CN115819426A - Plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity and preparation method and application thereof - Google Patents
Plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity and a preparation method and application thereof, belonging to the technical field of fluorescent probes, wherein the near-infrared fluorescent probe provided by the invention is an APMem fluorescent probe, and has the following structural formula:
wherein n =3, 6 or 9; also provides a preparation method and application of the fluorescent probe, and the fluorescent probe is prepared by introducing diazide bicyclo [2.2.2]Octane functional groups play a decisive key role in keeping the fluorescent probe on the cytoplasmic membrane for ultra-long retention. Therefore, the fluorescent probe is more suitable than the conventional aggregation-induced emission probe for imaging cytoplasmic membranesThe method is used for the ultra-long-time high-quality imaging of the plant cytoplasmic membrane, provides a sufficient time window for observing the dynamic physiological process of the plant cytoplasmic membrane in real time, and can be used for the specific imaging of the cytoplasmic membrane of different types and different plant species.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity, and a preparation method and application thereof.
Background
Cells play an important role in biological research as basic units of animal and plant structures and life activities. As an essential component of cells, the cytoplasmic membrane is an important barrier for isolating the intracellular and extracellular environments, and plays a key role in cell physiological processes such as cell migration, diffusion, uptake, exocytosis and substance transmembrane transport. Abnormalities in the structure of the cytoplasmic membrane are closely related to some physiological diseases, and thus the integrity of the cytoplasmic membrane is directly related to the integrity of biological cells.
At present, cytoplasmic membrane imaging mainly depends on two modes, namely fluorescent protein and organic small molecule fluorescent probe. Biological studies often use specific fluorescent proteins on the plasma membrane to track and monitor a range of physiological processes in living cells, but the construction of fluorescent proteins often requires cumbersome operations and is almost always accompanied by a decrease in fluorescence intensity during long-term fluorescence imaging. Incomplete maturation of the fluorophore of the fluorescent protein often compromises signal readout, and its large size also limits the function of site-specific labeling. Compared with the prior art, the organic small-molecule fluorescent probe has the advantages of ideal imaging effect, easiness in synthesis, simplicity in operation and the like, and can be regulated and controlled from the aspects of luminescent color, imaging specificity and the like. Most of the existing molecular fluorescent probes for imaging the cytoplasmic membrane can only realize short-time specific imaging aiming at the cytoplasmic membrane of the animal. The presence of the cell wall makes the cell structure of plants more complex than that of animal cells, so that few molecular fluorescent probes developed so far can be applied to long-term imaging of plant cell plasma membranes. Therefore, the near-infrared fluorescent probe based on the plant cytoplasmic membrane with high specificity and the preparation method and the application thereof are provided.
Disclosure of Invention
The APMem fluorescent probe realizes three-dimensional space imaging of plant cytoplasmic membranes and long-time high-retention high-specificity imaging for 10 hours in a multi-strategy cooperation mode, provides a valuable tool for monitoring dynamic physiological processes of the plant cytoplasmic membranes in real time, and can be used for specific imaging of the cytoplasmic membranes of different types and different plant species.
In order to achieve the purpose, the invention provides the following technical scheme:
a near-infrared fluorescent probe based on a plant cytoplasmic membrane with high specificity is an APMem fluorescent probe which has a diazide bicyclo [2.2.2] octane functional group and is (triphenylamine group) acrylonitrile pyridinium salt modified by diazide bicyclo [2.2.2] octane, and the structural formula of the near-infrared fluorescent probe is shown as follows:
The preparation method of the near-infrared fluorescent probe based on the plant cell plasma membrane with high specificity comprises the following steps:
a. synthesizing alkoxy substituted triphenylamine formaldehyde derivatives;
b. synthesizing alkoxy substituted (triphenylamine) acrylonitrile pyridine derivatives;
c. synthesizing bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivatives;
d. synthesizing (triphenylamine group) acrylonitrile pyridinium salt modified by diazido-bicyclo [2.2.2] octane, namely the target product APMem fluorescent probe.
Preferably, the specific operation of step a is as follows: under an inert atmosphere, cesium carbonate, alkoxy-substituted triphenylamine formaldehyde derivative, 4-bromobenzaldehyde and Pd (OAc) 2 And P (t-Bu) 3 Sequentially adding the mixture into toluene, uniformly mixing, reacting, cooling to room temperature, extracting with ethyl acetate, washing an organic layer with water for multiple times, drying with anhydrous sodium sulfate, and performing reduced pressure solvent evaporation and column chromatography purification treatment to obtain the alkoxy substituted triphenylamine formaldehyde derivative.
Preferably, in step a, cesium carbonate, alkoxy-substituted triphenylamine formaldehyde derivatives, 4-bromobenzaldehyde, pd (OAc) 2 And P (t-Bu) 3 The dosage ratio is as follows: 12.0 to 13.2mmol.
Preferably, the specific operation of step b is: adding sodium methoxide into methanol, stirring until the sodium methoxide is fully dissolved, vacuumizing, replacing with nitrogen, and repeating for multiple times; under inert atmosphere, sequentially adding 4-pyridine acetonitrile and alkoxy substituted triphenylamine formaldehyde derivatives, uniformly mixing, reacting, cooling to room temperature, extracting with ethyl acetate, washing an organic layer with water for multiple times, drying with anhydrous sodium sulfate, evaporating a solvent under reduced pressure, and purifying by column chromatography to obtain the alkoxy substituted (triphenylamine group) acrylonitrile pyridine derivatives.
Preferably, in the step b, the sodium methoxide, the 4-pyridine acetonitrile and the alkoxy substituted triphenylamine formaldehyde derivative are used in the following ratio: 2.66-3.06mmol.
Preferably, the specific operation of step c is: adding an alkoxy substituted (triphenylamine group) acrylonitrile pyridine derivative into dry acetonitrile, stirring until the mixture is fully dissolved, vacuumizing, replacing with nitrogen, and repeating for multiple times; adding 1,3-dibromopropane in an inert atmosphere, uniformly mixing, reacting, cooling to room temperature, evaporating the solvent under reduced pressure, washing, and removing residual solvent to obtain the bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivative.
Preferably, in the step c, the alkoxy substituted (triphenylamine) acrylonitrile pyridine derivative and 1,3-dibromopropane are used in the following ratio: 0.42-0.45mmol.
Preferably, the specific operation of step d is: adding bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivative into dry acetonitrile, stirring until the bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivative is fully dissolved, vacuumizing, replacing with nitrogen, repeating for many times, adding 1,4-diazido-bicyclo [2.2.2] octane under an inert atmosphere, mixing uniformly, reacting, cooling to room temperature, and evaporating a solvent under reduced pressure, washing and removing a residual solvent to obtain a target product APMem fluorescent probe; wherein, the dosage ratio of the bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivative to the 1,4-diazido bicyclo [2.2.2] octane is as follows: 1.2 to 1.5mmol.
The invention also provides an application of the near-infrared fluorescent probe based on the plant cytoplasmic membrane with high specificity in plant cytoplasmic membrane imaging.
Compared with the prior art, the invention has the beneficial effects that: the near-infrared APMem fluorescent probe designed based on 'multi-strategy cooperation' is characterized in that a key rigid steric hindrance group is introduced into the probe, namely: the introduction of diazido bicyclo [2.2.2] octane functional group plays a crucial key role in maintaining the ultra-long retention of the fluorescent probe on the cytoplasmic membrane, so that the APMem fluorescent probe shows high specificity and imaging over time for the cytoplasmic membrane of different cell types and different plant species. The long-term retention of the probe molecules on the plant cell plasma membrane is ensured by utilizing the similar affinity principle to target the plasma membrane, the self steric hindrance anti-permeation effect and the strong electrostatic interaction effect of multiple charges. Meanwhile, the APMem fluorescent probe has good water solubility, and the self-precipitation phenomenon in the application process of a biological system is avoided to a great extent. In the APMem fluorescent probe structure, the interference of the autofluorescence of plant cells is avoided or reduced to a great extent by constructing an aggregation-induced emission active molecular skeleton with near infrared emission. More importantly, its appropriate molecular size allows the probe to rapidly penetrate the cell wall with small pores, thereby smoothly and specifically targeting the cytoplasmic membrane. Thus, the APMem fluorescent probes are more suitable for high quality imaging of plant plasma membranes other than animal and human plasma membranes than previous aggregation-induced emission probes for plasma membrane imaging. On the premise of ensuring high-quality specific imaging performance, experimental results show that the maximum imaging time of the APMem fluorescent probe can reach 10 hours, which provides possibility for continuously monitoring relevant physiological processes based on cytoplasmic membranes for a long time. In addition, a series of experiments aiming at the imaging performance of the cytoplasmic membranes of different plants prove that the APMem fluorescent probe can be suitable for different cell types and different plant species, has extremely high universality, and the design strategy has certain wide applicability.
Drawings
FIG. 1A shows the UV-VIS absorption spectrum of fluorescent probe APMem-1 in DMSO solvent.
FIG. 1B shows the emission spectra of fluorescent probe APMem-1 in pure solid state and dispersed state.
FIG. 1C is a time-resolved fluorescence decay curve of a fluorescent probe APMem-1 in a pure solid state.
FIG. 1D is a time-resolved fluorescence decay curve of the dispersed state of the fluorescent probe APMem-1.
FIG. 2 is a fluorescence emission spectrum and a fluorescence photograph of different amounts of fluorescent probe APMem-1 added to the SDBS solution.
FIG. 3 is a three-dimensional space imaging diagram of laser confocal scanning of the root tip of an Arabidopsis seedling stained with a fluorescent probe APMem-1.
FIG. 4A is a graph of the photostability of Arabidopsis seedling root tip cell laser confocal scanning imaging stained with a fluorescent probe APMem-1.
FIG. 4B shows the change of fluorescence intensity of fluorescent probe APMem-1 plasma membrane imaging with irradiation time.
FIG. 5A is a global image of Arabidopsis thaliana seedling root cells stained with fluorescent probe APMem-1 taken under a laser scanning confocal microscope at different time periods.
FIG. 5B is a magnified image of the root cells of Arabidopsis seedlings stained with fluorescent probe APMem-1 taken under a laser scanning confocal microscope at different lengths.
FIG. 6 is a confocal laser scanning image of onion epidermal cells stained with fluorescent probe APMem-1.
FIG. 7 is a confocal laser scanning image of rice seedling root tip cell stained with fluorescent probe APMem-1.
FIG. 8 is a laser confocal scanning image (c, g, d, h) of tobacco leaves (a, e, b, f) stained with fluorescent probe APMem-1 and tobacco seedling root tip cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity, wherein the near-infrared fluorescent probe is an APMem fluorescent probe, the APMem fluorescent probe has a diazide bicyclo [2.2.2] octane functional group and is (triphenylamine group) acrylonitrile pyridinium salt modified by diazide bicyclo [2.2.2] octane, and the structural formula is shown as follows:
The APMem fluorescent probe is a probe molecule constructed based on 'multi-strategy cooperation'. The saturated alkyl chain is used for the targeting function of the cytoplasmic membrane, the introduction of two positive charges ensures good water solubility of the probe molecules, and the introduction of the rigid steric hindrance group diazide bicyclooctane can effectively prevent the probe molecules from penetrating through the cell membrane through the steric hindrance effect, so that the series of probe molecules can stay on the cytoplasmic membrane for a very long time.
Since fluorescent probes for long-term specific tracking of plant cell plasma membranes with aggregation-induced emission properties are designed and synthesized, the prerequisites for proper molecular size, longer emission wavelength and certain steric hindrance need to be met. In the method, a design principle of multi-strategy cooperation is provided, and the ultrashort-time long-term specific imaging of the plant cytoplasmic membrane is realized by introducing the synergistic effect of different functional groups.
(1) The alpha, beta-diaryl acrylonitrile structure with D-A effect and aggregation-induced emission characteristic is used as a molecular skeleton, and a cyano group is introduced into a central ethylene unit. The introduction of the cyano group not only contributes to the near infrared emission of the whole molecule and deep penetration and imaging, but also can change the linear structure of the central ethylene and make the molecular skeleton more rigid.
(2) The front end of the probe structure is introduced with a lipophilic unit with proper length to ensure strong interaction between the probe molecule and the cytoplasmic membrane, and simultaneously, the probe molecule can smoothly permeate from the small hole of the cell wall so as to target the cytoplasmic membrane.
(3) Two positive charges are introduced into the tail end of the probe structure to ensure that the probe has good water solubility, and the probe has larger steric hindrance under the action of a rigid group 1,4-diazide bicyclo [2.2.2] octane, so that the internalization of a cytoplasmic membrane of the probe is effectively prevented, and the imaging of the cytoplasmic membrane for ultra-long time is realized.
Based on the series of design principles, a water-soluble APMem fluorescent probe with induced emission characteristic for specific imaging of the plasma membrane of the near-infrared plant cell is synthesized.
The invention also provides a preparation method of the plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity, which comprises the following steps:
a. synthesizing alkoxy substituted triphenylamine formaldehyde derivatives;
b. synthesizing alkoxy substituted (triphenylamine) acrylonitrile pyridine derivatives;
c. synthesizing bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivatives;
d. synthesizing (triphenylamine group) acrylonitrile pyridinium salt modified by diazido-bicyclo [2.2.2] octane, namely the target product APMem fluorescent probe.
The above summary will be further described in detail with reference to specific examples.
Example 1:
the invention provides a preparation method of a plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity, wherein the APMem fluorescent probe (marked as APMem-1 fluorescent probe) has the structural formula as follows:
The synthetic route is as follows:
the preparation method of the APMem-1 fluorescent probe specifically comprises the following steps:
a. synthesis of 4- (4- (hexyloxydianilino)) benzaldehyde: cesium carbonate (3.9g, 12mmol), 4-hexyloxydianiline (0.538g, 2mmol), 4-bromobenzaldehyde (1.1g, 6mmol) and Pd (OAc) were reacted under a nitrogen atmosphere 2 (0.05 g) and P (t-Bu) 3 (1 mL) was added in succession to a 250mL three-necked flask containing 100mL of toluene. After mixing uniformly, the mixture was reacted at 110 ℃ for 48 hours. After cooling to room temperature, extraction with ethyl acetate and washing of the organic layer with water 3 times (3X 50 mL) and over anhydrous Na 2 SO 4 And (5) drying. Transferring the solution into a round-bottom flask, evaporating the solvent under reduced pressure, purifying by silica gel column chromatography, and eluting with an eluent of petroleum ether and ethyl acetate in a volume ratio of 2:1 to obtain a yellow-green gelatinous product 4- (4- (hexyloxydianilino)) benzaldehyde with the yield of 90%.
Product nmr data: 1 H NMR(600MHz,CDCl 3 ),δ(ppm):9.78(s,1H),7.67–7.63(m,2H),7.35–7.30(m,2H),7.19–7.09(m,5H),6.94(d,J＝8.8Hz,2H),6.91–6.87(m,2H),3.95(t,J＝6.5Hz,2H),1.82–1.75(m,2H),1.49–1.44(m,2H),1.37–1.33(m,4H),0.94–0.88(m,3H). 13 C NMR(151MHz,CDCl 3 ),δ(ppm):δ190.62,157.39,153.96,146.42,138.83,131.61,129.89,128.71,128.64,126.09,125.06,118.35,115.92,68.55,31.86,29.53,26.02,22.89,14.32。
b. synthesizing 4- (4-hexyloxy triphenylamine) acrylonitrile pyridine: sodium methoxide (0.145g, 2.66mmol) was added to a three-necked flask containing 30mL of methanol, and the mixture was stirred until it was sufficiently dissolved, evacuated, replaced with nitrogen, and repeated three times. Under a nitrogen atmosphere, 4-pyridylacetonitrile (0.24g, 2mmol) and 4- (4- (hexyloxydianilino)) benzaldehyde (0.5g, 1.34mmol) were added in this order, and the mixture was uniformly mixed and reacted at 80 ℃ for 5 hours. After cooling to room temperature, ethyl acetate was extracted, and the organic layer was washed 3 times with water (3X 50 mL) and passed over anhydrous Na 2 SO 4 And (5) drying. Transferring the solution into round-bottom flask, evaporating solvent under reduced pressure, purifying by silica gel column chromatography, and washing with petroleum ether and ethyl acetate eluate at volume ratio of 2:1The brown gelatinous product 4- (4-hexyloxy triphenylamino) acrylonitrile pyridine is obtained by removing, and the yield is 95%.
Product nmr data: 1 H NMR(400MHz,CDCl 3 ),δ(ppm):8.64(s,2H),7.80(d,J＝8.8Hz,2H),7.58(s,1H),7.52(d,J＝5.2Hz,2H),7.32(t,J＝7.9Hz,2H),7.17(d,J＝8.0Hz,2H),7.13(t,J＝7.9Hz,3H),6.97(d,J＝8.9Hz,2H),6.89(d,J＝8.9Hz,2H),3.96(t,J＝6.5Hz,2H),1.83–1.77(m,2H),1.50–1.45(m,2H),1.37–1.33(m,4H),0.92(t,J＝6.9Hz,3H). 13 C NMR(150MHz,CDCl 3 ),δ(ppm):157.04,151.38,150.41,146.20,144.39,142.65,138.61,131.53,129.57,128.30,125.50,124.57,124.31,119.52,118.85,117.95,115.65,103.68,68.29,31.59,29.26,25.75,22.61,14.20。
c. synthesizing p-bromopropyl-4- (hexyloxy triphenylamine) acrylonitrile pyridine: 4- (4-Hexytriphenylaminyl) Acrylonitrile pyridine (0.2g, 0.42mmol) was added to a three-necked flask with dry acetonitrile and stirred to dissolve well. Vacuumizing, replacing with nitrogen, and repeating for three times. 1,3-dibromopropane (1.28g, 6.3mmol) was added under nitrogen atmosphere, mixed well and reacted at 110 ℃ for 48h. After cooling to room temperature, the solvent was removed by evaporation under reduced pressure. The residue was washed with petroleum ether at least three times under sonication to remove most of the unreacted 1,3-dibromopropane. The residual solvent on the surface was evaporated under reduced pressure to give p-bromopropyl-4- (hexyloxytrianilino) acrylonitrile pyridine as a dark purple powder in 75% yield.
Product nmr data: 1 H NMR(400MHz,CDCl 3 ),δ(ppm):9.17(s,2H),8.46(s,1H),8.35(s,2H),8.07(d,J＝8.6Hz,2H),7.36(t,J＝7.7Hz,2H),7.19(t,J＝7.7Hz,3H),7.12(d,J＝8.4Hz,2H),6.89(t,4H),5.01(s,2H),3.96(t,J＝6.4Hz,2H),3.51(s,2H),2.67(s,2H),1.82–1.77(m,2H),1.49–1.44(m,2H),1.37–1.33(m,4H),0.91(t,J＝7.0Hz,3H). 13 C NMR(151MHz,CDCl 3 ),δ(ppm):157.68,153.72,152.21,151.45,145.04,144.15,137.42,134.57,129.83,128.49,126.37,125.95,123.37,122.46,117.72,117.23,115.76,97.31,68.33,58.78,33.68,31.58,29.21,29.06,25.73,22.62,14.07。
d. synthesis of diazidobicyclo [2.2.2] octane-modified (triphenylamine-based) acrylonitrile pyridinium salt: p-bromopropyl-4- (hexyloxytrianilino) acrylonitrile pyridine (0.8g, 1.2mmol) was charged into a three-necked flask containing dry acetonitrile and stirred until fully dissolved. Vacuumizing, replacing with nitrogen, and repeating the steps for three times. 1,4-diazidobicyclo [2.2.2] octane (0.4 g,3.6 mmol) was added under nitrogen atmosphere, mixed uniformly and reacted at 85 ℃ for 20h. In the reaction process, a dark purple precipitate is separated out from the solution. After the reaction was completed, it was cooled to room temperature, and the solvent was removed by evaporation under reduced pressure. The crude product was washed at least three times with a mixed solution of acetone and ethanol (acetone: ethanol = 100). The residual solvent on the surface is evaporated under reduced pressure to obtain the deep purple solid powder diazido bicyclo [2.2.2] octane modified (triphenylamino) acrylonitrile pyridinium salt, namely the target product APMem fluorescent probe, which is marked as APMem-1 fluorescent probe, and the yield is as follows: 70 percent.
Product nmr data: 1 H NMR(400MHz,MeOD-d 4 :DMSO-d6＝4:3(v/v)),δ(ppm):8.44(d,J＝6.4Hz,2H),7.46(m,3H),7.39(d,J＝6.5Hz,2H),7.33–7.22(m,6H),7.09(d,J＝8.9Hz,3H),6.93(d,J＝8.6Hz,2H),4.21(s,2H),4.07(t,J＝6.5Hz,2H),3.58–3.52(m,2H),3.46(m,12H),2.51–2.46(m,2H),1.84(m,2H),1.57–1.53(m,2H),1.45–1.42(m,4H),1.01(m,3H). 13 C NMR(150MHz,MeOD-d 4 ),δ(ppm):141.09,132.88,129.69,129.45,128.26,127.25,127.16,125.90,125.64,124.89,121.48,118.50,115.52,67.98,60.39,55.16,52.72,52.40,51.19,44.66,44.49,31.37,29.35,28.99,28.61,25.47,23.49,22.30,13.00。
example 2:
the invention provides a preparation method of a plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity, wherein the APMem fluorescent probe (marked as APMem-2 fluorescent probe) has the structural formula as follows:
The synthetic route is as follows:
the preparation method of the APMem-2 fluorescent probe specifically comprises the following steps:
a. synthesizing 4- (4-nonyloxy diphenylamine) benzaldehyde: cesium carbonate (4.1g, 12.6 mmol), 4-nonyloxydiphenylamine (0.65g, 2.1mmol), 4-bromobenzaldehyde (1.17g, 6.3mmol) and Pd (OAc) were reacted under nitrogen atmosphere 2 (0.06 g) and P (t-Bu) 3 (1.1 mL) were sequentially added to a 250mL three-necked flask containing 100mL of toluene, and the mixture was mixed well and reacted at 110 ℃ for 48 hours. After cooling to room temperature, extraction with ethyl acetate and washing of the organic layer with water 3 times (3X 50 mL) and over anhydrous Na 2 SO 4 And (5) drying. Transferring the solution into a round-bottom flask, evaporating the solvent under reduced pressure, purifying by silica gel column chromatography, and eluting with an eluent of petroleum ether and ethyl acetate in a volume ratio of 2:1 to obtain a yellow gelatinous product 4- (4-nonyloxy diphenylamine) benzaldehyde with a yield of 89%.
b. Synthesizing 4- (4-nonanoxy triphenylamine) acrylonitrile pyridine: sodium methoxide (0.155g, 2.86mmol) was added to a three-necked flask containing 30mL of methanol, stirred to dissolve sufficiently, evacuated, replaced with nitrogen, and repeated three times. Under nitrogen atmosphere, 4-pyridylacetonitrile (0.25g, 2.15mmol) and 4- (4-nonyloxydiphenylamine) benzaldehyde (0.60g, 1.44mmol) were added in this order, mixed uniformly, and reacted at 80 ℃ for 5 hours. After cooling to room temperature, extraction with ethyl acetate and washing of the organic layer with water 3 times (3X 50 mL) and over anhydrous Na 2 SO 4 And (5) drying. Transferring the solution into a round-bottom flask, evaporating the solvent under reduced pressure, purifying by silica gel column chromatography, and eluting with an eluent of petroleum ether and ethyl acetate at a volume ratio of 2:1 to obtain a brown gelatinous product, namely 4- (4-nonyloxytriphenylamine) acrylonitrile pyridine, wherein the yield is 90%.
c. Synthesizing p-bromopropyl-4- (nonanoxy triphenylamine) acrylonitrile pyridine: 4- (4-Nonoxytriphenylanilino) acrylonitrile pyridine (0.22g, 0.43mmol) was added to a three-necked flask containing dry acetonitrile, and stirred to be sufficiently dissolved. Vacuumizing, replacing with nitrogen, and repeating for three times. 1,3-dibromopropane (1.30g, 6.45mmol) was added under nitrogen atmosphere, mixed well and reacted at 110 ℃ for 48h. After cooling to room temperature, the solvent was removed by evaporation under reduced pressure. The residue was washed with petroleum ether at least three times under sonication to remove most of the unreacted 1,3-dibromopropane. The residual solvent on the surface was evaporated under reduced pressure to give p-bromopropyl-4- (nonanoyltriaminoanilino) acrylonitrile pyridine as a dark purple powder in 78% yield.
d. Synthesis of diazobicyclo [2.2.2] octane-modified (triphenylamino) acrylonitrile pyridinium salt: p-bromopropyl-4- (nonanoyloxytrianilino) acrylonitrile pyridine (0.93g, 1.3 mmol) was added to a three-necked flask containing dry acetonitrile and stirred until fully dissolved. Vacuumizing, replacing with nitrogen, and repeating the steps for three times. 1,4-diazido bicyclo [2.2.2] octane (0.44g, 3.9 mmol) is added under nitrogen atmosphere, mixed uniformly and reacted for 20h at 85 ℃. In the reaction process, a dark purple precipitate is separated out from the solution. After the reaction was completed, it was cooled to room temperature, and the solvent was removed by evaporation under reduced pressure. The crude product was washed at least three times with a mixed solution of acetone and ethanol (acetone: ethanol = 100) under ultrasonic conditions. The residual solvent on the surface is evaporated under reduced pressure to obtain the deep purple solid powder diazido bicyclo [2.2.2] octane modified (triphenylamino) acrylonitrile pyridinium salt, namely the target product APMem fluorescent probe, which is marked as APMem-2 fluorescent probe, and the yield is as follows: and 69 percent.
Example 3:
the invention provides a preparation method of a plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity, wherein the APMem fluorescent probe (marked as APMem-3 fluorescent probe) has the structural formula as follows:
The synthetic route is as follows:
the preparation method of the APMem-3 fluorescent probe specifically comprises the following steps:
a. synthesizing 4- (4-propoxydiphenylamine) benzaldehyde: cesium carbonate (4.3g, 13.2mmol), 4-propoxydiphenylamine (0.5g, 2.2mmol), 4-bromobenzaldehyde (1.8g, 6.6 mmol) and Pd (OAc) were reacted under a nitrogen atmosphere 2 (0.07 g) and P (t-Bu) 3 (1.2 mL) were sequentially added to a 250mL three-necked flask containing 100mL of toluene, and the mixture was mixed well and reacted at 110 ℃ for 48 hours. After cooling to room temperature, extraction with ethyl acetate and washing of the organic layer with water 3 times (3X 50 mL) and over anhydrous Na 2 SO 4 And (5) drying. Transferring the solution into a round-bottom flask, evaporating the solvent under reduced pressure, purifying by silica gel column chromatography, and eluting with an eluent of petroleum ether and ethyl acetate in a volume ratio of 10.
b. Synthesis of 4- (4-propoxytrianilino) Acrylonitrile pyridine: sodium methoxide (0.17g, 3.06mmol) was added to a three-necked flask containing 30mL of methanol, and the mixture was stirred until it was sufficiently dissolved, and then vacuum evacuation and nitrogen replacement were carried out, and the reaction was repeated three times. Under nitrogen atmosphere, 4-pyridine acetonitrile (0.27g, 2.3 mmol) and 4- (4-propoxydiphenylamine) benzaldehyde (0.51g, 1.54mmol) are added successively, mixed homogeneously and reacted at 80 deg.c for 5 hr. After cooling to room temperature, ethyl acetate was extracted, and the organic layer was washed 3 times with water (3X 50 mL) and passed over anhydrous Na 2 SO 4 And (5) drying. Transferring the solution into a round-bottom flask, evaporating the solvent under reduced pressure, and purifying by silica gel column chromatography to obtain a brown gelatinous product 4- (4-propoxytrianilino) acrylonitrile pyridine with the yield of 89%.
c. Synthesizing p-bromopropyl-4- (propoxytrianilino) acrylonitrile pyridine: 4- (4-propoxytrianilino) Acrylonitrile pyridine (0.19g, 0.45mmol) was added to a three-necked flask containing dry acetonitrile, and stirred until fully dissolved. Vacuumizing, replacing with nitrogen, and repeating the steps for three times. 1,3-dibromopropane (1.36g, 6.75mmol) is added under nitrogen atmosphere, mixed evenly and reacted for 48h at 110 ℃. After cooling to room temperature, the solvent was removed by evaporation under reduced pressure. The residue was washed with petroleum ether at least three times under sonication to remove most of the unreacted 1,3-dibromopropane. The residual solvent on the surface was evaporated under reduced pressure to give p-bromopropyl-4- (propoxytrianilino) acrylonitrylpyridine as a dark purple powder in 75% yield.
d. Synthesis of diazobicyclo [2.2.2] octane-modified (triphenylamino) acrylonitrile pyridinium salt: p-bromopropyl-4- (propoxytrianilino) acrylonitrylpyridine (0.95g, 1.5 mmol) was added to a three-necked flask with dry acetonitrile and stirred until fully dissolved. Vacuumizing, replacing with nitrogen, and repeating the steps for three times. 1,4-diazido bicyclo [2.2.2] octane (0.5 g,4.5 mmol) is added under nitrogen atmosphere, and after uniform mixing, the mixture reacts for 20h at 85 ℃. In the reaction process, a dark purple precipitate is separated out from the solution. After the reaction was completed, it was cooled to room temperature, and the solvent was removed by evaporation under reduced pressure. The crude product was washed at least three times with a mixed solution of acetone and ethanol (acetone: ethanol =100 by volume). The residual solvent on the surface is evaporated under reduced pressure to obtain the deep purple solid powder diazido bicyclo [2.2.2] octane modified (triphenylamino) acrylonitrile pyridinium salt, namely the target product APMem fluorescent probe, which is marked as APMem-3 fluorescent probe, and the yield is as follows: 65 percent.
Performance characterization
Characterization of UV-visible absorption spectrum and fluorescence emission spectrum was performed on APMem-1 fluorescent probe prepared in example 1. The test result of the ultraviolet-visible absorption spectrum of the APMem-1 fluorescent probe in a dimethyl sulfoxide (DMSO) solvent is shown in FIG. 1A, the APMem-1 fluorescent probe exists in a dark purple solid under sunlight, a visible light absorption band which is wider from 420nm to 660nm exists, and the maximum absorption peak is located at 525 nm. The fluorescence emission spectra are shown in FIG. 1B, and the emission spectra of APMem-1 fluorescence probe in pure solid state and in dispersed state are respectively measured, wherein the dispersed state of APMem-1 fluorescence probe is in molar ratio n APMem-1 :n Adamantane Dispersing the fluorescent probe in adamantane by the ratio of =1 and 50, wherein the test result shows that the pure solid state maximum emission peak wavelength of the APMem-1 fluorescent probe is 672nm and the service life is 1.8ns; the maximum emission peak wavelength of the APMem-1 fluorescent probe in a dispersed state is 688nm.
In addition, time-resolved fluorescence attenuation curves of the APMem-1 fluorescent probe in a pure solid state and a dispersed state are also tested, and the results are respectively shown in FIG. 1C and FIG. 1D, and the results show that the APMem-1 fluorescent probe in the pure solid state and the dispersed state has the same short service life of 1.8ns and 2.6ns respectively, and the experimental data prove that the luminescence is fluorescence.
The above characterization confirms the limited induced emission behavior of the APMem-1 fluorescent probe.
Since Sodium Dodecyl Benzene Sulfonate (SDBS) is an amphiphilic molecule consisting of a charged head and a long alkyl chain tail, a stable single-layer micelle can be spontaneously formed in an aqueous solution, and therefore, the SDBS can be used for simulating the structure of a cytoplasmic membrane to evaluate the fluorescent imaging performance of a fluorescent probe on the cytoplasmic membrane. The APMem-1 fluorescent probe emits almost no light in aqueous solution; when APMem-1 fluorescent probe was added to an aqueous solution containing 200.0. Mu.M SDBS, its red emission gradually increased. As shown in FIG. 2, the fluorescence intensity gradually increased as the amount of APMem-1 added was increased from 0.0. Mu.M to 20.0. Mu.M, and this sharp emission enhancement was visible to the naked eye under UV light. Due to the similar affinity principle, the APMem-1 fluorescent probe tends to be inserted into the monolayer micelle formed by SDBS after being added, so that these probe molecules will be confined within the micelle, resulting in a significant enhancement of emission, i.e. the fluorescence intensity of the probe molecules increases with increasing confinement intensity.
The invention also provides an application of the near-infrared fluorescent probe based on the plant cytoplasmic membrane with high specificity in the ultra-long-time high-quality imaging of the plant cytoplasmic membrane.
The following description will be made in detail by specific application examples.
Application example 1
The APMem-1 fluorescent probe is used for the arabidopsis root tip cytoplasmic membrane living body three-dimensional space imaging.
The specific implementation method comprises the following steps: the surface of wild type (Col-0) seeds of Arabidopsis thaliana was sterilized and soaked in dark at 4 ℃ for 3 days, and then sown to 0.5 XMurashige&Skoog (MS) 1.5% (w/v) on agar plates. Seedlings were grown in a climate-controlled growth chamber (22/20 ℃ day/night temperature, 16 h photoperiod and 80. Mu.E.s) -1 ·m -2 Light intensity) on a flat plate. Unless otherwise indicated, the study used five-day-old seedlings with healthy roots.
Arabidopsis seedlings were incubated with 10.0. Mu.MAPMem-1 at 22 ℃ for 5min. A fluorescence three-dimensional space imaging experiment was carried out on a LeicaTCS SP5 laser confocal scanning microscope (Germany) with an excitation wavelength of 561nm.
Under optimized conditions, namely APMem-1 (10.0 mu M) is used for treatment for 5min, and then a three-dimensional space staining image of the root tip of the arabidopsis thaliana seedling is taken by a LeicaTCS SP5 laser confocal scanning microscope. As shown in FIG. 3, this figure shows a three-dimensional aerial image and a two-dimensional scan slice image of the Arabidopsis root tip X, Y, Z axis. It can be clearly seen that there is a clear bright red signal across almost all the plasma membranes of the root tip and that the signal gradually increases from the center to the surface, consistent with the diffusion-controlled distribution of APMem-1. The three-axis cross-sectional staining image also confirmed the successful staining of cytoplasmic membranes of all cells at the root tip of arabidopsis seedlings, indicating that APMem-1 was able to stain the cytoplasmic membranes of all target plant cells within a short incubation time.
Application example 2
The APMem-1 fluorescent probe is used for long-time imaging of the plasma membrane of the root tip cell of Arabidopsis.
The specific implementation method and the instrument conditions are the same as those in application example 1.
In an ultralong imaging experiment of an APMem-1 fluorescent probe on a plasma membrane of a root tip cell of an arabidopsis seedling, the APMem-1 is continuously irradiated at 561nm by using an excitation source of a laser confocal microscope to study the light stability of the APMem-1. As shown in fig. 4A and 4B, no significant change in plasma membrane emission signal was observed for APMem-1 stained cells after continuous irradiation for up to 90min, and the bright red signal on the plasma membrane was well preserved under this continuous irradiation, indicating that APMem-1 has good photostability and is suitable for long-term imaging of the plasma membrane. As shown in FIGS. 5A and 5B, APMem-1 stained cytoplasmic membranes were subjected to imaging experiments for various times, taking Arabidopsis thaliana shoot root cells as an example. The cytoplasmic membrane can be clearly imaged at the root tip area cytoplasmic membrane over different time periods, and ultimately for imaging durations of up to 10 hours.
Application example 3
The APMem-1 fluorescent probe is used for imaging the plasma membrane of the onion epidermal cell.
The specific implementation method and the instrument conditions are the same as those in application example 1.
First, onion epidermal cells were treated with APMem-1 fluorescent probe (10.0. Mu.M) for 5 minutes, and as shown in FIG. 6, confocal laser microscopy images showed that the plasma membrane and cell wall of the cells observed gave clear red emission signals under the same instrument conditions. In the plasmolysis experiment, after 20 minutes of treatment with sucrose solution, it was clearly observed that all cells were sharply dehydrated and contracted into an ellipse-like shape, the cell wall and the cytoplasmic membrane were clearly separated, and all the cytoplasmic membranes separated from the surrounding cell wall exhibited continuous bright red signals, while the cell wall exhibited discontinuous signals, which strongly demonstrated that the APMem-1 fluorescent probe was able to effectively penetrate the cell wall to stain the cytoplasmic membrane of the onion epidermis with high specificity and brightness.
Application example 4
The APMem-1 fluorescent probe is used for rice root tip cytoplasmic membrane imaging.
The specific implementation method and the instrument conditions are the same as those in application example 1.
As shown in FIG. 7, this figure shows a high resolution fluorescence image of rice seedling root cells stained with APMem-1 fluorescent probe. The result shows that the cytoplasmic membranes of the root tip regions of the rice seedlings can be clearly and completely marked to be bright red by the APMem-1 fluorescent probe. Although their cell shapes differ by species differences, they are essentially evaluated from cytoplasmic membrane imaging and have no significant differences.
Application example 5
The APMem-1 fluorescent probe is used for imaging the tobacco leaf and the plasma membrane of the root tip cell.
The specific implementation method and the instrument conditions of the tobacco root tip cytoplasmic membrane imaging are the same as the application example 1; due to the waxy cuticle on the surface of the tobacco leaf, the tobacco leaf takes a long time (60 minutes) to achieve effective dyeing.
As shown in FIG. 8 (c, d, g, h), the four figures show high resolution fluorescence images of tobacco seedling root cells stained with APMem-1 fluorescent probe. Tobacco root cells displayed the ideal specific cytoplasmic membrane targeting red fluorescent signal under the APMem-1 marker.
Unlike tobacco root cells, leaf cells are composed of cells with different plant functions and complex structures. As shown in FIG. 8 (a, b, e, f), the four figures show laser scanning confocal microscope images of epidermal and guard cells on tobacco leaves stained with APMem-1. It can be seen that the red signal is clearly and completely marked on the cytoplasmic membranes of both the irregular-ripple epidermal cells and the semicircular-shaped guard cells. The images clearly show that the entire cytoplasmic membrane of both the larger epidermal cells and the smaller guard cells is specifically stained, and no significant signal is observed in other areas of both cells. This is because guard cells are scattered in leaf epidermal cells, control the opening and closing of pores, and are essentially a cellular structure composed of a membrane.
Application examples 1-5 show that the APMem fluorescent probe can be suitable for highly specific imaging of cytoplasmic membranes of different cell types and different plant species, and has long imaging time and high imaging quality.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (10)
1. A near-infrared fluorescent probe based on plant cytoplasmic membrane with high specificity is characterized in that: the near-infrared fluorescent probe is an APMem fluorescent probe which has a diazide bicyclo [2.2.2] octane functional group and is (triphenylamine group) acrylonitrile pyridinium salt modified by diazide bicyclo [2.2.2] octane, and the structural formula is shown as follows:
2. The method for preparing a near-infrared fluorescent probe based on the plasma membrane of a plant cell with high specificity as set forth in claim 1, wherein the near-infrared fluorescent probe comprises: the method comprises the following steps:
a. synthesizing alkoxy substituted triphenylamine formaldehyde derivatives;
b. synthesizing alkoxy substituted (triphenylamine) acrylonitrile pyridine derivatives;
c. synthesizing bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivatives;
d. synthesizing (triphenylamine group) acrylonitrile pyridinium salt modified by diazido-bicyclo [2.2.2] octane, namely the target product APMem fluorescent probe.
3. The method for preparing a near-infrared fluorescent probe based on the plant cell plasma membrane with high specificity as claimed in claim 2, wherein the near-infrared fluorescent probe comprises: the specific operation of the step a is as follows: under inert atmosphere, cesium carbonate, alkoxy-substituted triphenylamine formaldehyde derivative, 4-bromobenzaldehyde and Pd (OAc) 2 And P (t-Bu) 3 Sequentially adding the mixture into toluene, uniformly mixing, reacting, cooling to room temperature, extracting with ethyl acetate, washing an organic layer with water for multiple times, drying with anhydrous sodium sulfate, and performing reduced pressure solvent evaporation and column chromatography purification treatment to obtain the alkoxy substituted triphenylamine formaldehyde derivative.
4. The method for preparing a near-infrared fluorescent probe based on the plant cell plasma membrane with high specificity as claimed in claim 3, wherein the near-infrared fluorescent probe comprises: in said step a, cesium carbonate, alkoxy-substituted triphenylamine formaldehyde derivative, 4-bromobenzaldehyde, pd (OAc) 2 And P (t-Bu) 3 The dosage ratio is as follows: 12.0 to 13.2mmol.
5. The method for preparing a near-infrared fluorescent probe based on the plant cell plasma membrane with high specificity as claimed in claim 2, wherein the near-infrared fluorescent probe comprises: the specific operation of the step b is as follows: adding sodium methoxide into methanol, stirring until the sodium methoxide is fully dissolved, vacuumizing, replacing with nitrogen, and repeating for multiple times; under inert atmosphere, sequentially adding 4-pyridine acetonitrile and alkoxy substituted triphenylamine formaldehyde derivatives, uniformly mixing, reacting, cooling to room temperature, extracting with ethyl acetate, washing an organic layer with water for multiple times, drying with anhydrous sodium sulfate, evaporating a solvent under reduced pressure, and purifying by column chromatography to obtain the alkoxy substituted (triphenylamine group) acrylonitrile pyridine derivatives.
6. The method for preparing a plant cytoplasmic membrane near-infrared fluorescent probe based on high specificity as claimed in claim 5, wherein the method comprises the following steps: in the step b, the dosage ratio of the sodium methoxide, the 4-pyridine acetonitrile and the alkoxy substituted triphenylamine formaldehyde derivative is as follows: 2.66-3.06mmol.
7. The method for preparing a near-infrared fluorescent probe based on the plant cell plasma membrane with high specificity as claimed in claim 2, wherein the near-infrared fluorescent probe comprises: the specific operation of the step c is as follows: adding an alkoxy substituted (triphenylamine) acrylonitrile pyridine derivative into dry acetonitrile, stirring until the mixture is fully dissolved, vacuumizing, replacing with nitrogen, and repeating for multiple times; adding 1,3-dibromopropane in an inert atmosphere, uniformly mixing, reacting, cooling to room temperature, evaporating the solvent under reduced pressure, washing, and removing residual solvent to obtain the bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivative.
8. The method for preparing a near-infrared fluorescent probe based on the plant cell plasma membrane with high specificity as claimed in claim 7, wherein the near-infrared fluorescent probe comprises: in the step c, the dosage ratio of the alkoxy substituted (triphenylamine) acrylonitrile pyridine derivative to the 1,3-dibromopropane is as follows: 0.42-0.45mmol.
9. The method for preparing a near-infrared fluorescent probe based on the plant cell plasma membrane with high specificity as claimed in claim 2, wherein the near-infrared fluorescent probe comprises: the specific operation of the step d is as follows: adding bromopropyl (triphenylamine group) acrylonitrile pyridinium salt derivatives into dry acetonitrile, stirring until the bromopropyl (triphenylamine group) acrylonitrile pyridinium salt derivatives are fully dissolved, vacuumizing, replacing with nitrogen, repeating for multiple times, adding 1,4-diazido bicyclo [2.2.2] octane under the inert atmosphere, uniformly mixing, reacting, cooling to room temperature, and obtaining a target product APMem fluorescent probe after decompressing, evaporating a solvent, washing and removing a residual solvent; wherein, the dosage ratio of the bromopropyl (triphenylamine) acrylonitrile pyridinium salt derivative to the 1,4-diazido bicyclo [2.2.2] octane is as follows: 1.2 to 1.5mmol.
10. Use of the near-infrared fluorescent probe based on the plant cytoplasmic membrane with high specificity as set forth in claim 1 in plant cytoplasmic membrane imaging.
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