CN111333645B - Fluorescent probe for marking cytoskeleton - Google Patents
Fluorescent probe for marking cytoskeleton Download PDFInfo
- Publication number
- CN111333645B CN111333645B CN201811551510.2A CN201811551510A CN111333645B CN 111333645 B CN111333645 B CN 111333645B CN 201811551510 A CN201811551510 A CN 201811551510A CN 111333645 B CN111333645 B CN 111333645B
- Authority
- CN
- China
- Prior art keywords
- dac
- cytoskeleton
- bcome
- fluorescent probe
- volume ratio
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/06—Peri-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a fluorescent probe for cytoskeleton marking, which has the following chemical structure characteristics: the fluorescent probe is characterized in that a fluorophore matrix is a naphthalimide dye, one end of the naphthalimide dye is connected with a succinimide active group for labeling an antibody, and the specific structure of the fluorescent probe is shown as a formula (1). Compared with other common probes such as rhodamine, fluorescein and BODIPY, the probe has good light stability, is more suitable for imaging of a super-resolution microscope, is marked on an antibody corresponding to a cytoskeleton, and can mark the cytoskeleton and carry out clear imaging.
Description
Technical Field
The invention belongs to the field of biological analysis and detection, and particularly relates to a fluorescent probe for cytoskeleton marking.
Background
Cells are important targets of interest in the field of biology as a basic unit of life activities of organisms. The observation of the components inside cells by microscopic imaging techniques in order to obtain a more complete understanding of the sub-cellular structural functions has been a scientific problem that has been continuously and deeply explored by scientists for decades. The traditional optical microscope imaging is limited by the light wave diffraction effect, reaches the limit of resolution, and cannot observe and research the intracellular nano-scale organelles and cell components. In recent years, with the appearance of novel fluorescent molecules and the reformation and innovation of technologies such as digital development and the like, the laser scanning confocal fluorescence imaging technology is produced. The technology does not need to fix and inactivate the cells, continuously scans different sections of the fluorescence-labeled cells layer by laser, reconstructs a three-dimensional model by the obtained light slice through a computer, can accurately position the three-dimensional spatial arrangement of components in the living cells in real time, and greatly improves the microscopic imaging resolution, so that the technology is widely applied to exploring the rules of various life activities in the cells. The cytoskeleton with the intracellular diameter between 10 and 20 nanometers is used as an organelle discovered in the cell at the latest, and the diameter of the cytoskeleton can not be observed by a conventional optical microscope, so by means of a laser scanning confocal fluorescence imaging platform, scientists understand the structure and the function of the cytoskeleton and promote a new level in recent years.
The cytoskeleton is a complex network system consisting of proteins such as microfilaments, microtubules and intermediate fibers on the inner side of the nucleus and the inner side of the cell membrane of the eukaryotic cell, and has the main functions of mechanically supporting the normal morphological structure of the eukaryotic cell, protecting organelles from being damaged by external force of the cell and the like. Recent research evidence combined with confocal fluorescence imaging indicates that cytoskeleton is also involved in regulating and controlling multiple important vital activities in cells, such as chromosome traction during mitosis, providing directional tracks for intracellular substance transport and signal transduction, participating in apoptosis, assisting movement and migration of immune response cells in vivo, and being related to tissue invasion and metastasis of tumor cells. At present, the cytoskeleton function is not completely known, so that the cytoskeleton function is worthy of further exploration by means of higher-resolution imaging means. The method for dynamically observing cytoskeleton at present stage is mainly characterized in that specific fluorescent dye is used for marking the microfilament tubulin, exciting light with different wavelengths is used for processing light to enable the light to be focused on a certain focal plane of a cell, the dye positioned at the irradiation point is excited to emit fluorescence with corresponding wavelength, and then optical signals are received, analyzed and fed back by a computer, so that the positioning imaging of the fluorescence on the cytoskeleton is realized. To achieve the best signal-to-noise ratio and higher resolution for fluorescence imaging, increasing the intensity of the excitation light is the primary choice. However, the conventional fluorescent dye is affected by the photobleaching effect, continuous scanning of high-intensity excitation light is received in a confocal system, light absorption tends to be in a saturated state, molecules in an excited state in a fluorophore are irreversibly damaged, so that a fluorescent signal is greatly attenuated along with irradiation time, and positioning imaging of cytoskeletons is seriously affected. Therefore, there is a need to develop a novel photostable fluorescent molecular probe targeting cytoskeleton, reduce the photobleaching effect generated by fluorescent molecules during laser confocal imaging, and improve the high signal-to-noise ratio and high resolution of laser scanning confocal imaging of the cytoskeleton so as to realize continuous and dynamic exploration of more physiological functions of the cytoskeleton.
Disclosure of Invention
The invention provides a fluorescent probe for marking cytoskeleton, which has the stability higher than that of the traditional dyes such as rhodamine, fluorescein, BODIPY and the like, and the cytoskeleton protein marked by the fluorescent probe is imaged under a confocal microscope or a super-resolution microscope to obtain a clear structure.
The invention relates to a fluorescent probe for marking cytoskeleton, which has the following molecular structural formula:
a method for synthesizing a fluorescent probe for cytoskeleton marking comprises the following steps:
(1) synthesis of intermediate BCOMe-NBr:
4-bromo-5-nitro-1, 8-naphthalic anhydride, 4-aminobutyric acid ethyl ester hydrochloride and triethylamine are dissolved in absolute ethyl alcohol. Heating the reaction solution to 40-90 ℃, and stirring for 1-24 h. Cooling the reaction liquid to room temperature, removing the solvent under reduced pressure, separating by using a silica gel column, and removing the solvent under reduced pressure by using dichloromethane and petroleum ether in a volume ratio of 1: 0.25-6 or dichloromethane and methanol in a volume ratio of 1: 0-0.01 as an eluent to obtain an off-white solid BCOMe-NBr;
(2) synthesizing a probe BCOOH-DAC:
BCOMe-NBr, dissolved in ethylene glycol methyl ether, was added to cyclohexane diamine. The reaction solution is slowly heated to 140 ℃ of 100 ℃ and reacted for 10-24h under the protection of nitrogen. Removing the solvent under reduced pressure, separating by using a silica gel column, and removing the solvent by using dichloromethane and methanol in a volume ratio of 50-400: 1 as an eluent to obtain a brown yellow solid BCOMe-DAC;
BCOMe-DAC methanol, and 2M sodium hydroxide solution is added into the reaction solution dropwise. Reacting for 1-3h at room temperature, distilling under reduced pressure to remove methanol, filtering, washing with water, and drying to obtain BCOOH-DAC;
(3) synthesis of fluorescent probe with NHS active group
Dissolving BCOOH-DAC and DCC in dry N, N-dimethylformamide, and stirring at room temperature for 10-40 min. N-hydroxysuccinimide was dissolved in 1mL of dry N, N-dimethylformamide and added to the reaction solution. Decompressing and removing the solvent after 2-5h, separating by using a silica gel column, and removing the solvent by using dichloromethane and ethyl acetate in a volume ratio of 4-20: 1 as an eluent to obtain a fluorescent probe NHSB-DAC of the NHS active group;
in the step (1), the mass ratio of 4-bromo-5-nitro-1, 8-naphthalic anhydride, 4-ethyl aminobutyric acid hydrochloride and triethylamine is 1:1-3: 1-3; the volume ratio of the mass of the 4-bromo-5-nitro-1, 8-naphthalic anhydride to the volume of the ethanol is 1:20-80 g/mL;
in the step (2), the mass ratio of BCOMe-NBr to cyclohexanediamine is 1: 1-3; the volume ratio of the mass of BCOMe-NBr to ethylene glycol monomethyl ether is 10-50:1 g/mL;
the volume ratio of the mass of the BCOMe-DAC to the methanol is 10-20:1 g/mL; -the volume ratio of the mass of the BCOMe-DAC to the 2M sodium hydroxide solution is 10-20:1 g/mL; -the mass to water volume ratio of BCOMe-DAC is 10-20:1 g/mL;
in the step (3), the mass ratio of BCOOH-DAC, DCC and NHS is 1:1-5: 1-10; the ratio of the mass of BCOOH-DAC to the volume of N, N-dimethylformamide was 10-20:1 (g: mL).
The fluorescent probe for labeling the cytoskeleton is characterized by being used for labeling the cytoskeleton, and the labeling method comprises the following steps: (1) labeling cells with monoclonal antibodies corresponding to cytoskeletal proteins; (2) labeling fluorescent probes to corresponding polyclonal antibodies; (3) the fluorescently-labeled polyclonal antibody labels the monoclonal antibody, and then labels the corresponding cytoskeletal protein.
The method for labeling the cytoskeleton by the fluorescent probe is characterized by comprising the following steps: the cytoskeleton marked by the method is a microtubule, a microfilament and a middle fiber.
The method for labeling the cytoskeleton by the fluorescent probe is characterized by comprising the following steps: the cytoskeleton marked by the method can be used for confocal microscope or super-resolution microscope imaging.
The invention has the advantages and beneficial effects that:
the stability of the probe is stronger than that of the traditional dyes such as rhodamine, fluorescein, BODIPY and the like, and the marked cytoskeletal protein is used for imaging under a confocal microscope or a super-resolution microscope to obtain a clear structural image.
Drawings
FIG. 1 shows the hydrogen nuclear magnetic spectrum of NHSB-DAC as a fluorescent probe prepared in example 3;
FIG. 2 high resolution mass spectrum of the fluorescent probe NHSB-DAC prepared in example 3;
FIG. 3 is a photostability detection profile of the fluorescent probe NHSB-DAC prepared in example 3 as described in example 4.
FIG. 4 is an SDS-PAGE electrophoresis of the fluorescent probe NHSB-DAC prepared in example 3 labeled polyclonal antibody goat anti-mouse IgG described in example 5.
FIG. 5 is fluorescence confocal imaging of the NHSB-DAC labeled polyclonal antibody goat anti-mouse IgG labeled microfilament protein prepared in example 5 described in example 6.
FIG. 6 is fluorescence confocal imaging of the NHSB-DAC labeled polyclonal antibody goat anti-mouse IgG labeled tubulin prepared in example 5 described in example 7.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
Synthesis of intermediate 6- (N- (4-bromo-5-nitro-1, 8-naphthalimide)) ethyl aminobutyric acid (BCOMe-NBr)
4-bromo-5-nitro-1, 8-naphthalimide (1.00g,3.11mmol) was dissolved in 80mL of ethanol, and ethyl 4-aminobutyrate hydrochloride (1.00g, 6.00mmol) and 1.00g of triethylamine were added thereto. After 1 hour at 90 ℃, the solvent was distilled off under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane: petroleum ether: 3:1, V/V) to obtain 608mg of a white solid with a yield of 45%.
Synthesis of BCOOH-DAC
BCOMe-NBr (200mg, 0.46mmol) was dissolved in 20mL of ethylene glycol methyl ether, and 600mg of 1, 2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 100 ℃ and reacted for 24 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane: methanol 80:1, V/V) to give 103mg of a dark yellow solid in 53% yield.
BCOMe-DAC (80mg,0.19mmol) was dissolved in 40mL of methanol, and 8mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition, the reaction solution reacted at room temperature for 1h, the methanol was removed by distillation under reduced pressure, the turbid solution was filtered and the filter cake was washed with 8mL of water and dried to obtain BCOOH-DAC 65 mg with a yield of 87%.
Synthesis of NHSB-DAC
BCOOH-DAC (50mg,0.12mmol) and Dicyclohexylcarbodiimide (DCC) (50mg,0.24 mmol) were dissolved in 2.5mL of N, N-dimethylformamide and stirred at room temperature for 40 min. N-hydroxysuccinimide (50mg,0.44mmol) was dissolved in 1mL of N, N-dimethylformamide, and then added dropwise to the reaction solution. After 5h the solvent was removed under reduced pressure and separated on a silica gel column with dichloromethane: ethyl acetate 5:1 as eluent, and the solvent was removed to give 55mg of an earth yellow solid in 89% yield.
The detection shows that the structure of the dye is shown as the formula NHSB-DAC, the absorption wavelength of the dye in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield reaches 0.78.
Example 2
Synthesis of intermediate 6- (N- (4-bromo-5-nitro-1, 8-naphthalimide)) ethyl aminobutyric acid (BCOMe-NBr)
4-bromo-5-nitro-1, 8-naphthalimide (1.00g,3.11mmol) was dissolved in 20mL of ethanol, and ethyl 4-aminobutyrate hydrochloride (3.00g, 18.0mmol) and 3.00g of triethylamine were added thereto. After 24 hours of reaction at 40 ℃, the solvent was distilled off under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane: petroleum ether: 3:1, V/V) to give 500mg of a white solid in 37% yield. The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:
1H NMR(400MHz,CDCl3)δ8.71(d,J＝7.8Hz,1H),8.52(d,J＝7.9Hz,1H), 8.22(d,J＝7.9Hz,1H),7.93(d,J＝7.8Hz,1H),4.25(t,J＝7.1Hz,2H),4.10(q,J ＝7.1Hz,2H),2.44(t,J＝7.4Hz,2H),2.09(p,J＝7.3Hz,2H),1.24(t,J＝7.1Hz, 3H).13C NMR(101MHz,CDCl3)δ172.72,162.85,162.09,151.33,136.00,132.40, 131.30,130.57,125.65,124.24,123.56,122.36,121.24,60.53,40.11,31.82,23.20, 14.23.
the high resolution mass spectrum data is as follows: theoretical value C of high-resolution mass spectrum18H16BrN2O6[M+H]+435.0192, actual value 435.0193.
Synthesis of BCOOH-DAC
BCOMe-NBr (200mg, 0.46mmol) was dissolved in 4mL of ethylene glycol methyl ether, and 200mg of 1, 2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 140 ℃ and reacted for 10 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane: methanol 80:1, V/V) to give 86mg of a dark yellow solid in 44% yield. The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:
1H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.6Hz,2H),7.51(s,2H),6.82(d, J＝8.7Hz,2H),4.00(dt,J＝14.1,5.3Hz,4H),3.14(d,J＝8.8Hz,2H),2.30(t,J＝ 7.5Hz,2H),2.19(d,J＝11.7Hz,2H),1.89–1.80(m,2H),1.73(d,J＝6.8Hz,2H), 1.31(dt,J＝30.1,15.8Hz,4H),1.14(t,J＝7.1Hz,3H).13C NMR(101MHz, DMSO-d6)δ172.88,163.49,154.56,134.79,133.35,110.58,107.74,106.44,60.18, 59.48,38.55,32.07,31.80,23.75,23.63,14.53.
BCOMe-DAC (200mg,0.48mmol) was dissolved in 10mL of methanol, and 10mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition, the reaction solution reacted at room temperature for 3h, the methanol was removed by distillation under reduced pressure, the turbid solution was filtered and the filter cake was washed with 10mL of water and dried to obtain BCOOH-DAC 65 mg with a yield of 87%. The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:
1H NMR(400MHz,DMSO-d6)δ12.01(s,1H),8.04(d,J＝8.6Hz,2H),7.51(s, 2H),6.82(d,J＝8.7Hz,2H),3.99(dd,J＝9.2,4.6Hz,2H),3.15(d,J＝9.1Hz,2H), 2.21(dd,J＝16.7,9.3Hz,4H),1.88–1.76(m,2H),1.72(d,J＝8.0Hz,2H),1.42– 1.19(m,4H).13C NMR(101MHz,DMSO-d6)δ174.48,163.50,154.57,134.79, 133.36,110.58,107.76,106.47,59.50,47.97,33.82,32.08,31.90,25.79,24.93, 23.86,23.63.
synthesis of NHSB-DAC
BCOOH-DAC (20mg,0.05mmol) and Dicyclohexylcarbodiimide (DCC) (100mg,0.48 mmol) were dissolved in 2mL of N, N-dimethylformamide and stirred at room temperature for 10 min. N-hydroxysuccinimide (200mg,1.74mmol) was dissolved in 1mL of N, N-dimethylformamide, and then added dropwise to the reaction solution. After 2h the solvent was removed under reduced pressure and separated on a silica gel column with dichloromethane: ethyl acetate 5:1 as eluent, and the solvent was removed to give 19mg of an earthy yellow solid in 77% yield. The hydrogen spectrum of the nuclear magnetic spectrum is shown in figure 1, and the data of the hydrogen spectrum and the carbon spectrum of the nuclear magnetic spectrum are as follows:
1H NMR(400MHz,DMSO-d6)δ8.19–7.93(m,2H),7.53(s,2H),6.83(d,J＝ 8.7Hz,2H),4.05(t,J＝6.5Hz,2H),3.15(d,J＝9.2Hz,1H),2.80(s,4H),2.72(t,J ＝7.7Hz,2H),2.19(d,J＝11.4Hz,2H),1.97–1.88(m,2H),1.73(d,J＝7.2Hz, 2H),1.31(dt,J＝28.8,15.2Hz,4H).13C NMR(101MHz,DMSO-d6)δ170.66, 169.11,163.47,154.65,134.87,133.42,110.63,107.66,106.43,59.48,38.35,32.07, 28.69,25.90,23.73,23.63.
the high resolution mass spectrum is shown in fig. 2, and the specific data are as follows: theoretical value C of high-resolution mass spectrum26H27N4O6 [M+H]+491.1931, actual value 491.1981.
The detection shows that the structure of the dye is shown as the formula NHSB-DAC, the absorption wavelength of the dye in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield reaches 0.78.
Example 3
Synthesis of intermediate 6- (N- (4-bromo-5-nitro-1, 8-naphthalimide)) ethyl aminobutyric acid (BCOMe-NBr)
4-bromo-5-nitro-1, 8-naphthalimide (1.00g,3.11mmol) was dissolved in 20mL of ethanol, and ethyl 4-aminobutyrate hydrochloride (3.00g, 18.0mmol) and 3.00g of triethylamine were added thereto. After reacting at 60 ℃ for 18 hours, the solvent was distilled off under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane: petroleum ether: 3:1, V/V) to give 743mg of a white solid, yield 55%. The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:
1H NMR(400MHz,CDCl3)δ8.71(d,J＝7.8Hz,1H),8.52(d,J＝7.9Hz,1H), 8.22(d,J＝7.9Hz,1H),7.93(d,J＝7.8Hz,1H),4.25(t,J＝7.1Hz,2H),4.10(q,J ＝7.1Hz,2H),2.44(t,J＝7.4Hz,2H),2.09(p,J＝7.3Hz,2H),1.24(t,J＝7.1Hz, 3H).13C NMR(101MHz,CDCl3)δ172.72,162.85,162.09,151.33,136.00,132.40, 131.30,130.57,125.65,124.24,123.56,122.36,121.24,60.53,40.11,31.82,23.20, 14.23.
the high resolution mass spectrum data is as follows: theoretical value C of high-resolution mass spectrum18H16BrN2O6[M+H]+435.0192, actual value 435.0193.
Synthesis of BCOOH-DAC
BCOMe-NBr (200mg, 0.46mmol) was dissolved in 4mL of ethylene glycol methyl ether, and 200mg of 1, 2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 140 ℃ and reacted for 8 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane: methanol 80:1, V/V) to give 90mg of a dark yellow solid in a yield of 46%. The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:
1H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.6Hz,2H),7.51(s,2H),6.82(d, J＝8.7Hz,2H),4.00(dt,J＝14.1,5.3Hz,4H),3.14(d,J＝8.8Hz,2H),2.30(t,J＝ 7.5Hz,2H),2.19(d,J＝11.7Hz,2H),1.89–1.80(m,2H),1.73(d,J＝6.8Hz,2H), 1.31(dt,J＝30.1,15.8Hz,4H),1.14(t,J＝7.1Hz,3H).13C NMR(101MHz, DMSO-d6)δ172.88,163.49,154.56,134.79,133.35,110.58,107.74,106.44,60.18, 59.48,38.55,32.07,31.80,23.75,23.63,14.53.
BCOMe-DAC (200mg,0.48mmol) was dissolved in 10mL of methanol, and 10mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition, the reaction solution reacted at room temperature for 2h, the methanol was removed by distillation under reduced pressure, the turbid solution was filtered and the filter cake was washed with 10mL of water and dried to obtain 62 mg of BCOOH-DAC with a yield of 83%. The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:
1H NMR(400MHz,DMSO-d6)δ12.01(s,1H),8.04(d,J＝8.6Hz,2H),7.51(s, 2H),6.82(d,J＝8.7Hz,2H),3.99(dd,J＝9.2,4.6Hz,2H),3.15(d,J＝9.1Hz,2H), 2.21(dd,J＝16.7,9.3Hz,4H),1.88–1.76(m,2H),1.72(d,J＝8.0Hz,2H),1.42– 1.19(m,4H).13C NMR(101MHz,DMSO-d6)δ174.48,163.50,154.57,134.79, 133.36,110.58,107.76,106.47,59.50,47.97,33.82,32.08,31.90,25.79,24.93, 23.86,23.63.
synthesis of NHSB-DAC
BCOOH-DAC (20mg,0.05mmol) and Dicyclohexylcarbodiimide (DCC) (80mg,0.38 mmol) were dissolved in 2mL of N, N-dimethylformamide and stirred at room temperature for 10 min. N-hydroxysuccinimide (150mg,1.31mmol) was dissolved in 1mL of N, N-dimethylformamide, and then added dropwise to the reaction solution. After 2h the solvent was removed under reduced pressure and separated on a silica gel column with dichloromethane: ethyl acetate 5:1 as eluent, and the solvent was removed to give 20mg of an earthy yellow solid in 81% yield. The hydrogen spectrum of the nuclear magnetic spectrum is shown in figure 1, and the data of the hydrogen spectrum and the carbon spectrum of the nuclear magnetic spectrum are as follows:
1H NMR(400MHz,DMSO-d6)δ8.19–7.93(m,2H),7.53(s,2H),6.83(d,J＝ 8.7Hz,2H),4.05(t,J＝6.5Hz,2H),3.15(d,J＝9.2Hz,1H),2.80(s,4H),2.72(t,J ＝7.7Hz,2H),2.19(d,J＝11.4Hz,2H),1.97–1.88(m,2H),1.73(d,J＝7.2Hz, 2H),1.31(dt,J＝28.8,15.2Hz,4H).13C NMR(101MHz,DMSO-d6)δ170.66, 169.11,163.47,154.65,134.87,133.42,110.63,107.66,106.43,59.48,38.35,32.07, 28.69,25.90,23.73,23.63.
the detection shows that the structure of the dye is shown as the formula NHSB-DAC, the absorption wavelength of the dye in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield reaches 0.78.
Example 4
In vitro stability assay of fluorescent Probe NHSB-DAC prepared in example 3
NHSB-DAC, rhodamine 123, fluorescein, BODIPY were dissolved in PBS solution to a final concentration of 10. mu.M. The fluorescence intensity of the fluorescent material was measured at 0, 0.5, 1.5, 2, 3, 4, 6 and 8 hours under strong light irradiation. Fig. 3 was obtained with time as the abscissa and the normalized value of the maximum emission intensity as the ordinate.
NHSB-DAC, rhodamine 123, fluorescein, BODIPY stability test chart as shown in figure 3: the emission intensity of rhodamine 123, fluorescein and BODIPY is obviously weakened along with the time extension, while the weakened amplitude of the emission intensity of NHSB-DAC is obviously lower than that of the other three fluorescent dyes, which proves that the light stability is stronger.
Example 5
Example 3 preparation of fluorescent Probe NHSB-DAC-labeled polyclonal antibody and purification
NHSB-DAC dissolved in DMSO solution to make 1mM stock solution for use. mu.L of the NHSB-DAC stock solution was added to 100. mu.L of a solution containing polyclonal goat anti-mouse IgG (0.5mg/mL), allowed to stand at room temperature for 1h, and passed through Sephadex column G-25 to remove excess fluorescent small molecules. Polyclonal antibodies labeled NHSB-DAC were run on 12% SDS-PAGE and imaged first by UV irradiation and then stained with Coomassie Brilliant blue to give FIG. 4.
The fluorescence imaging of the fluorescent probe NHSB-DAC labeled polyclonal antibody is shown in FIG. 4: the first lane in FIG. 4 is protein marker; the second lane is NHSB-DAC labeled polyclonal antibody. Coomassie brilliant blue staining and bands under ultraviolet irradiation are consistent, and the fact that the fluorescent probe is marked on the polyclonal antibody is proved.
Example 6
NHSB-DAC labeled polyclonal antibody prepared in example 5 was used in microfilament protein fluorescence imaging experiments NHSB-DAC labeled polyclonal antibody was dissolved in aqueous solution to prepare 0.5mg/mL of stock solution for use. Hela cells (proliferating epidermal carcinoma cells) were plated in a petri dish containing 1mL of DMED high-sugar medium containing 10% fetal bovine serum, cultured at 37 ℃ and 5% carbon dioxide to a cell density of about 70%, the cells were gently washed with PBS buffer 2 times, fixed with 4% paraformaldehyde for 30min, washed with PBS 3 times after discarding the fixative, then permeabilized with 0.2% TritonX-100 for 20min, washed with PBS 3 times, each for 5min, then blocked with 5% BSA blocking solution for 20min, and washed with PBS 3 times. A solution of 200. mu.L PBS containing monoclonal antibodies against β -microfilamentin (about 10. mu.g/mL) was added and incubated overnight at 4 ℃. The next day, the cells were washed 3 times with PBS, and 200. mu.L PBS containing NHSB-DAC-labeled polyclonal antibody (about 10. mu.g/mL) was added and incubated at 37 ℃ for 3 hours. Finally, the plate was washed 3 times with PBS and imaged under a fluorescence confocal microscope to obtain FIG. 5.
The fluorescent image of NHSB-DAC labeled polyclonal antibody against microfilamentin is shown in FIG. 5: FIG. 5-a is an image of microfilamentin, FIG. 5-b is an image of nuclei labeled with nuclear commercial dye, FIG. 5-c is a superimposed image of FIGS. 5-a and 5-b, and FIG. 5-d is a bright field image.
Example 7
Fluorescence imaging experiment of NHSB-DAC-labeled polyclonal antibody on tubulin prepared in example 5 the NHSB-DAC-labeled polyclonal antibody was dissolved in aqueous solution to prepare a 0.5mg/mL stock solution for use. Hela cells (proliferating epidermal carcinoma cells) were plated in a petri dish containing 1mL of DMED high-sugar medium containing 10% fetal bovine serum, cultured at 37 ℃ and 5% carbon dioxide to a cell density of about 70%, the cells were gently washed with PBS buffer 2 times, fixed with 4% paraformaldehyde for 30min, washed with PBS 3 times after discarding the fixative, then permeabilized with 0.2% TritonX-100 for 20min, washed with PBS 3 times, each for 5min, then blocked with 5% BSA blocking solution for 20min, and washed with PBS 3 times. A solution of 200. mu.L PBS containing monoclonal antibody against a-tubulin (about 10. mu.g/mL) was added and incubated overnight at 4 ℃. The next day, the cells were washed 3 times with PBS, and 200. mu.L PBS containing NHSB-DAC-labeled polyclonal antibody (about 10. mu.g/mL) was added and incubated at 37 ℃ for 3 hours. Finally, the plate was washed 3 times with PBS and imaged under a fluorescence confocal microscope to obtain FIG. 6.
The fluorescence image of the NHSB-DAC labeled polyclonal antibody against tubulin is shown in FIG. 6: FIG. 6-a is an image of tubulin, FIG. 6-b is an image of nuclei labeled with nuclear commercial dye, FIG. 6-c is a superimposed image of FIGS. 6-a and 6-b, and FIG. 6-d is a bright field image.
Claims (8)
1. A fluorescent probe for cytoskeleton labeling, characterized in that the molecular structural formula of the probe is as follows:
2. the method of claim 1, wherein the method comprises the following steps:
(1) synthesis of intermediate BCOMe-NBr:
dissolving 4-bromo-5-nitro-1, 8-naphthalic anhydride, 4-ethyl aminobutyric acid hydrochloride and triethylamine in absolute ethyl alcohol, heating the reaction solution to 40-90 ℃, stirring for 1-24h, cooling the reaction solution to room temperature, removing the solvent under reduced pressure, separating by using a silica gel column, and separating by using dichloromethane and petroleum ether in a volume ratio of 1: 0.25-6 or a mixture of dichloromethane and petroleum ether in a volume ratio of 1: taking dichloromethane and methanol of 0-0.01 as an eluent, and removing the solvent under reduced pressure to obtain an off-white solid BCOMe-NBr;
(2) synthesizing a probe BCOOH-DAC:
dissolving BCOMe-NBr in ethylene glycol monomethyl ether, and adding cyclohexanediamine; slowly heating the reaction liquid to 140 ℃ at 100 ℃, and reacting for 10-24h under the protection of nitrogen; removing the solvent under reduced pressure, separating by using a silica gel column, and removing the solvent by using dichloromethane and methanol in a volume ratio of 50-400: 1 as an eluent to obtain a brown yellow solid BCOMe-DAC;
dissolving BCOMe-DAC in methanol, dropwise adding a 2M sodium hydroxide solution into the reaction solution, reacting at room temperature for 1-3h, carrying out reduced pressure distillation to remove the methanol, filtering, washing a filter cake with water, and drying to obtain BCOOH-DAC;
(3) synthesis of fluorescent probe with NHS active group
Dissolving BCOOH-DAC and DCC in dry N, N-dimethylformamide, and stirring at room temperature for 10-40 min; dissolving N-hydroxysuccinimide in 1mL of dry N, N-dimethylformamide, and adding the solution into the reaction solution; and (3) decompressing after 2-5h, removing the solvent, separating by using a silica gel column, and removing the solvent by using dichloromethane and ethyl acetate in a volume ratio of 4-20: 1 as an eluent to obtain the NHSB-DAC of the fluorescent probe with the NHS active group.
3. The method for synthesizing a fluorescent probe for cytoskeleton labeling according to claim 2, characterized in that in the step (1), the mass ratio of 4-bromo-5-nitro-1, 8-naphthalic anhydride, ethyl 4-aminobutyric acid hydrochloride and triethylamine is 1:1-3: 1-3; the volume ratio of the mass of the 4-bromo-5-nitro-1, 8-naphthalic anhydride to the volume of the ethanol is 1:20-80 g/mL.
4. The method for synthesizing a fluorescent probe for cytoskeleton labeling according to claim 2, wherein in the step (2), the mass ratio of BCOMe-NBr to cyclohexanediamine is 1: 1-3; the volume ratio of the mass of BCOMe-NBr to ethylene glycol monomethyl ether is 10-50:1 g/mL; the volume ratio of the mass of the BCOMe-DAC to the methanol is 10-20:1 g/mL;
the volume ratio of the mass of the BCOMe-DAC to the 2M sodium hydroxide solution is 10-20:1 g/mL; the ratio of the mass of the BCOMe-DAC to the volume of the water is 10-20:1 g/mL.
5. The method for synthesizing a fluorescent probe for cytoskeleton labeling according to claim 2, wherein in the step (3), the mass ratio of BCOOH-DAC, DCC and NHS is 1:1-5: 1-10; the volume ratio of the mass of BCOOH-DAC to the N, N-dimethylformamide is 10-20:1 g/mL.
6. Use of a fluorescent probe for cytoskeleton labelling according to claim 1, characterized in that the probe is used for cytoskeleton labelling by the following method:
(1) labeling cells with monoclonal antibodies corresponding to cytoskeletal proteins;
(2) labeling fluorescent probes to corresponding polyclonal antibodies;
(3) the fluorescently-labeled polyclonal antibody labels the monoclonal antibody, and then labels the corresponding cytoskeletal protein.
7. Use of a fluorescent probe for cytoskeletal labeling according to claim 6, wherein: the cytoskeleton marked by the method is a microtubule, a microfilament and a middle fiber.
8. Use of a fluorescent probe for cytoskeletal labeling according to claim 6, wherein: the cytoskeleton marked by the method is used for imaging by a confocal microscope or a super-resolution microscope.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811551510.2A CN111333645B (en) | 2018-12-18 | 2018-12-18 | Fluorescent probe for marking cytoskeleton |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811551510.2A CN111333645B (en) | 2018-12-18 | 2018-12-18 | Fluorescent probe for marking cytoskeleton |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111333645A CN111333645A (en) | 2020-06-26 |
| CN111333645B true CN111333645B (en) | 2021-10-15 |
Family
ID=71177566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811551510.2A Active CN111333645B (en) | 2018-12-18 | 2018-12-18 | Fluorescent probe for marking cytoskeleton |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111333645B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101304973A (en) * | 2005-09-15 | 2008-11-12 | 佩因赛普托药物公司 | Methods of modulating neurotrophin-mediated activity |
| CN107603269A (en) * | 2016-07-11 | 2018-01-19 | 华东理工大学 | A kind of fluorescent dye based on naphthalimide, its preparation method and application |
| CN108069902A (en) * | 2016-11-14 | 2018-05-25 | 中国科学院大连化学物理研究所 | The fluorescence probe of fat drips and its preparation and application in one kind mark and/or detection cell |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090082368A1 (en) * | 2007-09-24 | 2009-03-26 | Painceptor Pharma Corporation | Methods of modulating neurotrophin-mediated activity |
-
2018
- 2018-12-18 CN CN201811551510.2A patent/CN111333645B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101304973A (en) * | 2005-09-15 | 2008-11-12 | 佩因赛普托药物公司 | Methods of modulating neurotrophin-mediated activity |
| CN107603269A (en) * | 2016-07-11 | 2018-01-19 | 华东理工大学 | A kind of fluorescent dye based on naphthalimide, its preparation method and application |
| CN108069902A (en) * | 2016-11-14 | 2018-05-25 | 中国科学院大连化学物理研究所 | The fluorescence probe of fat drips and its preparation and application in one kind mark and/or detection cell |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111333645A (en) | 2020-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wurm et al. | Novel red fluorophores with superior performance in STED microscopy | |
| DeBiasio et al. | Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells. | |
| CN108815537A (en) | A kind of tumour cell targeting specific fluorescence probe and the preparation method and application thereof | |
| CN111334083B (en) | High-brightness high-stability active fluorescent dye and synthesis and application thereof | |
| Niu et al. | Novel two-photon fluorescent probe with high fluorescence quantum yields for tracking lipid droplets in biological systems | |
| Roubinet et al. | Photoactivatable rhodamine spiroamides and diazoketones decorated with “Universal Hydrophilizer” or hydroxyl groups | |
| CN111333642A (en) | Cell membrane fluorescent probe with high brightness, high stability and insensitivity to environment | |
| CN111333619B (en) | 488nm excited high-stability super-resolution fluorescent dye and synthesis and application thereof | |
| CN108641710A (en) | A kind of fluorescence probe and its preparation method and application of detection protein sulphur sulfhydrylation | |
| CN105823770B (en) | A kind of Raman labels probe of no optical interference and its preparation method and application | |
| CN111333645B (en) | Fluorescent probe for marking cytoskeleton | |
| Han et al. | A labeling strategy for living specimens in long-term/super-resolution fluorescence imaging | |
| WO2019034945A1 (en) | Dynamic host-guest interactive system | |
| WO2022027174A1 (en) | Cell nucleus fluorescent dye and dyeing method therefor | |
| Mertsch et al. | Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates | |
| CN112250700A (en) | BODIPY (boron dipyrromethene) protein misfolding probe Halo-BODIPY as well as preparation method and application thereof | |
| Fitter et al. | Single molecule fluorescence spectroscopy: a tool for protein studies approaching cellular environmental conditions | |
| US8530183B2 (en) | Zinc sensors for cellular imaging | |
| CN114470244B (en) | Preparation and use method of targeted lipid drop wash-free fluorescence imaging nano probe | |
| CN111333646B (en) | High-brightness high-stability wash-free SNAP-tag probe and preparation method and application thereof | |
| CN111333618B (en) | 488nm excited wash-free SNAP-tag probe and preparation method thereof | |
| EP3960817A1 (en) | Photoactivatable fluorescent dyes with hydrophilic caging groups and their use | |
| CN111333622B (en) | High-brightness and high-stability fluorescent dye containing active ester and synthesis and application thereof | |
| CN111333621B (en) | 488nm excited washing-free Halo-tag probe and synthesis and biological application thereof | |
| CN116987071B (en) | Lipid drop/lysosome dual-target ratio fluorescent probe capable of monitoring dynamic lipid phagocytosis process, preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |