with dichloromethane and saturated saline after the reaction is finished, collecting filtrate, distilling under reduced pressure to obtain a crude product, preparing a sample of the crude product, and separating by using a column chromatography column to obtain a compound 3, wherein the structural formula of the compound 3 is as follows:
s4, mixing the compound 3 obtained in the step S3, the compound A, tri (o-methylphenyl) phosphorus, Pd (OAc)2Mixing triethylamine and DMF, adding DMF, reacting, pouring the mixture into ice water after the reaction is finished, collecting red solid, and separating the obtained solid sample by using a column chromatography chromatographic column to obtain a compound of a formula I;
the compound a has the following structure:
the reaction scheme of the compound of formula I is shown in FIG. 1.
The invention also provides application of the compound with the structure shown in the formula I in detection of nitric oxide.
The invention also provides application of the compound with the structure shown in the formula I as a two-photon fluorescent probe. Preferably, the two-photon fluorescent probe has a lipid droplet localization function and can be used for detecting neuroinflammation.
The invention also provides a two-photon fluorescent probe which contains a compound with a structure shown in the formula I.
According to the embodiment of the invention, the single photon maximum absorption wavelength of the two-photon fluorescent probe is about 466 nm.
According to an embodiment of the present invention, the two-photon maximum absorption wavelength of the two-photon fluorescence probe is about 780 nm.
According to an embodiment of the present invention, the maximum emission wavelength of fluorescence of the two-photon fluorescent probe is about 630 nm.
According to an embodiment of the present invention, the stokes shift of the two-photon fluorescent probe is about 150 nm.
According to the embodiment of the invention, the two-photon fluorescent probe has a lipid droplet positioning function.
The invention also provides a method for detecting nitric oxide by using the two-photon fluorescent probe, which comprises the step of mixing Nitric Oxide (NO) or a target object to be detected containing the Nitric Oxide (NO) with the two-photon fluorescent probe.
According to the embodiment of the invention, the method further comprises the steps of mixing a to-be-tested sample of nitric oxide or a to-be-tested target object containing nitric oxide with the two-photon fluorescent probe, measuring the luminous intensity of the mixed solution, and calculating to obtain the concentration of nitric oxide. Wherein the concentration of the two-photon fluorescent probe in the sample to be tested is 5-20 μ M, such as 8-15 μ M, and exemplary 10 μ M.
Preferably, the concentration of the target to be detected is calculated by substituting into a concentration-dependent standard curve of the target to be detected.
According to an embodiment of the present invention, the method further comprises preparing a solution of the two-photon fluorescent probe and a solution of Nitric Oxide (NO) or a solution of a target to be measured containing Nitric Oxide (NO).
According to an embodiment of the invention, the method comprises in particular the steps of:
1) dissolving a compound with a structure shown in a formula I in DMSO to obtain a solution of the two-photon fluorescent probe;
2) preparing NO solutions with different concentrations or solutions of targets to be detected containing NO;
3) drawing a concentration-dependent standard curve of the NO solution or the target to be detected containing NO;
preferably, the step of plotting the concentration-dependent standard curve of the NO solution is as follows: taking two-photon fluorescent probe solution, taking one group of solution as a blank sample, adding NO solution with known concentration or solution of a target substance to be detected containing NO into the rest groups of solution respectively, mixing, incubating, measuring the luminous intensity of the mixed solution, taking the fluorescent intensity y of each group of mixed solution after adding the target substance solution to be detected as a vertical coordinate, taking the concentration x of the target substance solution to be detected as a horizontal coordinate, and making a concentration-dependent standard curve of the target substance to be detected containing NO or NO;
4) detecting the concentration of NO in the NO solution or the target to be detected containing NO;
preferably, the concentration of the target to be detected is specifically measured by the following steps: mixing the two-photon fluorescent probe solution with an NO solution with unknown concentration or a solution of an NO-containing target object to be detected, incubating, measuring the luminous intensity of the mixed solution, and substituting the luminous intensity into the NO concentration dependence standard curve drawn in the step 3) to obtain the concentration of NO.
According to the embodiment of the invention, the NO solution or the solution of the NO-containing object to be detected in the step 2) is obtained by mixing NO or the NO-containing object to be detected with a buffer solution;
preferably, the buffer solution can be selected from buffer solutions with pH values of 7-11; for example, the buffer solution may be selected from the group consisting of HEPES aqueous solution, MES buffer solution, Tris-HCl buffer, NaOH-H3BO3Buffer solution, NaCO3-NaHCO3Buffer solutions, phosphate buffer solutions, and the like;
according to an exemplary embodiment of the invention, the buffer solution is selected from Phosphate Buffered Saline (PBS) at pH 7.4;
according to an exemplary embodiment of the present invention, the NO solution is obtained by mixing NO with a PBS solution; the concentration of NO is more than 0 and not more than 20 μ M, preferably 1 to 20 μ M.
According to an embodiment of the present invention, the temperature of the incubation in step 4) may be selected from 30 to 50 ℃, preferably the temperature of the incubation is selected from 35 to 40 ℃, according to an exemplary embodiment of the present invention, the temperature of the incubation is 37 ℃;
the incubation time can be selected from 1-10 min, and preferably, the incubation time is selected from 1-5 min; according to an exemplary embodiment of the invention, the incubation time is 3 min;
preferably, the concentrations and volumes of the two-photon fluorescent probe solution and the NO solution with different concentrations can be mixed in any proportion.
The invention also provides the application of the detection method, which is used for detecting neuroinflammation;
preferably, the detection method is used for detecting nitric oxide reactive oxygen species of neuroinflammation.
The invention also provides a kit comprising the two-photon fluorescent probe.
The invention also provides a biosensor which comprises the two-photon fluorescent probe.
The invention also provides application of the two-photon fluorescent probe, the kit and/or the biosensor in detecting neuroinflammation.
The invention also provides application of the two-photon fluorescent probe, the kit and/or the biosensor in living cell or tissue imaging.
In the two-photon probe (marked as a probe SUN-TAN), the aromatic secondary amine is used as an NO identification domain, so that the interference of Ascorbic Acid (AA), dehydroascorbic acid (DHA) and Methylglyoxal (MGO) which are easily caused when o-phenylenediamine is used as the NO identification domain can be avoided, and the selectivity of the probe can be improved; the modified polyethylene glycol chain structure (amino triethylene glycol monomethyl ether) can increase the solubility of the probe and improve the biocompatibility of the probe in vivo, and is favorable for crossing a blood brain barrier to enter deep brain tissue; meanwhile, the triphenylamine structure is used as a lipid drop positioning group, so that targeted positioning to a neuroinflammation part is facilitated, and the detection accuracy is improved; and the naphthalimide structural unit is used as a fluorescent group, so that the naphthalimide structural unit has the advantages of high stability and high quantum yield, and is favorable for obtaining clearer imaging results.
The bimolecular fluorescent probe has the following action mechanism: when the probe SUN-TAN does not react with NO, electrons contained in the aromatic secondary amine group in the fluorescent probe molecule easily occupy electron holes generated by excitation of the naphthalimide structural unit, and an acceptor photoinduced electron transfer (a-PET) process is generated, so that the fluorescence of the SUAN-TAN is quenched. However, when SUA-TAN reacts with NO, the N-nitrosation reaction of the SUA-TAN and NO destroys the aromatic secondary amine structure, so that the electron hole process generated by the excitation of the electron transfer from the aromatic secondary amine group to the naphthalimide structural unit is blocked, the a-PET process is inhibited, and the fluorescence is recovered.
The invention has the beneficial effects that:
lipid droplets are an important class of organelles in eukaryotic cells, the formation of which is primarily derived from the body's own lipid metabolic pathways. Lipids in normal brain tissue are mainly used to synthesize cell membrane structures and are less stored in lipid droplets, so the number of lipid droplets in normal brain tissue is small. The inventor of the application finds that activated microglia excessively express various proinflammatory factors and are accompanied with accumulation of a large number of lipid droplets when the neuroinflammation reaction occurs, and based on the finding, the invention constructs a lipid droplet locked two-photon fluorescence probe to improve the targeting property for detecting neuroinflammation.
(1) The nitrogen monoxide two-photon lipid drop locking fluorescence probe SUN-TAN for detecting neuroinflammation has the advantages that the maximum single-photon absorption wavelength is 466nm, the maximum two-photon absorption wavelength is 780nm, the maximum fluorescence emission wavelength is 630nm, the probe has two-photon excitation property and larger Stokes shift (150nm), so that the self interference of organisms can be effectively reduced, and the accuracy of biological imaging is improved. The probe of the invention has the advantages of high fluorescence maximum response multiple, high sensitivity and good selectivity for NO response, and the like. Meanwhile, the probe of the invention has the advantages of simple synthesis, lipid drop positioning function and effective crossing of blood brain barrier, can realize accurate detection of neuroinflammation reaction,
(2) the probe has a lipid drop positioning function and can be accurately positioned at a nerve inflammation part; and the blood brain barrier can effectively cross the blood brain barrier so as to improve the definition of biological imaging.
Drawings
FIG. 1 is a synthesis scheme of the probe SUN-TAN of the present invention.
FIG. 2 shows the SUN-TAN of the probe of the present invention1H-NMR spectrum.
FIG. 3 shows the SUN-TAN of the probe of the present invention13C-NMR spectrum.
FIG. 4 is the HRMS spectrum of the SUN-TAN probe of the present invention.
FIG. 5 is a fluorescent spectrum of the response of SUN-TAN to NO in the probe of the present invention.
FIG. 6 is a graph showing the linear response of SUN-TAN to NO in the probe of the present invention.
FIG. 7 is a diagram showing the selectivity test of SUN-TAN, a probe of the present invention.
FIG. 8 is a graph showing the fluorescence stability of SUN-TAN, a probe of the present invention, in buffer solutions of different pH values.
FIG. 9 is a diagram showing the toxicity test of the probe SUN-TAN of the present invention in BV-2 cells.
FIG. 10 is a diagram showing the co-localization of SUN-TAN, a probe of the present invention, in BV-2 cells. Wherein: a is a SUN-TAN imaging structure of the probe, and a signal channel is a red channel (lambda)em＝550-750nm，λex780 nm); b is the imaging result of the dye BODIPY493/503, and the signal channel is a green channel (lambda)em＝500-530nm，λex488 nm); c is an overlapping image of a and b; d is the intensity correlation plot of SUN-TAN and BODIPY493/503 (both 10 μm scale).
FIG. 11 is a diagram showing the application of SUN-TAN, a probe of the present invention, in detecting neuroinflammation in BV-2 cells. Wherein: a is the cell bright field; b is a two-photon fluorescence image obtained by LPS treatment and culture for 6h at 200 mu g/mL, and then adding 10 mu M of probe SUN-TAN for incubation for 30 min; c is an overlapping image of a and b. Lambda [ alpha ]em＝550-750nm，λex780 nm; (all the scales are shown in the figure50 μm).
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
In the following examples of the present invention, the structural formula of compound a is:
the preparation method can be referred to as the following documents: YE M, HU W, HE M, et al, deep imaging for visualizing oxides in threaded primers, method disclosed in the conversion of the related between oxides and resistance to ceramic chemical communications [ J ], 56: 6233-.
Except for special instructions, the NO test in vitro with the probe SUN-TAN all takes PBS/1, 4-dioxane (7/3, v/v) with pH of 7.4 as a medium, and the concentration of the probe is 10 μ M.
In the present invention, mM represents mmol/L, and. mu.M represents. mu. mol/L.
Example 1
As shown in fig. 1, a method for synthesizing a nitric oxide two-photon lipid-droplet locked fluorescent probe (SUN-TAN) for detecting neuroinflammation comprises the following steps:
s1, synthesis of compound 1: 3g (14.71mmol) of 1-fluoro-4-nitrobenzene and 2g (12.26mmol) of aminotriglycol monomethyl ether were dissolved in 20mL of DMSO and the temperature was raised to 100 ℃ for reaction for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with dichloromethane and water, and the lower organic phase was collected and distilled under reduced pressure to obtain a crude product, which was then sampled and separated by column chromatography with an eluent of PE: DCM ═ 2:1(v/v), to obtain 2.8g of compound 1 with a yield of 80%.
S2, synthesis of compound 2: 2.8g (9.854mmol) of the compound 1 prepared in the step S1, 100mg of Pd/C powder and 20mL of hydrazine hydrate are sequentially added into a three-necked flask containing 80mL of ethanol, the temperature is raised to 80 ℃ under the protection of nitrogen, the reaction is carried out for 2 hours, and the reaction progress is tracked by TCL. After the reaction is finished, cooling the mixture to room temperature, and filtering under reduced pressure to remove Pd/C powder in the system. The filtrate was extracted with dichloromethane and saturated brine, the organic phase was collected and distilled under reduced pressure to give a crude product, which was sampled and separated by column chromatography using PE: PA ═ 1:1(v/v) as an eluent, to give 1.22g of compound 2 with a yield of 48.6%.
S3, synthesis of compound 3: 1.22g (4.79mmol) of the compound 2 obtained in step S2 and 1.1g (3.83mmol) of 4-bromo-1, 8-naphthalic anhydride were dissolved in 50mL of ethanol and reacted at 80 ℃ for 4 hours. After the reaction, the reaction mixture was extracted with dichloromethane and saturated brine, the filtrate was collected and distilled under reduced pressure to obtain a crude product, which was sampled and separated by column chromatography with an eluent PE: EA ═ 1:2(v/v), to obtain 1.02g of compound 3 with a yield of 53.2%.
S4, 0.5g (1.0mmol) of Compound 3 obtained in step S3, 0.4g (1.44mmol) of Compound A, 4mg (0.013mmol) of tris (o-methylphenyl) phosphorus, 5mg of Pd (OAc)20.93g triethylamine (9.2mmol) is added into the flask, after three times of charging and three times of pumping, 20mL DMF after drying is added, and the temperature is raised to 90 ℃ for reaction for 24 hours. After the reaction was completed, the mixture was poured into ice water, and a red solid was collected, and the obtained solid was sampled and separated by column chromatography using PE: PA ═ 1:2(v/v) to obtain 0.56g of a probe (SUN-TAN) with a yield of 80%.
The results of nuclear magnetic hydrogen spectroscopy of the probe (SUN-TAN) prepared in this example are shown in FIG. 2:1H NMR(600MHz，DMSO-d6)δ8.99(d，J＝8.7hz，1H)，8.52(d，J＝7.1hz，1H)，8.45(d，J＝7.8hz，1H)，8.23(d，J＝7.9hz，1H)，8.10(d，J＝16.0hz，1H)，7.95-7.88(m，1H)，7.77(d，J＝8.7hz，2H)，7.57(d，J＝16.0hz，1H)，7.36(t，J＝7.9hz，4H)，7.16-7.07(m，6H)，7.01(dd，J＝13.8，8.7hz，4H)，6.68(d，J＝8.8hz，2H)，3.61(dd，J＝7.5，4.1hz，2H)，3.59-3.57(m，2H)，3.57-3.56(m，2H)，3.54(dd，J＝5.7，3.9hz，2H)，3.45(dd，J＝5.7，3.8hz，2H)，3.27-3.23(m，5H)。
the carbon spectrum result of the probe (SUN-TAN) prepared in this example is shown in FIG. 3:13C NMR(151MHz，d6-DMSO)δ164.24，163.95，154.15，147.19，142.67，141.95，133.69，131.32，131.10，130.93，130.18，127.58，127.42，125.07，124.21，123.52，122.60，121.62，121.21，49.82，28.41。
the mass spectrum result of the probe (SUN-TAN) prepared in this example is shown in FIG. 4: HRMS m/z calcd for C45H41N3O5[M+H]+:704.3046found:704.3129。
Example 2
The fluorescent response experiment of the fluorescent probe molecule and NO comprises the following steps:
(1) 3.5mg of the probe (SUN-TAN) prepared in example 1 was dissolved in 1mL of DMSO, sonicated, mixed well, and 0.2mL of the above solution was put into a 1mL centrifuge tube, and 0.8mL of DMSO was added to prepare a 1mM probe stock solution.
(2) NO saturated solution (1.9mM) preparation: introducing nitrogen gas into 10mM PBS solution at room temperature for 30min to remove O in the solution2Then, the NO gas generated by the reaction of the sodium nitrite and the concentrated sulfuric acid is quickly led into the solution, the NO introducing time is about 40min, and then the NO saturated solution can be prepared and stored in a refrigerator at the temperature of 4 ℃. In use, the saturated NO solution is diluted to 1 mM.
(3) Taking 10 mu L of the probe stock solution prepared in the step (1), adding a proper amount of PBS/1, 4-dioxane (7/3, v/v) mixed solution with the pH value of 7.4, carrying out ultrasonic treatment, uniformly mixing, and quickly adding 0-20 mu L of NO solution (1mM) so that the final concentration of the probe in the test solution is 10 mu M, and the final concentrations of NO are 0 mu M, 1 mu M, 2 mu M, 5 mu M, 8 mu M, 12 mu M, 14 mu M and 20 mu M respectively. Then, the test solution is rapidly put into a constant temperature oscillator with the temperature of 37 ℃ and the rotating speed of 200r, and the oscillation reaction is carried out for 3 min. And finally, performing fluorescence detection by taking 471nm as single photon excitation wavelength, and determining a fluorescence spectrogram of each test solution. The results are shown in FIG. 5; the fluorescence intensity of each mixed solution after the addition of the NO solution was taken as the ordinate and the concentration of the NO solution was taken as the abscissa, and the results of plotting a standard curve are shown in FIG. 6.
FIG. 5 shows that the detection method established by the method of the present invention is capable of responding to NO in the concentration range of 0-20. mu.M, and the greater the concentration of NO, the higher the fluorescence intensity. The results in fig. 6 show that: when C is present(NO)At 0-20. mu.M, the fluorescence intensity (y) of the probe has a good linear relation with the concentration (x) of NO, and the linear equation is as follows: 65.38+242.64x (R)20.996), detection limit of 0.67 μ M (S/N is 3), and when C(NO)At 20. mu.M, the maximum fluorescence response was 312.
The results show that the two-photon lipid drop locked fluorescent probe (SUN-TAN) based on the naphthalimide and triphenylamine structure has higher detection sensitivity to NO, and can be used for high-sensitivity detection of Nitric Oxide (NO) of neuroinflammation.
Example 3
The anti-interference performance is one of the important indexes for measuring the practicability of the fluorescent probe. In order to examine the specific recognition performance of the probe (SUN-TAN) prepared by the invention on NO, the experimental method is as follows:
to 10. mu.M of the probe solutions prepared in example 1, 100. mu.M of metal ions were added, respectively (2 to 7 in FIG. 7 represent Zn, respectively)2+，Mn2+，Ba2+，Mg2+，Ca2+，Fe2+) 1.0mM of biological thiol (8-14 in FIG. 7 represent H, respectively)2S, Cys, Hcy, GSH, AA, DHA, MGO); 50 μ M active oxygen (15-17 in FIG. 7 represent H, respectively)2O2，·OH，ClO-) And 50. mu.M active nitrogen (18-19 in FIG. 7 represent HNO, ONOO, respectively-) And 20. mu.M NO (20 in FIG. 7), and then the test solution was quickly put into a constant temperature oscillator at 37 ℃ and 200r for shaking reaction for 3 min. And finally, performing fluorescence detection by taking 471nm as single photon excitation wavelength. The fluorescence emission spectra of each mixture was measured and processed with the software Origin, and the results are shown in FIG. 7. As is clear from the results in FIG. 7, the fluorescence intensity of the probe (SUN-TAN) was hardly affected before and after addition of other substances except NO. Thus indicating that the invention has been madeThe probe (SUN-TAN) has high selective recognition performance on NO response, and is expected to be used for high-sensitivity detection of Nitric Oxide (NO) of neuroinflammation.
Example 4
In order to examine the stability of the probe (SUN-TAN) prepared by the present invention, the change of the fluorescence intensity of the probe itself and the change of the fluorescence intensity of the probe after reaction with NO were observed by selecting PBS buffer systems with different pH values in this example. The specific method comprises the following steps:
PBS buffer system with pH 4.0-8.0 was prepared, and then 10. mu.M of the probe prepared in example 1 and 20. mu.M of NO were added to the PBS buffer, respectively. After the reaction is finished, 471nm is taken as a single photon excitation wavelength, and the fluorescence emission spectrum of the mixed solution before and after NO is added is measured. The results are shown in FIG. 8, which shows that: the fluorescence intensity value of the probe prepared by the invention is almost unchanged and tends to be stable within the pH value range of 4.0-8.0. Therefore, the probe (SUN-TAN) prepared by the invention has higher stability for NO detection, and can be used for NO imaging analysis under different cell microenvironments.
Example 5
To examine the biocompatibility of the probe to verify whether the probe can be used for live cell imaging analysis, this example was verified by toxicity test of the probe SUN-TAN in BV-2 cells. The specific experimental method is as follows:
placing BV-2 cells at 37 deg.C with 5% CO2In an atmosphere, BV-2 cells were seeded in 96-well plates and cultured for 24 h. Then adding different concentrations of SUN-TAN probe solution (0 μm, 5 μm, 10 μm, 15 μm and 25 μm, 1% DMSO DMEM), and culturing for 24 hr. Thereafter 20 μ L of MTT solution (5.0mg/mL) was added to each well separately, incubated for an additional 4h, and then 150 μ L of DMSO was added to dissolve the formazan. Finally, the absorbance of the solution at 490nm was measured using a microplate reader, and the cell survival rate (%) was calculated according to the following formula: a is the absorbance of the experimental group, A0Absorbance of the control group (0 μm).
As shown in FIG. 9, when the concentration of the probe reaches 25 μm, the survival rate of BV-2 cells is still maintained above 80%, thus indicating that the probe of the present invention has low cytotoxicity and good cell biocompatibility, and thus can be used for live cell imaging analysis.
Example 6
In order to verify the positioning ability of the probe of the present invention for lipid droplets, a co-localization experiment was performed on the two-photon fluorescence probe prepared in example 1 of the present invention. The specific experimental steps are as follows:
BV-2 cells were cultured in DMEM medium supplemented with 10% (v/v) newborn calf serum (Gibco), 100U/mL penicillin and 100. mu.g/mL streptomycin and placed at 37 ℃ with 5% CO2And (4) environmental atmosphere. Before imaging, BV-2 cells were passaged to NEST dishes and incubated for 4h with 300. mu.M oleic acid (agent that induces lipid droplet formation), followed by incubation for 30min with 10. mu.M probe SUN-TAN and the commercial lipid droplet localization dye BODIPY493/503 (1. mu.g/mL), followed by 3 washes with serum-free DMEM. And finally, carrying out two-photon confocal imaging by taking 780nm as an excitation wavelength. The results are shown in FIG. 10, which shows that: the probe SUN-TAN was accumulated mainly in the lipid droplet, and the Pearson co-localization coefficient reached 0.92, indicating that the probe SUN-TAN was able to localize in the lipid droplet.
Example 7
In this example, the probe SUN-TAN is used for detecting neuroinflammation in BV-2 cells, and the specific experimental steps are as follows:
before imaging, BV-2 cells were passaged to NEST dishes and 200. mu.g/mL LPS (inducing inflammation of BV-2 cells) was added, and incubation was continued for 6h, followed by addition of 10. mu.M of the SUN-TAN probe, incubation for 30min, and then washing 3 times with serum-free DMEM. And finally, carrying out two-photon confocal imaging by taking 780nm as an excitation wavelength. As shown in FIG. 11, the control group showed almost no fluorescence (a in FIG. 11), and the LPS-treated group showed strong fluorescence intensity and clear imaging (b in FIG. 11), thereby indicating that the probe SUN-TAN according to the present invention can be used for detecting neuroinflammation and has a good imaging effect.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (32)
1. A compound having the structure shown in formula I:
formula I.
2. A process for preparing a compound of formula I according to claim 1, comprising: reacting the compound 3 with a compound A to obtain a compound of a formula I;
the compound 3 has the following structure:
the compound A has the following structure:
3. the method of claim 2, wherein a base and a catalyst are added during the reaction.
4. The process according to claim 3, wherein the catalyst is palladium acetate (Pd (OAc)2) And tris (o-methylphenyl) phosphorus.
5. The method according to claim 3, wherein the base is an organic base or an inorganic base.
6. The method of claim 5, wherein the inorganic base is selected from at least one of the following compounds: hydrides, hydroxides, acetates, fluorides, phosphates, carbonates and bicarbonates of alkali metals or alkaline earth metals.
7. The process according to claim 6, wherein the inorganic base is selected from the group consisting of sodium amide, sodium hydride, sodium hydroxide, potassium hydroxide, sodium acetate, sodium phosphate, potassium fluoride, cesium fluoride, sodium carbonate, potassium bicarbonate, sodium bicarbonate and cesium carbonate.
8. The method of claim 5, wherein the organic base is selected from at least one of the following compounds: triethylamine, trimethylamine, N, N-diisopropylethylamine, tri-N-propylamine, tri-N-butylamine, tri-N-hexylamine, tricyclohexylamine, N-methylcyclohexylamine, N-methylpyrrolidine, N-methylpiperidine, N-ethylpiperidine, N, N-dimethylaniline, N-methylmorpholine, pyridine, 2, 3-or 4-methylpyridine, 2-methyl-5-ethylpyridine, 2, 6-dimethylpyridine, 2,4, 6-trimethylpyridine, 4-dimethylaminopyridine, quinoline, methylquinoline, N, N-tetramethylethylenediamine, N, N-dimethyl-1, 4-diazacyclohexane, N, N-diethyl-1, 4-diazacyclohexane, 1, 8-bis (dimethylamino) naphthalene, N-hexylamine, tri-N-hexylamine, N-methylcyclohexylamine, N-methylpyrrolidine, N-methylpyridine, 2, 6-methylpyridine, 2-methyl pyridine, 2-dimethylaminopyridine, quinoline, N, N, N-tetramethylethylenediamine, N-dimethyl-1, 4-diazacyclohexane, N-diethyl-1, 4-diazacyclohexane, 1, 8-bis (dimethylamino) naphthalene, N-methyl-2-methyl-ethyl-2-methyl-2-methyl-ethyl-2-methyl-2-methyl-2-methyl-2-methyl-ethyl-2-methyl-2-ethyl-methyl-2-methyl-2-methyl-2-methyl-2-ethyl-methyl-ethyl-2-methyl-2-ethyl-methyl-ethyl-methyl-2-ethyl-2-ethyl-2-methyl-2-methyl-2-methyl-2-methyl-2-methyl-2-, Diazabicyclooctane (DABCO), Diazabicyclononane (DBN), Diazabicycloundecane (DBU), butylimidazole and methylimidazole, lithium diisopropylamide, sodium methoxide, potassium tert-butoxide.
9. The preparation method according to claim 2, wherein the molar ratio of the compound 3 to the compound A is 1 (1-2).
10. The method according to claim 3, wherein the catalyst is used in an amount of 0.5 to 4% by mass based on the mass of the compound 3.
11. The preparation method according to claim 3, wherein the molar ratio of the compound 3 to the base is 1 (5-10).
12. The preparation method according to claim 3, wherein the reaction temperature is 60 to 100 ℃; the reaction time is 12-36 h.
13. The method of claim 2, wherein compound 3 is prepared by a process comprising: reacting the compound 2 with 4-bromo-1, 8-naphthalic anhydride to obtain the compound 3;
the compound 2 has the following structure:
14. the method according to claim 13, wherein the molar ratio of the compound 2 to 4-bromo-1, 8-naphthalic anhydride is 1 (0.5 to 1.0).
15. The preparation method of claim 13, wherein the reaction temperature is 60-100 ℃; the reaction time is 1-6 h.
16. The method of claim 13, wherein compound 2 is prepared by a process comprising: reacting the compound 1 with hydrazine hydrate to obtain a compound 2;
the compound 1 has the following structure:
17. the preparation method according to claim 16, wherein a catalyst is added in the reaction process, the catalyst is Pd/C powder, and the mass ratio of the catalyst to the compound 1 is 1 (20-40).
18. The preparation method according to claim 16, wherein the amount of hydrazine hydrate to the compound 1 is 1mL (0.1-0.3) g.
19. The method according to claim 16, wherein the reaction temperature is 60 to 100 ℃; the reaction time is 1-4 h.
20. The method of claim 16, wherein compound 1 is prepared by a process comprising: reacting 1-fluoro-4-nitrobenzene with aminotriglycol monomethyl ether to obtain a compound 1.
21. The method according to claim 20, wherein the molar ratio of 1-fluoro-4-nitrobenzene to aminotriglycol monomethyl ether is 1 (0.5 to 1).
22. The method according to claim 20, wherein the reaction temperature is 60 to 100 ℃; the reaction time is 4-8 h.
23. Use of a compound of formula I according to claim 1 for the preparation of a reagent for the detection of nitric oxide.
24. The use of the compound of formula I as defined in claim 1 for the preparation of a two-photon fluorescence probe, said two-photon fluorescence probe having lipid droplet localization function and being capable of being used for detecting neuroinflammation.
25. A two-photon fluorescent probe, which is the compound with the structure shown in the formula I in claim 1.
26. The two-photon fluorescent probe of claim 25, which has a single photon maximum absorption wavelength of about 466 nm.
27. The two-photon fluorescence probe of claim 25, wherein the two-photon absorption maximum wavelength of the two-photon fluorescence probe is about 780 nm.
28. The two-photon fluorescent probe of claim 25, wherein the two-photon fluorescent probe has a fluorescence maximum emission wavelength of about 630 nm.
29. The two-photon fluorescent probe of claim 25, wherein the stokes shift of the two-photon fluorescent probe is about 150 nm.
30. The two-photon fluorescence probe of claim 25, wherein the two-photon fluorescence probe has a lipid droplet localization function.
31. A kit comprising the two-photon fluorescent probe of claim 25.
32. A biosensor comprising the two-photon fluorescent probe of claim 25.
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