CN108864056B - Near infrared fluorescent compound and its preparation method and application with AIE performance - Google Patents
Near infrared fluorescent compound and its preparation method and application with AIE performance Download PDFInfo
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- CN108864056B CN108864056B CN201810879788.6A CN201810879788A CN108864056B CN 108864056 B CN108864056 B CN 108864056B CN 201810879788 A CN201810879788 A CN 201810879788A CN 108864056 B CN108864056 B CN 108864056B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- 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"
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- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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- 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
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- General Health & Medical Sciences (AREA)
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- General Physics & Mathematics (AREA)
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- Materials Engineering (AREA)
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- Optics & Photonics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The present invention relates to organic syntheses and new material technology field, more particularly, to the near infrared fluorescent compound and its preparation method and application with AIE performance.The fluorescent chemicals, using indoles salt as receptor, with pyrroles's donor and 2 of pyrroles conjugated structures different with 5 upper introducing donor can effective regulatory molecule fluorescent emission；Also, since the group on 1,2 and 5 forms certain distortion with pyrrole ring, compound AIE performance is imparted, the D- π-A effect of intramolecular makes molecule can be realized long-wave band and shines, emission wavelength has biggish Stokes shift between 600nm-750nm.The fluorescent chemicals are targeting group with indoles salt, may be implemented have many advantages, such as disposable, biological hypotoxicity and by force anti-light Bleachability in imaging process, and can be realized the real-time monitoring to mitochondria dyeing to various intracellular Mitochondrially targeted imagings.
Description
Technical Field
The invention relates to the technical field of organic synthesis and new materials, in particular to a near-infrared fluorescent compound with AIE performance, and a preparation method and application thereof.
Background
Fluorescent molecules have excellent properties in bioimaging, such as rapid response, excellent time resolution, super sensitivity, in-situ operability, ease of operation, good reproducibility, etc. (Wang D, Su H, Kwok R T K, et al. At present, fluorescent probes with aggregation-induced emission properties are widely applied in the biological field. Some reports about the AIE compounds and the application thereof in the biological field exist in the published prior art, such as the invention patent with the publication number of CN107200709A, and mainly disclose a fluorescent compound with aggregation-induced emission properties and the application thereof in the field of cell imaging; the invention patent with the publication number of CN105928920A mainly discloses a detection method based on aggregation-induced emission and aptamer; the invention patent with publication number CN105778894A mainly discloses a fluorescent reagent for detecting trace gamma-globulin, a preparation method and application thereof. Some of the currently reported bioluminescent probes are mostly concentrated in the yellow region or the red region. Since fluorescent dyes emitting light in the Near Infrared region can be used for deep tissue Imaging in biological Applications, have little light damage to biological structures, and can effectively reduce light scattering (Chen H J, Chen C Y, Chang E H, et al.S-Cis Diene formation: A New Bathochologic Shift Stratagene for Near-extracted fluorescent Dye and the Imaging Applications [ J ] Journal of the American Chemical Society,2018,140,5224 and 5234.), it is of great interest to develop AIE biological dyes having Near Infrared emission.
Both the metabolism and proliferative division of cells are energy-producing by mitochondria. Any strong changes in mitochondrial morphogenesis are indicative of diseases such as cancer, neurodegenerative diseases such as alzheimer's disease and parkinson's disease. Therefore, tracking mitochondrial changes is an important task that can provide insight into energy production, apoptosis and degenerative disorders in clinical studies (fusa S, Galluzzi L, Kroemer g. targeting mitochondia for cancer. [ J ]. Environmental & Molecular Mutagenesis,2010,51, 476-489.). The application of fluorescent dye in mitochondrial imaging is reported in the prior art which has been published, for example, in the patent of invention with publication number CN107922834A, mitochondrial autophagy process is monitored in real time by using fluorescent light-stabilized mitochondrial-specific bioprobes with AIE characteristics; the invention patent with publication number CN105874319A mainly discloses the use of positive charge AIE fluorophore to specifically detect and quantify cardiolipin and separate mitochondria and the manufacturing method of the AIE fluorophore. However, the presently reported fluorescent dyes and the commercialized mitochondrial dyes often have significant disadvantages, including small stokes shift, long incubation time and long washing time after cell staining. Prolonged incubation often results in non-specific targeting of cellular components and requires long washes to improve signal-to-noise ratio and remove strong residual signals in free dye. These processes can result in delayed real-time acquisition of mitochondrial data, reducing the accuracy of cellular imaging results. Although some mitochondrial targeting AIE probes have been synthesized and used for cellular imaging, most of them belong to red light emitting probes (less than 700 nm). These probes shorten the incubation time by 20min to 10min, overcoming the disadvantages of quenching and multiple washes. However, none of them can achieve all the characteristics of real-time imaging, no wash-out and photostability of mitochondria. Therefore, the development of the mitochondrial fluorescent dye with excellent performance has great application value.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a near-infrared fluorescent compound with AIE performance, which takes triphenyl pyrrole as a core, takes indole salt as an acceptor, introduces benzene rings or triphenylamine as donors at 2 and 5 positions of the pyrrole, can adjust the fluorescence emission wavelength of molecules, can realize luminescence in a near-infrared region, has larger Stokes shift, reduces light damage to biological structures, and has larger application value and prospect.
The second purpose of the invention is to provide a preparation method of the near-infrared fluorescent compound with AIE performance, which has the advantages of simple operation, mild conditions and high yield.
The third purpose of the invention is to provide an application of the near infrared fluorescent compound with AIE performance in targeted imaging of mitochondria, and the fluorescent compound has the characteristics of real-time imaging, ultralow cytotoxicity, no washing, photobleaching resistance and the like.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a near infrared fluorescent compound with AIE performance has a structural formula as follows:
wherein,the R is1Selected from alkyl, said R2Is selected from Any one of the above.
The fluorescent compound provided by the invention takes triphenyl pyrrole as a core, takes indole salt as an acceptor, introduces benzene rings or triphenylamine as donors at 2 and 5 positions of pyrrole, can adjust the fluorescence emission wavelength of molecules, can realize luminescence in a near infrared region, has larger Stokes shift, reduces photodamage to biological structures, and has larger application value and prospect. And the ester group at the pyrrole 1 position provides good biocompatibility, can be modified to introduce various targeting groups, and has potential application value.
Preferably, said R is1Is selected from-CnH2n+1Any one of, wherein n is selected from a positive integer of 1 to 5. Such as, the R1Selected from methyl or ethyl, etc.
The group is adopted to provide certain biocompatibility for the fluorescent compound.
The compound of the invention takes indole salt as an acceptor, takes a pyrrole donor and donors of introducing benzene ring or triphenylamine at 2 site and 5 site of the pyrrole, can effectively adjust the fluorescence emission wavelength of molecules, can realize luminescence in a near infrared region, has larger Stokes shift, and reduces the light damage to a biological structure.
The invention also provides a preparation method of the near-infrared fluorescent compound with AIE performance, and the synthetic route is as follows:
wherein said X comprises any one of iodine and bromine.
Preferably, the preparation method comprises the following steps:
and carrying out reflux reaction on the compound A and indole salt in a solvent to obtain a compound B, adding a hexafluorophosphate aqueous solution, and stirring for reaction to obtain the fluorescent compound.
Preferably, the solvent includes one or both of ethanol and methanol.
Preferably, the preparation method comprises the following steps:
heating and refluxing the compound A and indole salt in ethanol and/or methanol for 12-30h, removing ethanol and/or methanol, and dissolving with tetrahydrofuran; adding saturated hexafluorophosphate water solution, stirring at normal temperature for 0.2-1 hr, removing solvent, and purifying to obtain the fluorescent compound. The normal temperature is 25 +/-5 ℃.
Preferably, the hexafluorophosphate salt includes any one of potassium hexafluorophosphate and sodium hexafluorophosphate.
Preferably, the molar ratio of compound a to indole salt is 1: 1 (1-1.2), preferably 1: 1 (1.05-1.1).
Preferably, the purification method comprises: purification by column chromatography on silica gel eluting with dichloromethane and methanol at a volume ratio of (15-5): 1, preferably 10: 1.
Preferably, the synthetic route of the compound A is as follows:
preferably, the synthesis step of the compound A comprises: compound C is added to the admixture with POCl3In DMF solution, and reacting at room temperature for 8-20 h.
Preferably, when said R is2Is composed ofThe synthetic route of the compound C is as follows:
preferably, when said R is2Is composed ofThe synthetic route of the compound C is as follows:
. Wherein, in the first step, the reaction is carried out for 8-20h, such as 12h, under reflux conditions, and the molar ratio of the reactant 4-aminobenzoate to the reactant 2, 5-dimethoxytetrahydrofuran is 1: 0.8-1.2, preferably 1: 1; the second step is carried out at room temperature for 8-20h, such as 12h, and the molar ratio of the reactant 1-bromopyrrolidine-2, 5-dione to the reactant 1-ethyl-p-benzoate-based pyrrole is 1: 1 (1-1.1), such as 1: 1.02 and the like; in the third step, the reactant and catalyst Pd (PPh)3)4Dissolving in acetonitrile, adding saturated potassium carbonate aqueous solution, and stirring at 80 + -5 deg.C under nitrogen for 18-30h to obtain a molar ratio of 1: 2.5 (1: 2.5) between 1-ethyl p-benzoate-2.5-dibromopyrrole and (4- (diphenylamino) phenyl) boronic acid (1: 2.25).
Preferably, when said R is2Is composed ofThe synthetic route of the compound C is as follows:
the invention also provides an application of the near-infrared fluorescent compound with the AIE performance in targeted imaging of mitochondria.
Preferably, in targeted imaging of mitochondria, the rate of staining of mitochondria is modulated by modulating the structure of the donor in the fluorescent compound.
Preferably, the mitochondria include any one of mitochondria of HeLa, MCF-7, HepG2 and NIH3T 3. The near-infrared fluorescent compound with the AIE performance has universal target imaging on mitochondria of various cells.
Compared with the prior art, the invention has the beneficial effects that:
(1) the fluorescent compound provided by the invention has the advantages that indole salt is used as an acceptor, and the pyrrole donor and donors with different conjugated structures introduced into the 2-position and the 5-position of pyrrole can effectively regulate and control the fluorescence emission of molecules; in addition, the compound AIE performance is endowed by the twisted structures formed by the groups on the 1,2 and 5 positions and the pyrrole ring, and the molecule can realize long-wave-band luminescence under the strong D-pi-A action, the luminescence wavelength is between 600nm and 750nm, and the compound has larger Stokes shift;
(2) the fluorescent compound provided by the invention takes indole salt as a targeting group, can realize targeted imaging of mitochondria in various cells, and has the advantages of no washing, low biological toxicity, strong photobleaching resistance and the like in the imaging process; the dyeing rate of mitochondria can be adjusted due to different donor structures, so that real-time imaging of mitochondrial dyeing can be realized;
(3) the fluorescent compound provided by the invention is simple in preparation process, easy to operate and low in cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the emission wavelengths of fluorescent compounds TPP-1, TPP-2 and TPP-3 prepared in the examples of the present invention;
FIG. 2 is a graph showing the tendency of fluorescence of fluorescent compounds TPP-1, TPP-2 and TPP-3 prepared in the examples of the present invention in mixed solutions of water and DMSO in different ratios;
FIG. 3 is a diagram showing the cellular fluorescence imaging of the co-staining of HeLa cells with the fluorescent compound TPP-2 prepared in the example of the present invention and the commercial dye Mitotricker Green; wherein A and D are the dyeing sites of the TPP-2, B and E are the dyeing sites of the commercial dye, and C and F are the sites where the TPP-2 and the commercial dye are co-dyed.
FIG. 4 is a graph showing the fluorescence images of cells stained with HeLa, MCF-7, HepG2 and NIH3T3 respectively by the fluorescent compounds TPP-1, TPP-2 and TPP-3 prepared in the example of the present invention; wherein A-L are cytofluorescence imaging images of various cells in a dark field, and a-L are cytofluorescence imaging images in a bright field; the scales in FIG. 4 are all 25 μm;
FIG. 5 is a graph showing the real-time staining of HeLa cells by fluorescent compounds TPP-1, TPP-2 and TPP-3 prepared in the example of the present invention;
FIG. 6 is a graph showing the cellular fluorescence images of HeLa cells under continuous illumination by the fluorescent compound TPP-2 and the commercial dye Mitotractor Red prepared in the example of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a near-infrared fluorescent compound with AIE performance, which has the following structural formula:
wherein, R is1Selected from alkyl, said R2Is selected from Any one of the above.
In a preferred embodiment of the present invention, R is1Is selected from-CnH2n+1Any one of, wherein n is selected from a positive integer of 1 to 5. Such as, the R1Selected from methyl or ethyl, etc. By adjusting the starting materials to obtain different R1Different requirements are fulfilled.
The invention also provides a preparation method of the near-infrared fluorescent compound with AIE performance, and the synthetic route is as follows:
wherein said X comprises any one of iodine and bromine.
In a preferred embodiment of the present invention, the preparation method comprises the steps of:
and carrying out reflux reaction on the compound A and indole salt in a solvent to obtain a compound B, adding a hexafluorophosphate aqueous solution, and stirring for reaction to obtain the fluorescent compound.
In a preferred embodiment of the present invention, the solvent includes one or both of ethanol and methanol.
In a preferred embodiment of the present invention, the preparation method comprises the steps of:
heating and refluxing the compound A and indole salt in ethanol and/or methanol for 12-30h, removing ethanol and/or methanol, and dissolving with tetrahydrofuran; adding saturated hexafluorophosphate water solution, stirring at normal temperature for 0.2-1 hr, removing solvent, and purifying to obtain the fluorescent compound. The normal temperature is 25 +/-5 ℃.
In a preferred embodiment of the present invention, the hexafluorophosphate salt includes any one of potassium hexafluorophosphate and sodium hexafluorophosphate.
In a preferred embodiment of the present invention, the method of purification comprises: purification by column chromatography on silica gel eluting with dichloromethane and methanol at a volume ratio of (15-5): 1, preferably 10: 1.
In a preferred embodiment of the invention, the molar ratio of compound A to indole salt is 1: 1 (1-1.2), preferably 1: 1 (1.05-1.1).
In a preferred embodiment of the present invention, the synthetic route of compound a is as follows:
in a preferred embodiment of the present invention, the step of synthesizing compound a comprises: compound C is added to the admixture with POCl3In DMF solution, and reacting at room temperature for 8-20 h.
In a preferred embodiment of the present invention, when R is2Is composed ofThe synthetic route of the compound C is as follows:
in a preferred embodiment of the present invention, when R is2Is composed ofThe synthetic route of the compound C is as follows:
in a preferred embodiment of the present invention, when R is2Is composed ofThe synthetic route of the compound C is as follows:
the invention also provides an application of the near-infrared fluorescent compound with the AIE performance in targeted imaging of mitochondria.
In a preferred embodiment of the invention, in targeted imaging of mitochondria, the rate of staining of mitochondria is modulated by adjusting the structure of the donor in the fluorescent compound.
In a preferred embodiment of the present invention, the mitochondria include any one of mitochondria of HeLa, MCF-7, HepG2 and NIH3T 3. The near-infrared fluorescent compound with the AIE performance has universal target imaging on mitochondria of various cells.
In the following examples, the reagents and equipment information used are as follows:
ethanol, analytical grade, Beijing chemical reagent factory;
CuCl, analytical grade, J & K company;
POCl3analytically pure, western reagent;
THF, analytical grade, beijing chemical reagent factory;
Pd(PPh3)4analytically pure, J&Company K;
potassium hexafluorophosphate, analytical grade, alatin;
potassium carbonate, analytically pure, J & K company;
DMSO, analytical pure, beijing chemical reagent factory;
DMF, analytical grade, beijing chemical reagent factory;
mitotracker Red, cell culture grade, department of hundred (Baxter, austria);
mitotracker Green, cell culture grade, department of hundred (Baxter, austria);
a nuclear magnetic resonance spectrometer model Mercury-Plus 400, manufactured by Varian corporation, USA;
the fluorescence spectrometer is of a Hitachi F-7000 model, and the manufacturer is Hitachi corporation in Japan;
the mass spectrometer is of a model Q-exact, and the manufacturer is Sammer Feishel science and technology company;
laser scanning confocal microscope, model Leica TCS SP5, Leica company, Germany.
Example 1
This example provides a process for the preparation of three compounds a, by the following steps:
1、TPP-CHO-1preparation of
(1) Adding 5.02mmol of 1, 2-diphenylethylene diyne, 20.08mmol of ethyl p-aminobenzoate and 0.5mmol of CuCl into a 100mL polymerization tube, vacuumizing and filling nitrogen for three times, and reacting at 120 ℃ for 24 hours under the protection of nitrogen; after the reaction is finished, dissolving the raw materials by using dichloromethane, washing the raw materials for three times by using water, washing the raw materials for three times by using a hydrochloric acid aqueous solution with the mass fraction of 5%, drying the raw materials by using anhydrous magnesium sulfate, and removing the solvent to obtain a crude product; separating and purifying the crude product by using dichloromethane and petroleum ether according to the volume ratio of 1: 2 to obtain 1-ethyl p-benzoate-2, 5-diphenylpyrrole;
(2) at 0 deg.C, 270. mu.L of POCl3Slowly dropwise adding into 24mL of DMF, stirring at room temperature for 1h, adding 2.0mmol of 1-ethyl p-benzoate-2, 5-diphenylpyrrole into the solution3Reacting in DMF solution at room temperature for 12h, extracting with dichloromethane and water after the reaction is finished, adding anhydrous magnesium sulfate to remove water after the extraction, filtering, and removing the solvent to obtain a crude product; the crude product is separated and purified by eluent of dichloromethane and petroleum ether at the volume ratio of 1: 2 to obtain TPP-CHO-1.
TPP-CHO-1 was characterized using a nuclear magnetic resonance spectrometer and a mass spectrometer with the following data:
1H NMR(400MHz,DMSO-d6)δ(ppm)：9.58(s,1H),7.83(d,J＝7.9Hz,2H),7.37-7.27(m,6H),7.25(d,J＝7.5Hz,4H),7.14(d,J＝5.6Hz,2H),6.91(s,1H),4.27(q,J＝6.9Hz,2H),1.28(t,J＝6.9Hz,3H)。
MS(ESI+): molecular formula C26H21NO3M/z: calculated value [ M + Na]+418.14 test value [ M + Na]+＝417.71。
2、TPP-CHO-2Preparation of
(1) Dissolving 10mmol of ethyl 4-aminobenzoate and 10mmol of 2, 5-dimethoxy tetrahydrofuran in 25mL of acetic acid, and carrying out reflux reaction for 12 h; after the reaction is finished, cooling to room temperature, removing the solvent, dissolving the residual substances in dichloromethane, washing with water for three times, extracting with dichloromethane, removing water with anhydrous magnesium sulfate, filtering, and removing the solvent to obtain a crude product; separating and purifying the crude product by eluent of dichloromethane and petroleum ether at a volume ratio of 1: 6 to obtain 1-ethyl terephthalate pyrrole;
(2) dissolving 5mmol of 1-bromopyrrolidine-2, 5-diketone in 25mL of DMF, adding 5.10mmol of 1-ethyl terephthalate pyrrole prepared in the step (1), and reacting at room temperature for 12 h; after the reaction is finished, washing with water for three times, extracting with dichloromethane, then removing water with anhydrous magnesium sulfate, filtering, and removing the solvent to obtain a crude product; separating and purifying the crude product by using dichloromethane and petroleum ether at a volume ratio of 1: 6 to obtain 1-ethyl p-benzoate-2, 5-dibromopyrrole;
(3) taking 2mmol of 1-ethyl p-benzoate-2, 5-dibromopyrrole, 4.5mmol of (4- (diphenylamino) phenyl) boric acid and 0.08mmol of Pd (PPh)3)4Dissolving in 20mL of acetonitrile, adding 5mL of saturated potassium carbonate aqueous solution, and stirring and reacting at 80 ℃ for 24 hours under the protection of nitrogen; after the reaction is finished, cooling to room temperature, removing the solvent, extracting residual substances by using water and dichloromethane, then removing water by using anhydrous magnesium sulfate, filtering, and removing the solvent to obtain a crude product; separating and purifying the crude product by eluting with dichloromethane and petroleum ether at a volume ratio of 1: 6 to obtain ethyl 4- (2, 5-bis (4- (diphenylamino) phenyl) -1H-pyrrol-1-yl) benzoate;
(4) at 0 deg.C, 270. mu.L of POCl3Slowly added dropwise to 24mL of DMF, stirred at room temperature for 1H, 2.0mmol of ethyl 4- (2, 5-bis (4- (diphenylamino) phenyl) -1H-pyrrol-1-yl) benzoate was added to the above-mentioned solution of POCl3In DMF solution, reacting at room temperature for 12h, extracting with dichloromethane and water after the reaction is finished, and extractingAdding anhydrous magnesium sulfate to remove water, filtering, and removing the solvent to obtain a crude product; separating and purifying the crude product by eluent of dichloromethane and petroleum ether at a volume ratio of 1: 2 to obtain TPP-CHO-2.
TPP-CHO-2 was characterized using a nuclear magnetic resonance spectrometer and a mass spectrometer with the following data:
1H NMR(400MHz,CDCl3)δ(ppm)：9.76(s,1H),7.95(d,J＝8.0Hz,2H),7.30-7.21(m,9H),7.12-6.99(m,14H),6.96(d,J＝8.3Hz,2H),6.93-6.85(m,6H),4.39(q,J＝7.1Hz,2H),1.41(t,J＝7.2Hz,3H)。
ms (maldi): molecular formula C50H39N3O3M/z: calculated value [ M]+729.30 test value [ M]+＝729.07。
3、TPP-CHO-3Preparation of
(1) Adding 5.02mmol of 1, 2-di-p-bromophenyl ethylene diyne, 20.08mmol of ethyl p-aminobenzoate and 0.5mmol of CuCl into a 100mL polymerization tube, vacuumizing and filling nitrogen for three times, and reacting at 120 ℃ for 24 hours under the protection of nitrogen; after the reaction is finished, dissolving the raw materials by using dichloromethane, washing the raw materials for three times by using water, washing the raw materials for three times by using a hydrochloric acid aqueous solution with the mass fraction of 5%, drying the raw materials by using anhydrous magnesium sulfate, and removing the solvent to obtain a crude product; separating and purifying the crude product by using dichloromethane and petroleum ether according to the volume ratio of 1: 2 to obtain 1-ethyl p-benzoate-2, 5-di-p-bromophenyl pyrrole;
(2) 2mmol of 1-ethyl p-benzoate-2, 5-di-p-bromophenyl pyrrole, 4.5mmol of (4- (diphenylamino) phenyl) boronic acid and 0.08mmol of Pd (PPh)3)4Dissolving in 20mL of acetonitrile, adding 5mL of saturated potassium carbonate aqueous solution, and stirring and reacting at 80 ℃ for 24 hours under the protection of nitrogen; after the reaction is finished, cooling to room temperature, removing the solvent, extracting residual substances by using water and dichloromethane, then removing water by using anhydrous magnesium sulfate, filtering, and removing the solvent to obtain a crude product; separating and purifying the crude product with eluent of dichloromethane and petroleum ether at volume ratio of 1: 6To 4- (2, 5-bis (4'- (diphenylamino) - [1,1' -biphenyl)]-4-yl) -1H-pyrrol-1-yl) benzoic acid ethyl ester;
(3) at 0 deg.C, 270. mu.L of POCl3Slowly dropwise added to 24mL of DMF, stirred at room temperature for 1h, and 2.0mmol of 4- (2, 5-bis (4'- (diphenylamino) - [1,1' -biphenyl)]-4-yl) -1H-pyrrol-1-yl) benzoic acid ethyl ester to the previously described dissolved POCl3Reacting in DMF solution at room temperature for 12h, extracting with dichloromethane and water after the reaction is finished, adding anhydrous magnesium sulfate to remove water after the extraction, filtering, and removing the solvent to obtain a crude product; the crude product is separated and purified by eluent of dichloromethane and petroleum ether at the volume ratio of 1: 2 to obtain TPP-CHO-3.
TPP-CHO-3 was characterized using a nuclear magnetic resonance spectrometer and a mass spectrometer with the following data:
1H NMR(400MHz,CDCl3):δ(ppm)：9.79(s,1H),7.92(d,J＝8.1Hz,2H),7.53-7.46(m,3H),7.46-7.38(m,5H),7.32-7.17(m,11H),7.16-7.07(m,16H),7.07-6.97(m,5H),4.34(q,J＝7.2Hz,2H),1.36(t,J＝7.2Hz,3H)。
ms (apci): molecular formula C62H47N3O3M/z: calculated value [ M + H]+882.36 test value [ M + H]+882.37。
Example 2
This example provides TPP-1The preparation method comprises the following steps:
0.6mmol of TPP-CHO-1 prepared in example 1 and 0.65mmol of 1-ethyl-2, 3, 3-trimethyl-3H-indol-1-ium are taken to be refluxed and reacted in 15mL of absolute ethyl alcohol for 24 hours; after the reaction was complete, the reaction mixture was cooled to room temperature, the solvent was removed, the solid was collected, dissolved in 5mL of THF, and 5mL of saturated KPF was added6After stirring for 30min, the solvent was removed and purified by silica gel column chromatography eluting with dichloromethane and methanol at a volume ratio of 10: 1 to give TPP-1.
TPP-1 was characterized using a nuclear magnetic resonance spectrometer and a mass spectrometer with the following data:
1H NMR(400MHz,DMSO-d6)δ(ppm)：7.99-7.85(m,3H),7.86-7.75(m,2H),7.68(s,1H),7.63-7.50(m,2H),7.50-7.41(m,4H),7.33-7.26(m,7H),7.20(d,J＝6.9Hz,2H),4.58(q,J＝7.3Hz,2H),4.28(q,J＝8.9,6.8Hz,2H),1.53(s,6H),1.42(t,J＝7.3Hz,3H),1.29(t,J＝6.8Hz,3H)。
MS(ESI+): molecular formula C39H37N2O2 +M/z: calculated value [ M]+565.2849 test value [ M]+＝565.2846。
Example 3
This example provides TPP-2With reference to example 2, except that: TPP-CHO-1 from example 2 was replaced with an equimolar amount of TPP-CHO-2.
TPP-2 was characterized using a nuclear magnetic resonance spectrometer and a mass spectrometer with the following data:
1H NMR(400MHz,DMSO-d6)δ(ppm)：8.02-7.90(m,3H),7.82(t,J＝7.5Hz,2H),7.66-7.49(m,3H),7.46-7.25(m,11H),7.20-7.11(m,4H),7.10-7.02(m,8H),6.99(d,J＝7.8Hz,4H),6.92(d,J＝8.0Hz,2H),6.84(d,J＝8.1Hz,2H),4.56(d,J＝7.0Hz,2H),4.31(q,J＝6.9Hz,2H),1.59(s,6H),1.42(t,J＝6.9Hz,3H),1.32(t,J＝7.0Hz,3H)。
MS(ESI+): molecular formula C64H56N3O2 +M/z: calculated value [ M + H]+899.4367 test value [ M + H]+＝899.4293。
Example 4
This example provides TPP-3With reference to example 2, except that: TPP-CHO-1 from example 2 was replaced with an equimolar amount of TPP-CHO-3.
TPP-2 was characterized using a nuclear magnetic resonance spectrometer and a mass spectrometer with the following data:
1H NMR(400MHz,DMSO-d6)δ(ppm)：8.05-7.89(m,3H),7.86-7.71(m,5H),7.68(d,J＝8.0Hz,2H),7.65-7.56(m,5H),7.56-7.40(m,4H),7.39-7.29(m,10H),7.25(d,J＝7.6Hz,2H),7.15-6.97(m,10.2Hz,16H),4.59(q,J＝6.4Hz,2H),4.28(q,J＝6.4Hz,2H),1.57(s,6H),1.43(t,J＝6.4Hz,3H),1.28(t,J＝7.2Hz,3H)。
MS(ESI+): molecular formula C75H63N4O2 +M/z: calculated value [ M]+1051.4945 test value [ M]+＝1051.4933。
Example 5
The fluorescence emission spectra of the compounds TPP-1, TPP-2 and TPP-3 prepared in examples 2-4 in the solid state were measured by a fluorescence spectrometer, and the fluorescence intensities in mixed solutions of water and DMSO at different ratios were measured, and the results are shown in FIG. 1 and FIG. 2, respectively. As can be seen from FIG. 1, the emission wavelengths of TPP-1, TPP-2 and TPP-3 are 600nm, 724nm and 677nm, respectively. As can be seen from FIG. 2, the fluorescence intensity of TPP-1 is maintained at a low value at water contents of 0% to 90%; when the water content reaches 99%, molecules aggregate, and the fluorescence intensity is obviously increased; TPP-2 has a low fluorescence intensity at a water content of 0-50%, and a fluorescence intensity increasing with an increase in water content due to molecular aggregation at a water content of 50-70%, and a fluorescence decreasing due to the generation of a less fluorescent aggregate at a water content of more than 70%; TPP-3 has a low fluorescence intensity at a water content of 0-40%, and a low fluorescence intensity at a water content of 40-60%, wherein the fluorescence intensity increases with the increase of the water content due to the increase of the fluorescence due to the aggregation of molecules, and the fluorescence decreases due to the generation of aggregates with weak fluorescence when the water content is more than 60%; the results show that the near-infrared fluorescent compound prepared by the invention has obvious aggregation-induced emission performance.
Example 6
To demonstrate targeting of TPP-1, TPP-2 and TPP-3 prepared in examples 2-4 to mitochondria, the commercially available mitochondrial dye Mitotracker G was usedreen, exemplified by the fluorescent compound TPP-2 prepared in example 3, co-stained HeLa cells with Mitotracker Green, and placed in a medium containing HeLa cells at a concentration of 10-7In mol/L TPP-2 and Mitotracker Green dye in medium at 37 ℃ and 5% CO2Overnight. As shown in FIG. 3, A and D are the staining sites for TPP-2, B and E are the staining sites for the commercial dye Mitotracker Green, C and F are the cytofluorescence imaging images co-stained with both, and the Green fluorescence sites from Mitotracker Green match well with the red fluorescence of TPP-2. The near infrared fluorescent compound with the AIE performance prepared by the invention can specifically dye mitochondria.
Example 7
HeLa, MCF-7, HepG2 and NIH3T3 cells were seeded in phi 20mm glass-bottomed cell culture dishes (7.2X 10 cells per dish), respectively5±0.05×105Individual cells). At 37 ℃ 5% CO2After overnight incubation in a humidified incubator, the medium was removed and the concentration was 10-7And (3) dyeing the samples in mol/L DMEM with TPP-1, TPP-2 and TPP-3 for 5 min. After staining, without washing, fluorescence imaging of TPP-1, TPP-2 and TPP-3 in living cells was clearly observed directly with a Leica TCSSP5 laser scanning confocal microscope, as shown in FIG. 4. In FIG. 4, A-L show the images of fluorescence of various cells in the dark field, and FIGS. a-L show the images of fluorescence of various cells in the bright field. As can be seen from the figure, the fluorescent compounds prepared in the examples of the present invention have universality for mitochondrial staining of cells and have no-wash function.
Example 8
HeLa cells were stained in DMEM containing TPP-1, TPP-2 and TPP-3 as described in the present invention, as shown in FIG. 5. The result shows that the TPP-1 has the fastest structure and the fastest speed of entering cells, thereby having the fastest staining speed to mitochondria and stronger fluorescence intensity; the TPP-2 dyeing speed is moderate, so that the whole process of dyeing mitochondria can be clearly observed; TPP-3 has a relatively slow rate of entry into cells and relatively few molecules of fluorescence due to the relatively complex structure of the molecule, resulting in relatively slow staining rate and relatively weak fluorescence. Therefore, the invention also shows that the fluorescent compounds with different molecular structures can regulate and control the dyeing rate of mitochondria, and the real-time dyeing process of the mitochondria can be observed.
Example 9
Placing Hela cells in 10-7Staining in cell culture solution containing TPP-2 at mol/L for 5min and 10- 7Staining was performed for 30min in cell culture medium with the mitochondrial commercial dye Mitotracker Red in mol/L. Then, the mitochondria were continuously irradiated under an ultraviolet lamp, and the change of mitochondrial staining by continuous irradiation was observed by a laser scanning confocal microscope, as shown in fig. 6. The results show that the commercial dye mitotracker red almost completely disappeared after 1h of continuous illumination; and TPP-2 has small change of fluorescence intensity after dyeing of mitochondria is continuously illuminated for 6h, which shows that the mitochondria dye related in the invention has strong photobleaching resistance.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (16)
1. The near infrared fluorescent compound with AIE performance is characterized in that the structural formula is as follows:
wherein, R is1Selected from alkyl, said R2Is selected from Any one of the above.
2. The near-infrared fluorescent compound with AIE properties according to claim 1, wherein R is1Is selected from-CnH2n+1Any one of, wherein n is selected from a positive integer of 1 to 5.
3. The near infrared fluorescent compound having AIE properties according to claim 2, wherein n is 2.
4. A method for preparing near-infrared fluorescent compounds with AIE properties according to any of claims 1-3, characterized in that the synthetic route is as follows:
wherein X is any one of iodine and bromine.
5. The method of claim 4, wherein the method comprises the steps of:
and carrying out reflux reaction on the compound A and indole salt in a solvent to obtain a compound B, adding a hexafluorophosphate aqueous solution, and stirring for reaction to obtain the fluorescent compound.
6. The method of claim 5, wherein the solvent is one or both of ethanol and methanol.
7. The method of claim 4, wherein the method comprises the steps of: heating and refluxing the compound A and indole salt in ethanol and/or methanol for 12-30h, removing ethanol and/or methanol, and dissolving with tetrahydrofuran; adding saturated hexafluorophosphate water solution, stirring at normal temperature for 0.2-1 hr, removing solvent, and purifying to obtain the fluorescent compound.
8. The method for preparing a near-infrared fluorescent compound having AIE property according to any one of claims 5 to 7, wherein the hexafluorophosphate is any one of potassium hexafluorophosphate and sodium hexafluorophosphate.
9. The method of claim 8, wherein the molar ratio of compound a to indole salt is 1: 1 (1-1.2).
10. The method of claim 8, wherein the purifying step comprises: purifying by silica gel column chromatography, eluting with dichloromethane and methanol at a volume ratio of (15-5): 1.
11. The method for preparing near-infrared fluorescent compounds with AIE properties according to any of claims 5-7, characterized in that the synthetic route of compound a is as follows:
12. the method of claim 11, wherein the step of synthesizing compound a comprises: compound C is added to the admixture with POCl3In DMF solution, and reacting at room temperature for 8-20 h.
13. The method of claim 11, wherein R is the same as R in the near infrared fluorescent compound having AIE properties2Is composed ofThe synthetic route of the compound C is as follows:
or, when said R is2Is composed ofThe synthetic route of the compound C is as follows:
or, when said R is2Is composed ofThe synthetic route of the compound C is as follows:
14. use of a near infrared fluorescent compound with AIE properties according to any one of claims 1 to 3 for non-diagnostic and therapeutic purposes in targeted imaging of mitochondria.
15. Use according to claim 14, characterized in that in targeted imaging of mitochondria the rate of staining of mitochondria is modulated by adjusting the structure of the donor in the fluorescent compound.
16. The use of claim 15, wherein the mitochondria is any one of the mitochondria of HeLa, MCF-7, HepG2 and NIH3T 3.
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