CN111333643A - High-brightness, high-light stability and environmental insensitivity nuclear fluorescent probe - Google Patents

High-brightness, high-light stability and environmental insensitivity nuclear fluorescent probe Download PDF

Info

Publication number
CN111333643A
CN111333643A CN201811550629.8A CN201811550629A CN111333643A CN 111333643 A CN111333643 A CN 111333643A CN 201811550629 A CN201811550629 A CN 201811550629A CN 111333643 A CN111333643 A CN 111333643A
Authority
CN
China
Prior art keywords
probe
nitro
nuclear
morpholine
bromo
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.)
Granted
Application number
CN201811550629.8A
Other languages
Chinese (zh)
Other versions
CN111333643B (en
Inventor
徐兆超
乔庆龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian Institute of Chemical Physics of CAS
Original Assignee
Dalian Institute of Chemical Physics of CAS
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Dalian Institute of Chemical Physics of CAS filed Critical Dalian Institute of Chemical Physics of CAS
Priority to CN201811550629.8A priority Critical patent/CN111333643B/en
Publication of CN111333643A publication Critical patent/CN111333643A/en
Application granted granted Critical
Publication of CN111333643B publication Critical patent/CN111333643B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/08—Naphthalimide dyes; Phthalimide dyes
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • 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
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
    • 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)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Materials Engineering (AREA)
  • Engineering & Computer Science (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention provides a nuclear fluorescent probe with high brightness, high light stability and insensitive environment, and a naphthalimide probe which can be used for nuclear fluorescent imaging. Researches show that the probe has strong rigidity in molecules due to the cyclohexanediamine structure introduced into the 4-, 5-position of the naphthalimide parent body, the nonradiative transition process can be effectively inhibited, and the molar extinction coefficient of the dye in ethanol reaches 46443M‑1cm‑1The quantum yield reaches 0.73, and the brightness and the light stability are high; the probe structure has strong rigidity and obvious resonance type dye environment insensitivityThe nature of the sensation; in addition, the probe has good rigidity and flatness, can be inserted into a nucleic acid chain in a cell nucleus, has proper lipid solubility, and can quickly permeate a cell membrane, so that the rapid and accurate positioning of the cell nucleus can be realized, the cell nucleus can be quickly marked, and the probe is applied to the fields of cell nucleus fluorescence imaging and the like.

Description

High-brightness, high-light stability and environmental insensitivity nuclear fluorescent probe
Technical Field
The invention belongs to the technical field of fluorescence imaging, and particularly relates to a nuclear fluorescence probe with high brightness, high light stability and insensitivity to environment.
Background
Because of the importance of the nucleus in the transcription of genetic material, the labeling of the nucleus is of vital importance in biological as well as in many pathological studies. With the continuous and deep research on cell nucleuses, the requirements of researchers on the properties and functions of cell nuclear fluorescent dyes are continuously improved, and particularly, the rapid rise of the super-resolution fluorescence imaging technology promotes the development and application of novel cell nuclear fluorescent dyes with high stability and high brightness.
Currently, commonly used nuclear fluorescent dyes include: (1) SYTO/POPO series and the like (based on cyanine dyes), the labeling reagent can be inserted into a DNA double strand, can be excited by visible light, and has high fluorescence brightness; however, the fluorescent labeling dye has positive charges, cannot accurately label cell nuclei, and has extremely poor light stability. (2) The DAPI and Hoechst series of most common nuclear dyes have the excitation wavelength of 360nm of ultraviolet light, so that the damage to cells is large, and the most suitable excitation light cannot be matched with laser of a fluorescence imaging technology (generally, a laser with the shortest wavelength of 405 nm). In addition, the nuclear dye has wide fluorescence emission spectrum (easy color cross), poor cell membrane permeability and long dyeing time (usually more than 30 minutes), and is strictly limited in living cell dyeing experiments. The above nuclear fluorescent dyes are difficult to meet the requirements of super-resolution technology on fluorescent stability, fluorescent brightness, long-time dyeing and other performances, so that the development of the nuclear fluorescent dyes with high brightness, high stability and capability of rapidly marking cell nuclei is urgent.
Disclosure of Invention
The invention provides a nuclear fluorescent probe with high brightness, high light stability and insensitive environment, a naphthalimide fluorescent probe, which introduces a cyclohexanediamine structure at the 4-, 5-position of a naphthalimide matrix and simultaneously increases the rigidity and the flatness of a dye; a basic morpholine ring was introduced as a nuclear targeting group. The research finds that the dye has high brightness and excellent light stability, can quickly penetrate through cells and is marked in cell nucleus with high selectivity, and can be further used for fluorescence imaging. The dye also has environmental insensitivity, very small spectral property difference in different environments and very good imaging accuracy.
The invention relates to a high-brightness high-light stability and environment-insensitive cell nucleus fluorescent probe, which has the structural formula as follows:
Figure BDA0001910544810000021
a synthesis method of a nuclear fluorescent probe with high brightness, high light stability and insensitivity to environment comprises the following steps:
Figure BDA0001910544810000022
the specific synthesis steps are as follows:
the method comprises the following steps: synthesis of intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide (Lyso-NBr)
Dissolving 4-bromo-5-nitro-1, 8-naphthalic anhydride in 10-40mL of ethanol, dropwise adding N- (2-aminoethyl) morpholine, heating to 50-70 ℃ for reaction for 1-10h, distilling under reduced pressure to remove the solvent, and separating the residue by a silica gel column to obtain an intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide.
Step two: synthesis of Nuclear Probe Nu-DAC
Dissolving the intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide in ethylene glycol monomethyl ether, adding 1, 2-cyclohexanediamine, slowly heating the reaction solution to 60-90 ℃, and reacting for 10-24 h. Removing ethylene glycol monomethyl ether under reduced pressure, and separating the residue by a silica gel column to obtain the nuclear probe Nu-DAC.
In the first step, the mass ratio of the 4-bromo-5-nitro-1, 8-naphthalic anhydride to the N- (2-aminoethyl) morpholine is 1:0.3-6, and the mass to volume ratio of the 4-bromo-5-nitro-1, 8-naphthalic anhydride to the ethanol is 1:5-80 g/mL.
In the second step, the mass ratio of the N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8 naphthalimide to the 1, 2-cyclohexanediamine is 1:0.3-30, and the volume ratio of the mass of the N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8 naphthalimide to the ethylene glycol monomethyl ether is 1:5-200 g/mL.
The nuclear fluorescent probe can specifically mark the nucleus and realize fluorescent imaging in living cells and living bodies.
The application of the nuclear fluorescent probe with high brightness, high light stability and insensitive environment in the fields of fluorescence imaging, molecular probes and fluorescence sensing.
The invention has the following features:
the probe has the advantages of low price of synthetic raw materials, simple method, easy derivation and the like.
The probe has a cyclohexanediamine structure introduced into 4-, 5-position of naphthalimide matrix, so that molecules have strong rigidity, a non-radiation transition process can be effectively inhibited, and the molar extinction coefficient of the dye in ethanol reaches 46443M-1cm-1The quantum yield reaches 0.73, and the brightness and the light stability are high.
The probe structure has strong rigidity and obvious resonance type dye environment insensitivity.
The probe has good rigidity and flatness, can be inserted into a nucleic acid chain in a cell nucleus, has proper lipid solubility, and can quickly permeate a cell membrane, so that the probe can realize quick and accurate positioning of the cell nucleus, and can be applied to interaction research of the cell nucleus and other organelles, apoptosis research and the like.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of the nuclear probe Nu-DAC prepared in example 1;
FIG. 2 is a high resolution mass spectrum of the nuclear probe Nu-DAC prepared in example 1;
FIG. 3 is a normalized fluorescence excitation and emission spectrum of the nuclear probe Nu-DAC prepared in example 1 in ethanol, with the abscissa being the wavelength, the ordinate being the normalized fluorescence intensity and absorption intensity, and the concentration of the fluorescent probe being 10 μ M;
FIG. 4 is a graph showing the change in the maximum fluorescence emission intensity of the nuclear probe Nu-DAC prepared in example 1 and the commercial dye fluorescein after laser irradiation in PBS buffer (20mM, pH 7.4) for various periods of time, with the abscissa being the laser irradiation time and the ordinate being the relative fluorescence intensity, i.e., the ratio of the maximum fluorescence emission intensity to the initial maximum fluorescence emission intensity;
FIG. 5 is a normalized UV absorption spectrum of the nuclear dye Nu-DAC prepared in example 1 in different solvents, with the abscissa being the wavelength, the ordinate being the normalized UV absorption intensity, and the concentration of the fluorescent probe being 10 μ M;
FIG. 6 is a normalized fluorescence emission spectrum of the nuclear dye Nu-DAC prepared in example 1 in different solvents, with the abscissa being the wavelength, the ordinate being the normalized fluorescence emission intensity, and the concentration of the fluorescent probe being 10 μ M;
FIG. 7 is a graph of the fluorescence images of the nuclear dye Nu-DAC prepared in example 1 in different cell lines (CHO Chinese hamster ovary cells, HT29 human colon cancer cells, HeLa cervical cancer cells, MCF-7 breast cancer cells, C3A human liver cancer cells, PC3 prostate cancer cells);
FIG. 8 is a photograph of a real-time staining image of the nuclear dye Nu-DAC prepared in example 1 and a commercial nuclear fluorescent dye (Hoechst33342) in Chinese hamster ovary Cells (CHO).
Detailed Description
Example 1
A method for synthesizing a nuclear dye Nu-DAC.
Synthesis of intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide (Lyso-NBr):
Figure BDA0001910544810000041
4-bromo-5-nitro-1, 8-naphthalimide (0.50g,1.56mmol) was dissolved in 40mL of ethanol, N- (2-aminoethyl) morpholine (609mg, 4.68mmol) was added dropwise thereto, the reaction was carried out at 70 ℃ for 3 hours, the solvent was distilled off under reduced pressure, and the residue was separated by a silica gel column (dichloromethane/methanol ═ 80/1, V/V) to give Lyso-NBr as a yellowish solid 0.12g in 55% yield.
The nuclear magnetic spectrum hydrogen spectrum data is 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),4.33(t,J＝6.6Hz,2H),3.71–3.57(m,4H),2.70(t,J＝6.5Hz,2H),2.57(s,4H).
the nuclear magnetic spectrum carbon spectrum data are as follows:
13C NMR(101MHz,CDCl3)δ162.85,162.08,151.34,136.00,132.36,131.25,130.63,125.73,124.22,123.57,122.44,121.29,67.02,55.94,53.81,37.66.
the high resolution mass spectrum data is as follows:
HRMS(ESI)：m/z：[M+H]+: calculated values: 434.0352, Experimental value: 434.0386.
the structure of the compound is shown as the above formula Lyso-NBr through detection.
Synthesis of nuclear dye Nu-DAC:
Figure BDA0001910544810000051
Lyso-NBr (100mg, 0.23mmol) was dissolved in 10mL of ethylene glycol methyl ether, and 1, 2-cyclohexanediamine (300mg,0.69mmol) was added thereto. After the reaction solution was slowly heated to 90 ℃ and reacted for 18 hours, ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane/methanol-50/1, V/V) to obtain 48mg of a yellow solid with a yield of 50%.
The nuclear magnetic spectrum hydrogen spectrum is shown in figure 1, and the specific data is as follows:
1H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.6Hz,2H),7.52(s,2H),6.83(d,J＝8.7Hz,2H),4.10(t,J＝7.0Hz,2H),3.62–3.51(m,4H),3.15(d,J＝9.2Hz,2H),2.45(s,4H),2.19(d,J＝11.9Hz,2H),1.73(d,J＝7.1Hz,2H),1.44–1.20(m,4H).
the nuclear magnetic spectrum carbon spectrum data are as follows:
13C NMR(101MHz,DMSO-d6)δ163.39,154.58,134.78,133.35,110.59,107.75,106.45,66.66,59.50,56.42,53.89,36.50,32.08,23.64.
the high resolution mass spectrum is shown in fig. 2, and the specific data is as follows:
HRMS(ESI):m/z:[M+H]+calculating the following values: 421.2240, actual value: 421.2240.
through detection, the structure of the material is shown as the formula Nu-DAC, and the molar extinction coefficient in ethanol reaches 46443M-1cm-1The quantum yield reaches 0.73, and the method has high brightness and light stability and can accurately position the cell nucleus of the living cell.
The dyes to be detected are respectively dissolved in dimethyl sulfoxide solution to prepare 2mM mother liquor of different dyes, test solutions with different concentrations are prepared according to requirements, and the fluorescence spectrum change and the cell nucleus fluorescence imaging of the test solutions are detected.
Nu-DAC in ethanol spectral test. 20 mu LNu-DAC mother liquor is added into 4mL ethanol to prepare 10 mu M fluorescent probe test solution, and ultraviolet and fluorescence spectrum tests are carried out.
The absorption spectrum and the fluorescence spectrum of Nu-DAC in ethanol are shown in figure 3; the concentration of the fluorescent dye is 10 mu M, and the detection shows that the molar extinction coefficient of Nu-DAC in ethanol reaches 46443M-1cm-1The quantum yield reaches 0.73, and the probe has high brightness.
The method comprises the following steps of dissolving fluorescein in 0.1mmol/L NaOH solution, dissolving Nu-DAC, MitoTracker Green and BODIPY in Hepes buffer solution to prepare a test sample with the concentration of 10 mu M, placing the solution to be tested in a quartz cuvette with the concentration of 1 × 1 × 3cm, and using a 500W tungsten lamp as a light source for illumination.
The results of the photostability test of Nu-DAC are shown in FIG. 4, and the fluorescent probe concentration is 10 μ M, which is the ratio of the maximum fluorescence emission intensity to the initial maximum fluorescence emission intensity of Nu-DAC and some commercial dyes after laser irradiation at different times. After 10h of continuous irradiation, the fluorescence intensities of MitoTracker Green and BODIPY were reduced to the initial values of 40%, 60% and 65%, respectively, while the fluorescence intensity of Nu-DAC remained essentially unchanged, indicating that Nu-DAC has better stability relative to common commercial dyes.
Nu-DAC in different solvents (acetonitrile, dimethyl sulfoxide, ethanol, water) ultraviolet absorption spectrum test. And (3) adding 20 mu LNu-DAC mother liquor into 4mL of solvent to be tested respectively each time to prepare 10 mu M of fluorescent probe test solution, and testing the absorption spectrum.
Normalized ultraviolet absorption spectrum in different solvents (acetonitrile, dimethyl sulfoxide, ethanol, water) is shown in FIG. 5, wherein the concentration of the fluorescent probe is 10 μ M; in a solvent with large difference in polarity, the ultraviolet absorption wavelength and the intensity of Nu-DAC have no obvious change, and the Nu-DAC is proved to be an environment-insensitive dye.
Nu-DAC in different solvents (acetonitrile, dimethyl sulfoxide, ethanol, water) fluorescence emission spectroscopy test. And (3) adding 20 mu LNu-DAC mother liquor into 4mL of solvent to be tested respectively each time to prepare 10 mu M of fluorescent probe test solution, and testing the fluorescence spectrum.
The normalized fluorescence emission spectra in different solvents (acetonitrile, dimethyl sulfoxide, ethanol, water) are shown in FIG. 6, wherein the concentration of the fluorescent probe is 10 μ M; in a solvent with large difference in polarity, the emission wavelength and the intensity of Nu-DAC have no obvious change, and the Nu-DAC is proved to be an environment-insensitive dye.
Example 2
A method for synthesizing a nuclear dye Nu-DAC.
Synthesis of intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide (Lyso-NBr):
Figure BDA0001910544810000071
4-bromo-5-nitro-1, 8-naphthalimide (0.50g,1.56mmol) was dissolved in 40mL of ethanol, N- (2-aminoethyl) morpholine (0.15g, 0.96mmol) was added dropwise thereto, the mixture was heated to 50 ℃ and reacted for 8 hours, the solvent was distilled off under reduced pressure, and the residue was separated by a silica gel column (dichloromethane/methanol ═ 80/1, V/V) to give Lyso-NBr as a solid in the form of a yellowish earth (0.10 g, 85% yield).
The nuclear magnetic spectrum hydrogen spectrum data is 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),4.33(t,J＝6.6Hz,2H),3.71–3.57(m,4H),2.70(t,J＝6.5Hz,2H),2.57(s,4H).
the nuclear magnetic spectrum carbon spectrum data are as follows:
13C NMR(101MHz,CDCl3)δ162.85,162.08,151.34,136.00,132.36,131.25,130.63,125.73,124.22,123.57,122.44,121.29,67.02,55.94,53.81,37.66.
the high resolution mass spectrum data is as follows:
HRMS(ESI)：m/z：[M+H]+: calculated values: 434.0352, Experimental value: 434.0386.
the structure of the compound is shown as the above formula Lyso-NBr through detection.
Synthesis of nuclear dye Nu-DAC:
Figure BDA0001910544810000081
Lyso-NBr (100mg, 0.23mmol) was dissolved in 10mL ethylene glycol methyl ether, and 1, 2-cyclohexanediamine (3g,6.9mmol) was added thereto. After the reaction solution was slowly heated to 90 ℃ and reacted for 18 hours, ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane/methanol-50/1, V/V) to obtain 60mg of a yellow solid in 73% yield.
The nuclear magnetic spectrum hydrogen spectrum is shown in figure 1, and the specific data is as follows:
1H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.6Hz,2H),7.52(s,2H),6.83(d,J＝8.7Hz,2H),4.10(t,J＝7.0Hz,2H),3.62–3.51(m,4H),3.15(d,J＝9.2Hz,2H),2.45(s,4H),2.19(d,J＝11.9Hz,2H),1.73(d,J＝7.1Hz,2H),1.44–1.20(m,4H).
the nuclear magnetic spectrum carbon spectrum data are as follows:
13C NMR(101MHz,DMSO-d6)δ163.39,154.58,134.78,133.35,110.59,107.75,106.45,66.66,59.50,56.42,53.89,36.50,32.08,23.64.
the high resolution mass spectrum is shown in fig. 2, and the specific data is as follows:
HRMS(ESI):m/z:[M+H]+calculating the following values: 421.2240, actual value: 421.2240.
through detection, the structure of the material is shown as the formula Nu-DAC, and the molar extinction coefficient in ethanol reaches 46443M-1cm-1The quantum yield reaches 0.73, and the method has high brightness and light stability and can accurately position the cell nucleus of the living cell.
Example 3
A method for synthesizing a nuclear dye Nu-DAC.
Synthesis of intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide (Lyso-NBr):
Figure BDA0001910544810000091
4-bromo-5-nitro-1, 8-naphthalimide (0.50g,1.56mmol) was dissolved in 40mL of ethanol, N- (2-aminoethyl) morpholine (3.0g, 23.4mmol) was added dropwise thereto, the mixture was allowed to react at 70 ℃ for 5 hours, the solvent was distilled off under reduced pressure, and the residue was separated by a silica gel column (dichloromethane/methanol ═ 80/1, V/V) to give Lyso-NBr as a solid in the form of a yellowish earth (0.07 g, 72% yield).
The nuclear magnetic spectrum hydrogen spectrum data is 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),4.33(t,J＝6.6Hz,2H),3.71–3.57(m,4H),2.70(t,J＝6.5Hz,2H),2.57(s,4H).
the nuclear magnetic spectrum carbon spectrum data are as follows:
13C NMR(101MHz,CDCl3)δ162.85,162.08,151.34,136.00,132.36,131.25,130.63,125.73,124.22,123.57,122.44,121.29,67.02,55.94,53.81,37.66.
the high resolution mass spectrum data is as follows:
HRMS(ESI)：m/z：[M+H]+: calculated values: 434.0352, Experimental value: 434.0386.
the structure of the compound is shown as the above formula Lyso-NBr through detection.
Synthesis of nuclear dye Nu-DAC:
Figure BDA0001910544810000101
Lyso-NBr (100mg, 0.23mmol) was dissolved in 10mL of ethylene glycol methyl ether, and 1, 2-cyclohexanediamine (30mg,0.08mmol) was added thereto. After the reaction mixture was slowly heated to 60 ℃ and reacted for 18 hours, ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated by means of a silica gel column (dichloromethane/methanol-50/1, V/V) to obtain 26mg of a yellow solid with a yield of 36%.
The nuclear magnetic spectrum hydrogen spectrum is shown in figure 1, and the specific data is as follows:
1H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.6Hz,2H),7.52(s,2H),6.83(d,J＝8.7Hz,2H),4.10(t,J＝7.0Hz,2H),3.62–3.51(m,4H),3.15(d,J＝9.2Hz,2H),2.45(s,4H),2.19(d,J＝11.9Hz,2H),1.73(d,J＝7.1Hz,2H),1.44–1.20(m,4H).
the nuclear magnetic spectrum carbon spectrum data are as follows:
13C NMR(101MHz,DMSO-d6)δ163.39,154.58,134.78,133.35,110.59,107.75,106.45,66.66,59.50,56.42,53.89,36.50,32.08,23.64.
the high resolution mass spectrum is shown in fig. 2, and the specific data is as follows:
HRMS(ESI):m/z:[M+H]+calculating the following values: 421.2240, actual value: 421.2240.
through detection, the structure of the material is shown as the formula Nu-DAC, and the molar extinction coefficient in ethanol reaches 46443M-1cm-1The quantum yield reaches 0.73, and the method has high brightness and light stability and can accurately position the cell nucleus of the living cell.
The dyes are respectively dissolved in DMSO solution to prepare 2mM mother liquor of different dyes, test solutions with different concentrations are prepared according to requirements, and the fluorescence spectrum change and cell and in-vivo cell nucleus fluorescence imaging of the test solutions are detected.
Example 4
Nu-DAC is used for detecting the fluorescence imaging after staining different cell lines (CHO Chinese hamster ovary cells, HT29 human colon cancer cells, HeLa cervical cancer cells, MCF-7 breast cancer cells, C3A human liver cancer cells and PC3 prostate cancer cells). Dissolving 1 μ LNu-DAC mother liquor in 2mL cell culture solution, 37 deg.C, 5% CO2After incubation for 15 minutes, fluorescence confocal imaging was performed.
Confocal fluorescence imaging after incubation of cell culture solutions of different cell lines (CHO Chinese hamster ovary cells, HT29 human colon cancer cells, HeLa cervical cancer cells, MCF-7 breast cancer cells, C3A human liver cancer cells, PC3 prostate cancer cells) with a final Nu-DAC concentration of 2 μ M for 10 minutes is shown in FIG. 7: nuclei of different cell lines can be stained by Nu-DAC, and green fluorescence is presented. Namely, Nu-DAC can carry out specific marking on cell nucleus in different cell lines and has high signal-to-noise ratio.
Nu-DAC was detected by real-time staining imaging with a commercial nuclear fluorescent dye (Hoechst33342) in Chinese Hamster Ovary (CHO). 0.5 mu LNu-DAC stock solution and 1 mu LHoechst33342 stock solution were dissolved in 2mL CHO medium, and fluorescence confocal imaging was performed after incubation for 0, 1,2, and 3 minutes.
Confocal fluorescence imaging is shown in FIG. 8 after incubation of CHO cells with Nu-DAC, commercial dye Hoechst33342 at a final concentration of 2 μ M for 0, 1,2, 3 minutes; Nu-DAC has fluorescence signals generated in the cell nucleus within 1 minute, and can specifically stain the cell nucleus in the CHO cell within 3 minutes; while Hoechst33342, a commercial dye, does not reflect a significant fluorescence signal at 3 minutes and does not stain the nucleus effectively. Nu-DAC is proved to have higher cell permeability, can rapidly dye cell nucleus, and has the dyeing rate far higher than that of the current common commercial cell nucleus fluorescent dye.

Claims (5)

1. A high-brightness high-light stability and environmental insensitivity cell nucleus fluorescent probe is characterized in that: the structural formula of the compound is shown as follows,
Figure FDA0001910544800000011
2. the method of claim 1, wherein the method comprises the steps of: the specific method for synthesizing is as follows:
the method comprises the following steps: synthesis of intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide
Dissolving 4-bromo-5-nitro-1, 8-naphthalic anhydride in 10-40mL of ethanol, dropwise adding N- (2-aminoethyl) morpholine, heating to 50-70 ℃, reacting for 1-10h, distilling under reduced pressure to remove the solvent, and separating the residue by a silica gel column to obtain an intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide;
step two: synthesis of Nuclear Probe Nu-DAC
Dissolving the intermediate N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8-naphthalimide in ethylene glycol monomethyl ether, adding 1, 2-cyclohexanediamine, slowly heating the reaction solution to 60-90 ℃, and reacting for 10-24 h. Removing ethylene glycol monomethyl ether under reduced pressure, and separating the residue by a silica gel column to obtain the nuclear probe Nu-DAC.
3. The method for synthesizing a high brightness, high light stability, environmentally insensitive nuclear fluorescent probe as claimed in claim 2, wherein: in the first step, the mass ratio of the 4-bromo-5-nitro-1, 8-naphthalic anhydride to the N- (2-aminoethyl) morpholine is 1:0.3-6,
the mass-volume ratio of the 4-bromo-5-nitro-1, 8-naphthalic anhydride to the ethanol is 1:5-80 g/mL.
4. The method of claim 2 for synthesizing a high brightness, high light stability, environmentally insensitive nuclear fluorescent probe, comprising: in the second step, the mass ratio of the N- (2-morpholine) ethyl-4-bromine-5-nitro-1, 8-naphthalimide to the 1, 2-cyclohexanediamine is 1:0.3-30,
the volume ratio of the mass of the N- (2-morpholine) ethyl-4-bromo-5-nitro-1, 8 naphthalimide to the volume of the ethylene glycol monomethyl ether is 1:5-200 g/mL.
5. The use of the high brightness, high light stability, environmentally insensitive nuclear fluorescent probe of claim 1 in the fields of fluorescence imaging, molecular probes and fluorescence sensing.
CN201811550629.8A 2018-12-18 2018-12-18 High-brightness, high-light stability and environmental insensitivity nuclear fluorescent probe Active CN111333643B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811550629.8A CN111333643B (en) 2018-12-18 2018-12-18 High-brightness, high-light stability and environmental insensitivity nuclear fluorescent probe

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811550629.8A CN111333643B (en) 2018-12-18 2018-12-18 High-brightness, high-light stability and environmental insensitivity nuclear fluorescent probe

Publications (2)

Publication Number Publication Date
CN111333643A true CN111333643A (en) 2020-06-26
CN111333643B CN111333643B (en) 2021-11-09

Family

ID=71179456

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811550629.8A Active CN111333643B (en) 2018-12-18 2018-12-18 High-brightness, high-light stability and environmental insensitivity nuclear fluorescent probe

Country Status (1)

Country Link
CN (1) CN111333643B (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104804725A (en) * 2014-01-28 2015-07-29 中国科学院大连化学物理研究所 Fluorescent probe for detecting H2S in lysosome and its synthesis method and use
CN106008342A (en) * 2016-05-20 2016-10-12 济南大学 Fluorescent probe for detecting formaldehyde in cytolysosome and preparation method thereof
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
CN108610289A (en) * 2018-04-23 2018-10-02 李新元 A kind of quick high-selectivity hypersensitive hypobromous acid fluorescence probe
CN108774171A (en) * 2018-07-24 2018-11-09 姜懿珊 A kind of preparation and application of real-time Sensitive Detection hypochlorous acid fluorescence probe
CN108949161A (en) * 2018-09-28 2018-12-07 台州学院 A kind of Ratiometric fluorescent probe compound and application thereof for detecting hypobromous acid

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104804725A (en) * 2014-01-28 2015-07-29 中国科学院大连化学物理研究所 Fluorescent probe for detecting H2S in lysosome and its synthesis method and use
CN106008342A (en) * 2016-05-20 2016-10-12 济南大学 Fluorescent probe for detecting formaldehyde in cytolysosome and preparation method thereof
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
CN108610289A (en) * 2018-04-23 2018-10-02 李新元 A kind of quick high-selectivity hypersensitive hypobromous acid fluorescence probe
CN108774171A (en) * 2018-07-24 2018-11-09 姜懿珊 A kind of preparation and application of real-time Sensitive Detection hypochlorous acid fluorescence probe
CN108949161A (en) * 2018-09-28 2018-12-07 台州学院 A kind of Ratiometric fluorescent probe compound and application thereof for detecting hypobromous acid

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
乔庆龙: "萘酰亚胺类染料荧光构效关系研究及生物应用", 《中国优秀博硕士学位论文全文数据库(博士) 工程科技I辑》 *

Also Published As

Publication number Publication date
CN111333643B (en) 2021-11-09

Similar Documents

Publication Publication Date Title
Zhang et al. A BODIPY-based fluorescent dye for mitochondria in living cells, with low cytotoxicity and high photostability
Li et al. An acid catalyzed reversible ring-opening/ring-closure reaction involving a cyano-rhodamine spirolactam
Huo et al. A turn on fluorescent sensor for cyanide based on ICT off in aqueous and its application for bioimaging
CN107089937B (en) The fluorescence probe and its preparation method and application of Mitochondrially targeted measurement viscosity
CN108069967B (en) Fluorescent probe for intracellular protein labeling and synthetic method and application thereof
CN104629754B (en) Carbazoles Ratio-type pH fluorescent probe and its preparation method and application
Li et al. A near-infrared fluorescent probe for Cu2+ in living cells based on coordination effect
CN102702768A (en) Novel red BODIPY fluorescent dye and preparation method and application thereof
CN111100476A (en) Synthesis and application of pH fluorescent probe
CN111334081B (en) High-brightness multi-color imaging fluorescent probe for lipid drop cell nucleus
CN106543226A (en) A kind of preparation and application for positioning mitochondrial ATP fluorescent probes
CN103710021B (en) Fluorescent dye with nitrobenzimidazole as RNA (ribonucleic acid) recognition group as well as preparation method and application of fluorescent dye
CN111333642A (en) Cell membrane fluorescent probe with high brightness, high stability and insensitivity to environment
CN104650610A (en) Asymmetric near-infrared BODIPY fluorescent dye as well as preparation method and application thereof
CN111333643B (en) High-brightness, high-light stability and environmental insensitivity nuclear fluorescent probe
CN114276356B (en) Mitochondria-targeted fluorescent probe and synthesis method and application thereof
CN109280017A (en) A kind of two-photon fluorescence Golgi localization agent and its preparation method and application
CN102516793B (en) One-class bi-benzyl pentaMethyl cyanine fluorescent dye as well as preparation method and application thereof
CN111334076B (en) High-brightness and high-light-stability cell nucleus fluorescent probe
CN111334080B (en) Carbonic anhydrase fluorescent probe with high brightness and high light stability
CN111333574B (en) Carbonic anhydrase detection fluorescent probe with high brightness and high light stability
Yin et al. Preparation, regulation and biological application of a Schiff base fluorescence probe
CN111334074A (en) High-brightness and high-stability mitochondrial fluorescent dye
CN111334084B (en) Mitochondrial fluorescent dye with high brightness, high stability and high permeability
CN111334072B (en) Mitochondrial super-resolution fluorescent dye with high brightness and high stability

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