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CN112945912A - High-brightness large-Stokes-shift wash-free lipid-droplet fluorescent probe and synthetic method and application thereof - Google Patents

High-brightness large-Stokes-shift wash-free lipid-droplet fluorescent probe and synthetic method and application thereof Download PDF

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CN112945912A
CN112945912A CN201911257581.6A CN201911257581A CN112945912A CN 112945912 A CN112945912 A CN 112945912A CN 201911257581 A CN201911257581 A CN 201911257581A CN 112945912 A CN112945912 A CN 112945912A
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徐兆超
陈婕
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention develops a high-brightness large-Stokes-displacement wash-free lipid drop probe and a synthesis method and application thereof, and has the structural characteristics that carbonyl and nitrogenous alkyl functional groups are introduced into naphthalene rings. The fluorescence quantum yield of the probe in a protic solvent such as water is less than 0.01, and the fluorescence quantum yield in an aprotic solvent such as dichloromethane can reach 0.5. The probe has high brightness and larger Stokes displacement, can quickly permeate through a cell membrane and is enriched in lipid droplets, realizes the wash-free fluorescence imaging of the lipid droplets, and has wide application prospect in the research of cell biology.

Description

High-brightness large-Stokes-shift wash-free lipid-droplet fluorescent probe and synthetic method and application thereof
Technical Field
The invention belongs to the field of fluorescent probes and imaging, and particularly relates to a structure and a synthetic method of a high-brightness and large-Stokes-displacement wash-free lipid drop fluorescent probe and application of the structure and the synthetic method in the field of fluorescent imaging.
Background
Lipid droplets are the storage place of neutral lipid in cells, and the interaction between the lipid droplets and other organelles regulates important physiological processes such as lipid synthesis and lipid metabolism in cells, and are closely related to diseases such as obesity and diabetes. Monitoring its morphology and interaction with various organelles is therefore essential for a profound understanding of complex cellular life activities. The fluorescent probe provides a powerful tool for life science research due to the characteristics of high sensitivity and high selectivity.
The Stokes shift is the difference between the emission wavelength and the excitation wavelength in the fluorescence spectrum, and is an important parameter for evaluating the performance of the fluorescent probe. Most fluorescent probes have only a small stokes shift (50-90nm), whereas commercial lipid droplet probes, such as BODIPY 493, have only a stokes shift of ten and a few nanometers, and these molecules are susceptible to interference of excitation light when used for lipid droplet fluorescence imaging, reducing the imaging accuracy. Therefore, there is a need to develop a high brightness, large stokes shift probe to meet the requirement of live cell lipid droplet imaging.
Disclosure of Invention
The invention develops a naphthalene-derived fluorescent probe, and by introducing carbonyl and a nitrogenous alkyl structure on a naphthalene ring, the fluorescent quantum yield of the probe in a protic solvent such as water is less than 0.01, and the fluorescent quantum yield in an aprotic solvent such as dichloromethane can reach 0.5. Researches show that the probe has higher brightness and larger Stokes displacement, ensures that the probe has higher brightness during imaging, effectively reduces the background during imaging, can quickly penetrate through cells and is enriched in lipid droplets, and realizes the fluorescence imaging of the lipid droplets by means of a confocal fluorescence microscope.
The technical scheme adopted by the invention is as follows:
a high-brightness large-Stokes-shift wash-free fat-drop fluorescent probe takes naphthalene as a structural unit, and the structural formula of the probe is as follows:
Figure BDA0002310708710000021
Figure BDA0002310708710000022
or
Figure BDA0002310708710000023
Figure BDA0002310708710000024
Or
Figure BDA0002310708710000025
Wherein n and m are integers of 0-3.
Meanwhile, the invention also provides a general synthetic method of the lipid droplet probe, which comprises the following synthetic steps:
Figure BDA0002310708710000026
Figure BDA0002310708710000027
or
Figure BDA0002310708710000028
Figure BDA0002310708710000029
Or
Figure BDA00023107087100000210
Wherein n and m are integers of 0-3.
The washing-free lipid drop fluorescent probe with high brightness and large Stokes displacement is synthesized by the following specific steps:
(1) synthesis of intermediate 5-bromo-1-naphthyl ester or 5-bromo-1-naphthylamide derivative (P-1)
Dissolving 5-bromo-1-naphthoic acid in thionyl chloride, heating to 70-90 ℃, stirring for reaction for 8-12 hours, and removing the solvent under reduced pressure to obtain a light yellow solid; dissolving the light yellow solid in dichloromethane, adding corresponding alcohol or amine, reacting at room temperature for 1-12 hours, removing the solvent under reduced pressure, and separating and purifying by a silica gel chromatographic column to obtain an intermediate P-1;
(2) synthesis of lipid drop probe 5-N-substituted-1-naphthyl ester or 5-N-substituted-1-naphthylamide (P-2)
Dissolving the intermediate P-1 obtained in the step (1), tris (dibenzylidene-BASE acetone) dipalladium (0), 3-dicyclohexyl phosphorus-2, 4, 6-triisopropyl biphenyl and cesium carbonate in dry dioxane under the atmosphere of nitrogen, adding corresponding amine, heating to 80-110 ℃, stirring, reacting for 4-12 hours, removing the solvent under reduced pressure, and separating and purifying by a silica gel chromatographic column to obtain the fat drop probe 5-N-substituted-1-naphthyl ester or 5-N-substituted-1-naphthamide.
The amine is a primary or secondary amine.
In the step (1): the mass-to-volume ratio of the 5-bromo-1-naphthoic acid to the thionyl chloride is 1:10-50 g/mL; the mass ratio of the 5-bromo-1-naphthoic acid to the corresponding alcohol or amine is 1: 0.2-20; the mass-to-volume ratio of the 5-bromo-1-naphthoic acid to the dichloromethane is 1:20-100 g/mL.
In the step (2): the mass ratio of the intermediate P-1 to tris (dibenzylidene-BASE acetone) dipalladium (0), 3-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl and cesium carbonate is 1:0.1-1:0.3-1: 2-10; the mass-to-volume ratio of the intermediate P-1 to the corresponding amine is 1:1-5 g/mL; the mass-to-volume ratio of the intermediate P-1 to the dioxane is 1:10-100 g/mL.
The probe can specifically mark lipid drops in living cells and realize fluorescence imaging.
The invention has the advantages and beneficial effects that:
the probe has the advantages of low price of synthetic raw materials, simple method, easy derivation and the like.
The probe introduces carbonyl and nitrogen-containing alkyl structure on naphthalene ring, so that the fluorescence quantum yield of the probe in protic solvent such as water is less than 0.01, and the fluorescence quantum yield in aprotic solvent such as dichloromethane can reach 0.5. And the probe has larger Stokes displacement, so that the interference of exciting light during imaging is avoided. .
The probe can realize accurate positioning of lipid droplets in cells, and can be used for research in the fields of imaging, sensing, detection and the like of lipid droplets and various lipids in cells or tissues.
Drawings
FIG. 1: nuclear magnetic hydrogen spectrum of the lipid droplet probe F-1 prepared in example 1;
FIG. 2: nuclear magnetic carbon spectrum of lipid droplet probe F-1 prepared in example 1;
FIG. 3: the lipid drop probe F-3 prepared in the example 3 is normalized in dichloromethane for fluorescence excitation and emission spectrogram, the abscissa is wavelength, the ordinate is normalized fluorescence intensity and absorption intensity, and the concentration of the fluorescent probe is 10 mu M; (ii) a
FIG. 4: nuclear magnetic hydrogen spectrum of the lipid droplet probe F-5 prepared in example 5;
FIG. 5: nuclear magnetic carbon spectrum of lipid droplet probe F-5 prepared in example 5;
FIG. 6: a high resolution mass spectrum of the lipid droplet probe F-5 prepared in example 5;
FIG. 7: nuclear magnetic hydrogen spectrum of the lipid droplet probe F-6 prepared in example 6;
FIG. 8: confocal fluorescence microscopy images of HeLa cells of the lipid droplet probe F-2 prepared in example 2.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1.
Synthesis of lipid droplet Probe F-1.
When in use
Figure BDA0002310708710000041
When n is 1, the synthesis route of the lipid droplet probe F-1 is as follows.
The synthetic route and the product structure of the intermediate P-Z-1 are as follows:
Figure BDA0002310708710000042
weighing 5-bromo-1-naphthoic acid (500mg,2mmol), dissolving in 5mL thionyl chloride, heating to 80 ℃, stirring for reaction for 8h, cooling the reaction solution to room temperature, and distilling under reduced pressure to remove thionyl chloride to obtain a light yellow solid. The solid was dissolved by adding 3mL of dichloromethane, ethanol (9.2g, 0.2mol) was added, the reaction was carried out at room temperature for 12 hours, then the solvent was removed under reduced pressure, and the residue was separated by means of a silica gel column (petroleum ether/ethyl acetate: 5/1, V/V) to give 455mg of the product as a pale yellow viscous liquid with a yield of 82%.
The high resolution mass spectrum data is as follows:
HRMS(ESI)：m/z：[M+H]+: calculated values: 279.0021, Experimental value: 279.0012.
the structure of the compound is shown as the formula P-Z-1 through detection.
The synthetic route and the product structure of the lipid droplet probe F-1 are as follows.
Figure BDA0002310708710000051
Intermediate P-Z-1(50mg,0.18mmol), tris (dibenzylidene-BASE acetone) dipalladium (0) (16.5mg,0.018mmol), 3-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (25.6mg,0.054mmol), cesium carbonate (176mg,0.54mmol) were dissolved in dry dioxane under nitrogen atmosphere, 0.4M ammonia in dioxane (450 μ L,1.8mmol) was added, the reaction mixture was cooled to room temperature after 12h of reaction at 80 ℃, the solvent was removed under reduced pressure, and the residue was separated by a silica gel column (petroleum ether/ethyl acetate 5/1, V/V) to give 27mg of the product, a pale yellow viscous liquid, a yield of 70%.
The nuclear magnetic spectrum hydrogen spectrum is shown in the following figure 1, and the specific data is as follows:
1H NMR(400MHz,CDCl3)δ8.28(d,J＝8.7Hz,1H),8.10(d,J＝7.1Hz,1H),8.04(d,J＝8.5Hz,1H),7.48–7.44(m,1H),7.42–7.38(m,1H),6.84(d,J＝7.4Hz,1H),4.46(q,J＝7.1Hz,2H),4.14(s,2H),1.45(t,J＝7.1Hz,3H).
the nuclear magnetic spectrum carbon spectrum is shown in the following figure 2, and the specific data is as follows:
13C NMR(101MHz,CDCl3)δ168.04,142.46,132.15,129.68,128.36,128.14,125.58,124.24,123.36,116.70,110.36,61.09,14.40.
through detection, the structure of the fluorescent probe is shown as the formula F-1, and the fluorescent probe can be accurately positioned in a living cell lipid drop for fluorescence imaging.
Example 2.
Synthesis of lipid droplet Probe F-2.
When in use
Figure BDA0002310708710000052
The synthetic route and product structure of the lipid droplet probe P-2 are as follows.
The synthetic route and the product structure of the intermediate P-Z-1 are as follows:
Figure BDA0002310708710000061
weighing 5-bromo-1-naphthoic acid (500mg,2mmol), dissolving in 5mL thionyl chloride, heating to 80 ℃, stirring for reaction for 10h, cooling the reaction liquid to room temperature, and distilling under reduced pressure to remove thionyl chloride to obtain a light yellow solid. The solid was dissolved by adding 3mL of dichloromethane, ethanol (9.2g, 0.2mol) was added, the reaction was carried out at room temperature for 12 hours, then the solvent was removed under reduced pressure, and the residue was separated by means of a silica gel column (petroleum ether/ethyl acetate: 5/1, V/V) to give 470mg of the product as a pale yellow viscous liquid with a yield of 85%.
The high resolution mass spectrum data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 279.0021, Experimental value: 279.0012.
the structure of the compound is shown as the formula P-Z-1 through detection.
The synthetic route and the product structure of the lipid droplet probe F-2 are as follows.
Figure BDA0002310708710000062
Intermediate P-Z-1(50mg,0.18mmol), tris (dibenzylidene-BASE acetone) dipalladium (0) (16.5mg,0.018mmol), 3-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (25.6mg,0.054mmol), cesium carbonate (176mg,0.54mmol) were dissolved in dry dioxane under nitrogen atmosphere, azetidine (500 μ L,7.4mmol) was added, the temperature was raised to 80 ℃ to react for 4h, the reaction solution was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was separated by a silica gel column (petroleum ether/ethyl acetate 8/1, V/V) to give 40mg of product, yellow viscous liquid, 88% yield.
The high resolution mass spectrum data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 256.1338, Experimental value: 256.1374.
through detection, the structure of the fluorescent probe is shown as the formula F-2, and the fluorescent probe can be accurately positioned in a living cell lipid drop for fluorescence imaging.
Example 3
Synthesis of lipid droplet Probe F-3.
When in use
Figure BDA0002310708710000071
The synthetic route and product structure of the lipid droplet probe P-3 are as follows.
The synthetic route and the product structure of the intermediate P-Z-2 are as follows:
Figure BDA0002310708710000072
weighing 5-bromo-1-naphthoic acid (500mg,2mmol), dissolving in 5mL thionyl chloride, heating to 80 ℃, stirring for reaction for 10h, cooling the reaction liquid to room temperature, and distilling under reduced pressure to remove thionyl chloride to obtain a light yellow solid. The solid was dissolved by adding 3mL of dichloromethane, methanol (5.7g, 0.2mol) was added, the reaction was carried out at room temperature for 12 hours, then the solvent was removed under reduced pressure, and the residue was separated by means of a silica gel column (petroleum ether/ethyl acetate: 5/1, V/V) to give 435mg of the product as a pale yellow viscous liquid with a yield of 82%.
The high resolution mass spectrum data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 264.9864, Experimental value: 264.9845.
the structure of the compound is shown as the formula P-Z-2 through detection.
The synthetic route and the product structure of the lipid droplet probe F-2 are as follows.
Figure BDA0002310708710000081
Intermediate P-Z-2(50mg,0.19mmol), tris (dibenzylidene-BASE acetone) dipalladium (0) (16.5mg,0.018mmol), 3-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (25.6mg,0.054mmol), cesium carbonate (176mg,0.54mmol) were dissolved in dry dioxane under nitrogen atmosphere, piperidine (197 μ L,2mmol) was added, the temperature was raised to 80 ℃ to react for 4h, the reaction solution was cooled to room temperature, the solvent was removed under reduced pressure, the residue was separated by a silica gel column (petroleum ether/ethyl acetate ═ 8/1, V/V) to give 31.7mg, a yellow viscous liquid, yield 62%.
The high resolution mass spectrum data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 270.1494, respectively; experimental values: 270.1491.
through detection, the structure of the fluorescent probe is shown as the formula F-3, and the fluorescent probe can be accurately positioned in a living cell lipid drop for fluorescence imaging.
Dissolving a probe to be detected in a dimethyl sulfoxide solution to prepare 2mM mother liquor, preparing test solutions with different concentrations according to requirements, and detecting the spectrum and cell imaging of the test solutions.
F-3 spectroscopic test in dichloromethane. And adding 20 mu LF-3 mother liquor into 4mL dichloromethane to prepare 10 mu M fluorescent probe test solution, and testing the ultraviolet absorption spectrum and the fluorescence emission spectrum.
The normalized ultraviolet absorption spectrum and fluorescence emission spectrum of F-3 in dichloromethane are shown in FIG. 4, wherein the concentration of the fluorescent probe is 10 μ M, and the quantum yield of F-3 in dichloromethane reaches 0.5, so that the brightness is higher.
The maximum absorption wavelength of F-3 in dichloromethane is 350nm, the maximum fluorescence emission wavelength is 541nm, the Stokes shift is 191nm, and the probe has larger Stokes shift.
Example 4.
F-4 Synthesis of lipid droplet Probe.
When in use
Figure BDA0002310708710000091
The synthetic route and product structure of the lipid droplet probe F-4 are as follows. .
The synthetic route and the product structure of the intermediate P-NH-1 are as follows:
Figure BDA0002310708710000092
weighing 5-bromo-1-naphthoic acid (500mg,2mmol), dissolving in 3mL thionyl chloride, heating to 80 ℃, stirring for reaction for 10h, cooling the reaction liquid to room temperature, and distilling under reduced pressure to remove thionyl chloride to obtain a light yellow solid. The solid was dissolved by adding 8mL of dichloromethane, methylamine (70mg, 2mmol) was added, the reaction was carried out at room temperature for 1 hour, then the solvent was removed under reduced pressure, and the residue was separated by means of a silica gel column (petroleum ether/ethyl acetate ═ 1/1, V/V) to give 498mg of the product as a white solid powder with a yield of 87%.
The high resolution mass spectrum data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 264.0024, Experimental value: 264.0056.
the structure of the compound is shown as the formula P-NH-1 through detection.
The synthetic route and the product structure of the lipid droplet probe F-4 are as follows.
Figure BDA0002310708710000093
Under a nitrogen atmosphere, intermediate P-NH-1(50mg,0.18mmol), tris (dibenzylidene-BASE acetone) dipalladium (0) (165mg,0.18mmol), 3-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (256mg,0.54mmol), and cesium carbonate (586mg,1.8mmol) were dissolved in dry dioxane, propylamine (250 μ L,3mmol) was added, the temperature was raised to 110 ℃ to react for 8 hours, the reaction liquid was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was separated by a silica gel column (petroleum ether/ethyl acetate ═ 8/1, V/V) to give 34mg of a product as a pale yellow solid in 78% yield.
The high resolution mass spectrum data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 243.1497, Experimental value: 243.1416.
through detection, the structure of the fluorescent probe is shown as the formula F-4, and the fluorescent probe can be positioned in lipid droplets of living cells for fluorescence imaging.
Example 5.
F-5 Synthesis of lipid droplet Probe.
When in use
Figure BDA0002310708710000101
R2When n is 1 and m is 1, the synthetic route and the product structure of the lipid droplet probe F-5 are as follows. .
The synthetic route and the product structure of the intermediate P-NN-1 are as follows:
Figure BDA0002310708710000102
weighing 5-bromo-1-naphthoic acid (500mg,2mmol), dissolving in 5mL thionyl chloride, heating to 90 ℃, stirring for reaction for 6h, cooling the reaction solution to room temperature, and distilling under reduced pressure to remove thionyl chloride to obtain a light yellow solid. The solid was dissolved by adding 8mL of dichloromethane, diethylamine (1.46g, 20mmol) was added, the reaction was carried out at room temperature for 3 hours, then the solvent was removed under reduced pressure, and the residue was separated by silica gel column (petroleum ether/ethyl acetate-1/2, V/V) to give 452mg of the product as a white solid powder, yield 74%.
The high resolution mass spectrum data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 306.0494, Experimental value: 306.0488.
the structure of the compound is shown as the formula P-NN-1 after detection.
The synthetic route and the product structure of the lipid droplet probe F-5 are as follows.
Figure BDA0002310708710000111
Under a nitrogen atmosphere, intermediate P-NN-1(50mg,0.18mmol), tris (dibenzylidene-BASE acetone) dipalladium (0) (83mg,0.09mmol), 3-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (142mg,0.3mmol), and cesium carbonate (293mg,0.9mmol) were dissolved in dry dioxane, a 0.4M dioxane solution of ammonia (450 μ L,1.8mmol) was added, the reaction mixture was heated to 110 ℃ for 12 hours, the reaction solution was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was separated by a silica gel column (petroleum ether/ethyl acetate ═ 8/1, V/V) to give 30mg of the product as a pale yellow solid in 69% yield.
The nuclear magnetic spectrum hydrogen spectrum is shown in the following figure 4, and the specific data is as follows:
1H NMR(400MHz,CDCl3)δ7.74(d,J＝8.4Hz,1H),7.36–7.31(m,1H),7.28(dd,J＝6.9,1.0Hz,1H),7.21(dd,J＝13.2,5.9Hz,1H),7.15(d,J＝8.3Hz,1H),6.70(dd,J＝7.2,0.8Hz,1H),4.18–4.05(m,2H),3.79(td,J＝13.9,7.0Hz,1H),3.42(dq,J＝13.8,7.0Hz,1H),3.05–2.93(m,2H),1.28(t,J＝7.1Hz,3H),0.90(t,J＝7.1Hz,3H).
the nuclear magnetic spectrum carbon spectrum is shown in the following figure 5, and the specific data is as follows:
13C NMR(101MHz,CDCl3)δ170.57,142.50,135.69,130.48,127.38,124.03,123.66,123.23,121.59,115.54,110.19,43.05,38.97,14.24,13.04.
the high resolution mass spectrum is shown in fig. 6, and the specific data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 243.1497, Experimental value: 243.1469.
through detection, the structure of the fluorescent probe is shown as the formula F-5, and the fluorescent probe can be positioned in lipid droplets of living cells for fluorescence imaging.
Example 6
F-6 Synthesis of lipid droplet Probe.
When in use
Figure BDA0002310708710000121
The synthetic route and product structure of the lipid droplet probe F-6 are as follows. .
The synthetic route and the product structure of the intermediate P-NH-2 are as follows:
Figure BDA0002310708710000122
weighing 5-bromo-1-naphthoic acid (500mg,2mmol), dissolving in 3mL thionyl chloride, heating to 80 ℃, stirring for reaction for 10h, cooling the reaction liquid to room temperature, and distilling under reduced pressure to remove thionyl chloride to obtain a light yellow solid. The solid was dissolved by adding 8mL of dichloromethane, ethylamine (90mg, 2mmol) was added, the reaction was carried out at room temperature for 1 hour, then the solvent was removed under reduced pressure, and the residue was separated by means of a silica gel column (petroleum ether/ethyl acetate ═ 1/1, V/V) to give 515mg of the product as a white solid powder in 91% yield.
The high resolution mass spectrum data is as follows:
hrms (esi): m/z: [ M + H ] +: calculated values: 278.0181, Experimental value: 278.0193.
the structure of the compound is shown as the formula P-NH-2 through detection.
The synthetic route and the product structure of the lipid droplet probe F-6 are as follows.
Figure BDA0002310708710000123
Under a nitrogen atmosphere, the intermediates P-NH-2(50mg,0.18mmol), tris (dibenzylidene-BASE acetone) dipalladium (0) (165mg,0.18mmol), 3-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (256mg,0.54mmol), cesium carbonate (586mg,1.8mmol) were dissolved in dry dioxane, azetidine (500 μ L,7.4mmol) was added, the temperature was raised to 110 ℃ to react for 8h, the reaction solution was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was separated by a silica gel column (petroleum ether/ethyl acetate ═ 8/1, V/V) to give 30mg of the product as a pale yellow solid in 72% yield.
The nuclear magnetic spectrum hydrogen spectrum is shown in the following figure 7, and the specific data is as follows:
1H NMR(400MHz,CDCl3)δ7.92(d,J＝8.6Hz,1H),7.60(d,J＝8.4Hz,1H),7.40(d,J＝6.9Hz,1H),7.30(t,J＝8.0Hz,1H),7.23(t,J＝7.8Hz,1H),6.49(d,J＝7.6Hz,1H),5.99(s,1H),4.03(t,J＝7.2Hz,4H),3.48–3.39(m,2H),2.35–2.25(m,2H),1.16(t,J＝7.2Hz,3H).
through detection, the structure of the fluorescent probe is shown as the formula F-6, and the fluorescent probe can be positioned in lipid droplets of living cells for fluorescence imaging.
Example 7.
F-2 fluorescence imaging microscopic imaging experiment after living cell staining. The probe to be detected is dissolved in dimethyl sulfoxide solution to prepare 2mM mother liquor. Dissolving 1 μ L F-2 mother liquor in 1mL cell culture solution at 37 deg.C，5％CO2Confocal fluorescence microscopy was performed after 15 minutes of incubation.
Fluorescent micrographs of HeLa cells incubated with a final concentration of 2. mu.M F-2 for 15 minutes were clearly visible as circular lipid droplets in the HeLa cells, as shown in FIG. 8.

Claims (7)

1.一类高亮度、大斯托克斯位移的免洗脂滴探针，其特征在于，其结构如下：1. the no-cleaning lipid droplet probe of a class of high brightness, large Stokes displacement, is characterized in that, its structure is as follows:
Figure FDA0002310708700000011
Figure FDA0002310708700000011
 R1为
Figure FDA0002310708700000012
 R1 is
Figure FDA0002310708700000012
 R2为
Figure FDA0002310708700000013
 R2 is
Figure FDA0002310708700000013
 其中，n、m为0-3整数。Wherein, n and m are integers from 0 to 3. 2.根据权利要求1所述的脂滴探针，其特征在于，该脂滴探针通过在萘环上引入羰基及含氮烷基功能团得到。2 . The lipid droplet probe according to claim 1 , wherein the lipid droplet probe is obtained by introducing carbonyl and nitrogen-containing alkyl functional groups into the naphthalene ring. 3 . 3.一种如权利要求1所述的高亮度、大斯托克斯位移的免洗脂滴探针的合成方法，其特征在于，具体步骤如下：3. a synthetic method of the no-cleaning lipid drop probe of high brightness as claimed in claim 1, large Stokes displacement, is characterized in that, concrete steps are as follows: (1)中间体5-溴-1-萘酯或5-溴-1-萘酰胺衍生物(P-1)的合成(1) Synthesis of intermediate 5-bromo-1-naphthyl ester or 5-bromo-1-naphthylamide derivative (P-1) 将5-溴-1-萘甲酸溶于氯化亚砜中，升温至70-90℃搅拌反应8-12小时后减压除去溶剂，得到淡黄色固体；将淡黄色固体溶于二氯甲烷中，加入对应的醇或胺，室温反应1-12小时后减压除去溶剂，硅胶色谱柱分离提纯得到中间体P-1；Dissolve 5-bromo-1-naphthoic acid in thionyl chloride, heat up to 70-90°C and stir for 8-12 hours, remove the solvent under reduced pressure, and obtain a light yellow solid; dissolve the light yellow solid in dichloromethane , add the corresponding alcohol or amine, react at room temperature for 1-12 hours, then remove the solvent under reduced pressure, and separate and purify by silica gel column to obtain intermediate P-1; (2)脂滴探针5-N-取代-1-萘酯或5-N-取代-1-萘酰胺(P-2)的合成(2) Synthesis of lipid droplet probe 5-N-substituted-1-naphthyl ester or 5-N-substituted-1-naphthylamide (P-2) 在氮气氛围下，将步骤(1)得到的中间体P-1、三(二亚苄-BASE丙酮)二钯(0)、3-二环己基磷-2,4,6-三异丙基联苯、碳酸铯溶于干燥的二氧六环中，加入对应的胺，升温至80-110℃搅拌，反应4-12小时后减压除去溶剂，硅胶色谱柱分离提纯得到脂滴探针5-N-取代-1-萘酯或5-N-取代-1-萘酰胺。Under nitrogen atmosphere, the intermediate P-1, tris(dibenzylidene-BASEacetone)dipalladium(0), 3-dicyclohexylphosphorus-2,4,6-triisopropyl obtained in step (1) were Biphenyl and cesium carbonate were dissolved in dry dioxane, the corresponding amine was added, the temperature was raised to 80-110 °C and stirred, the solvent was removed under reduced pressure after the reaction for 4-12 hours, and the lipid droplet probe 5 was obtained by separation and purification on a silica gel column. -N-substituted-1-naphthyl ester or 5-N-substituted-1-naphthalene amide. 4.如权利要求3中所述的合成方法，其特征在于，所述胺为伯胺或仲胺。4. The synthetic method as claimed in claim 3, wherein the amine is a primary amine or a secondary amine. 5.如权利要求3中所述的合成方法，其特征在于，步骤(1)中：5-溴-1-萘甲酸与氯化亚砜的质量与体积比为1:10-50g/mL；5-溴-1-萘甲酸与对应的醇或胺的质量比为1:0.2-20；5-溴-1-萘甲酸与二氯甲烷的质量与体积比为1:20-100g/mL。5. synthetic method as described in claim 3, is characterized in that, in step (1): the mass to volume ratio of 5-bromo-1-naphthoic acid and sulfur oxychloride is 1:10-50g/mL; The mass ratio of 5-bromo-1-naphthoic acid to the corresponding alcohol or amine is 1:0.2-20; the mass to volume ratio of 5-bromo-1-naphthoic acid to dichloromethane is 1:20-100 g/mL. 6.如权利要求3中所述的合成方法，其特征在于，步骤(2)中：中间体P-1与三(二亚苄-BASE丙酮)二钯(0)、3-二环己基磷-2,4,6-三异丙基联苯、碳酸铯的质量比为1:0.1-1:0.3-1:2-10；中间体P-1与对应胺的质量与体积比为1:1-5g/mL；中间体P-1与二氧六环的质量与体积比为1:10-100g/mL。6. synthetic method as described in claim 3, is characterized in that, in step (2): intermediate P-1 and tris (dibenzylidene-BASE acetone) dipalladium (0), 3-dicyclohexyl phosphorus The mass ratio of -2,4,6-triisopropylbiphenyl and cesium carbonate is 1:0.1-1:0.3-1:2-10; the mass-to-volume ratio of intermediate P-1 and the corresponding amine is 1: 1-5 g/mL; the mass-to-volume ratio of intermediate P-1 and dioxane is 1:10-100 g/mL. 7.一种如权利要求1所述的高亮度、大斯托克斯位移的免洗脂滴探针在荧光成像、荧光传感、生化检测等领域的应用。7 . The application of the no-wash lipid droplet probe with high brightness and large Stokes shift according to claim 1 in the fields of fluorescence imaging, fluorescence sensing, and biochemical detection. 8 .
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