CN110093153B - Positioning Golgi body near-infrared fluorescent probe and synthetic method and application thereof - Google Patents
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Abstract
The invention relates to a positioning Golgi body near-infrared fluorescent probe and a synthetic method and application thereof. The structural formula of the near-infrared fluorescent probe is shown as the formula I:
the Golgi apparatus is located by the precise binding of its small molecule inhibitor to the enzyme, and the location is shown by the fluorescence of the near infrared fluorophore Cy 7. The interference of the self fluorescence of the biological tissue is avoided, and the detection process is not interfered by pH, ROS, RNS and metal ions.
Description
Technical Field
The invention belongs to the technical field of fluorescence detection, and particularly relates to a positioning Golgi body near-infrared fluorescent probe, and a synthetic method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
In recent years, bio-imaging technology has become a powerful tool for detecting various vital activities. The small molecular fluorescent probe has the characteristics of high sensitivity, simplicity in operation and the like, so that the small molecular fluorescent probe is widely applied to the aspect of biological imaging. According to the existing report, most of the absorption and emission wavelengths of the small-molecule fluorescent dyes are located in the visible light region, which greatly limits the application of the small-molecule fluorescent dyes in biological detection. However, the near infrared fluorescent dye has the advantages of less interference of background light, strong penetrating capability of biological tissues, small light damage to the biological tissues and the like, thereby attracting much attention.
Two commercial probes for locating the Golgi are available at present, namely a Golgi Red fluorescent probe (Golgi-Tracker Red), and the maximum excitation and emission wavelengths are 589nm and 617nm respectively; golgi Green fluorescent probes (Golgi-Tracker Green) have maximum excitation and emission wavelengths of 505nm and 511nm, respectively. The inventor finds that the two fluorescent probes have shorter wavelengths and are easily interfered by the self background fluorescence of biological tissues in biological imaging experiments.
Disclosure of Invention
In view of the problems in the prior art, an object of the present invention is to provide a near-infrared fluorescent probe for locating golgi, and a synthetic method and applications thereof. The invention provides a near-infrared fluorescent probe which is based on a small-molecule inhibitor of sphingomyelin synthetase 1 and takes Cy7 as a fluorophore to locate Golgi. The invention has the advantages of simple preparation, accurate positioning and the like. It is noted that the present invention avoids interference of autofluorescence of biological tissue by near infrared fluorescence. By using the invention, the intracellular Golgi body positioning is successfully realized.
In order to solve the technical problems, the technical scheme of the invention is as follows:
in a first aspect, the invention provides a positioning Golgi near-infrared fluorescent probe, which has a structural formula shown in formula I:
the invention utilizes the accurate combination of the small molecule inhibitor and the enzyme to position the Golgi apparatus, and the fluorescence of the near infrared fluorophore Cy7 is used for displaying the positioning. Cy7 is a near infrared dye with a longer wavelength to avoid background fluorescence.
The enzyme inhibitor is bound to other molecules to increase the molecular weight of the enzyme inhibitor itself, thereby enlarging the molecular structure, and the enzyme inhibitor is not always bound smoothly because the enzyme inhibitor is affected by the cell environment during the binding with the sphingomyelin synthase 1. The sphingomyelin synthase 1 is distributed only in a small area on the reverse side of the Golgi apparatus, so that the enzyme inhibitor binding to sphingomyelin synthase 1 requires accurate access to the corresponding site for binding. Therefore, the invention provides a probe capable of accurately positioning the Golgi apparatus.
The enzyme inhibitor is combined with the sphingomyelin synthase 1 through alpha-cyanamide, the combination site of the enzyme inhibitor can affect the combination of the enzyme inhibitor and enzyme if the combination site is covered by a fluorescent molecular structure, and the combination of the enzyme inhibitor and the sphingomyelin synthase 1 can be easily affected due to the larger molecular chain of the compound after the enzyme inhibitor is combined with other molecules to generate a macromolecule.
In a second aspect, the present invention provides a method for preparing the structure represented by formula i, comprising the following steps:
(1) taking 5-nitro salicylaldehyde as an initial raw material, and carrying out halogenation reaction on the initial raw material and benzyl bromide under the condition of taking potassium carbonate as alkali to obtain a compound 2;
(2) simultaneously carrying out nucleophilic addition reaction on the compound 2 and aniline respectively, and carrying out nucleophilic substitution reaction on the compound 2 and TMSCN to obtain a compound 3;
(3) compound 3 with SnCl2·2H2Carrying out reduction reaction on O to obtain a compound 4;
(4) nucleophilic substitution reaction of the compound 4 and Cy7 is carried out to obtain the structure shown in the formula I.
The reaction route is as follows:
preferably, the condensation reaction in the step (1) is performed under the condition of refluxing at 75-85 ℃ for 2-4 h; preferably, the volume of the benzyl bromide corresponding to 1g of 5-nitrosalicylaldehyde is 770-790 mu L; preferably, the mass ratio of the 5-nitro salicylaldehyde to the potassium carbonate is 1: 1.6-1.7; preferably, the 5-nitro salicylaldehyde takes acetonitrile as a solvent, and the volume of acetonitrile corresponding to 1g of 5-nitro salicylaldehyde is 7-9 mL; preferably, after the reaction, filtration, washing with ethyl acetate, extraction with saturated NaCl, and concentration, compound 2 is obtained.
Preferably, the reaction conditions in step (2) are room temperature; preferably, the compound 2 uses dichloromethane as solvent, and 1g of the compound 2 is pThe volume of the dichloromethane is 7-8 mL; preferably, the volume of aniline corresponding to 1g of compound 2 is 350-; preferably, the catalyst of the reaction is I2Compounds 2 and I2The mass ratio of (1): 9-11; preferably, after the reaction is finished, the mixture is purified by thin layer chromatography, and the eluent ratio is n-hexane: ethyl acetate 2-3: 1 to give compound 3; preferably, the reaction conditions of the step (2) are room temperature, and the reaction time is 4-6 h.
And (3) after purification in the step (2), forming a black spot under 254nm illumination, and forming a compound 2 in a non-light part under 365nm illumination.
Preferably, the solvent of the compound 3 in the step (3) is a mixed solution of ethanol and concentrated hydrochloric acid, the volume of ethanol corresponding to 1g of the compound 3 is 13-15mL, and the volume of concentrated hydrochloric acid corresponding to 1g of the compound 3 is 27-29 mL; preferably, compound 3 is reacted with SnCl2·2H2The mass ratio of O is 2.4-2.6; preferably, SnCl2·2H2Adding O for four times, wherein each time is separated by half an hour; preferably, the reaction condition of the step (3) is that the temperature is 40-50 ℃, and the reaction time is 11-13 h; preferably, saturated Na is used after the reaction in the step (3) is finished2CO3Adjusting the alkalinity, extracting with ethyl acetate, and concentrating to obtain the compound 4.
Preferably, the solvent of compound 4 in step (4) is DMF, and the volume of the solvent corresponding to 1g of compound 4 is 30-40 mL; preferably, the mass ratio of compound 4 to Cy7 is 3-4: 1; preferably, the reaction temperature in the step (4) is 70-90 ℃, and the reaction time is 11-13 h; preferably, after the reaction in step (4), purifying by thin layer chromatography, wherein the eluent ratio is ethyl acetate: methanol 9-11: 1.
in the present invention, it was found that the reaction of compound 4 with Cy7 is more favorable in the above-mentioned mass ratio range, and the reaction activity is increased in the above-mentioned temperature range. The reaction of the compound 4 with Cy7 is more favorable for the reaction at the mass ratio of 3:1, and the reaction activity is highest at 80 ℃.
And (4) purifying by thin layer chromatography to obtain a blue band as a final product. The probe is a blue substance under natural light, and can be directly distinguished after purification.
According to the invention, 5-nitro salicylaldehyde is used as a reaction raw material, the nitro group is reduced to generate an amino group, the amino group and chlorine in a Cy7 compound undergo a nucleophilic substitution reaction, and the compound 4 and Cy7 are combined to obtain the probe.
The inventors selected 5-nitro salicylaldehyde as the reaction raw material with the purpose of: the nitro is introduced into the fixed position of the enzyme inhibitor, and the position of the nitro has certain influence on the combination of the enzyme inhibitor and the sphingomyelin synthetase 1.
The near-infrared fluorescent probe is applied to the detection of the Golgi apparatus in the positioning cell.
The invention has the beneficial effects that:
the near-infrared fluorescent probe is used as an inhibitor of sphingomyelin synthase 1, the sphingomyelin synthase 1 is an enzyme positioned at the reverse side of Golgi apparatus, the inhibitor is combined with the sphingomyelin synthase 1 on the surface of the Golgi apparatus to obtain the position of the Golgi apparatus in the cell, and the positioning of the Golgi apparatus in the cell is realized by combining the display effect of Cy7 fluorescence.
The near-infrared fluorescent probe has the advantages of simple preparation and accurate positioning, and avoids the interference of the self-fluorescence of the biological tissue through the near-infrared fluorescence;
the near-infrared fluorescent probe has selectivity, and the detection process is not interfered by pH, ROS, RNS and metal ions.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the invention and not to limit the invention.
FIG. 1 is a graph of optical properties in Experimental example 1 of the present invention, in which a is an ultraviolet-visible spectrum, b is a fluorescence-excitation and emission wavelength spectrum distribution curve, c is a fluorescence-pH spectrum distribution curve, and d is a fluorescence-interference factor spectrum distribution curve;
FIG. 2 is a mass spectrum of a probe prepared in example 1 of the present invention;
FIG. 3 is a fluorescence mapping chart of Experimental example 2 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Sphingomyelin synthase 1 (SMS 1) is a subtype of sphingomyelin synthase (SMS) and is located on the opposite side of the golgi membrane.
The invention will be further illustrated by the following examples
Example 1
1g of 5-nitrosalicylaldehyde was dissolved in 8mL of acetonitrile, and 1.654g of potassium carbonate and 780. mu.L of benzyl bromide were added and reacted at 80 ℃ under reflux for 3 hours. After the reaction is finished, filtering, washing with ethyl acetate, extracting with saturated NaCl, and concentrating to obtain a compound 1; 0.257g of Compound 1 was dissolved in 2mL of dichloromethane, and 92. mu.L of aniline, 188. mu.L of TMSCN, 0.0257g I2 were added thereto, followed by stirring at room temperature for 5 hours. After the reaction is finished, purifying by using a thin layer chromatography, wherein the eluent ratio is n-hexane: ethyl acetate ═ 2.5: 1, obtaining a compound 2 which is a black spot under 254nm illumination and a dark part under 365nm illumination; 0.359g of Compound 2 was dissolved in 5mL of ethanol and 10mL of concentrated hydrochloric acid, and 0.9g of SnCl was added in portions2·2H2O, reacting for 12 hours at 45 ℃, and using saturated Na after the reaction is finished2CO3Adjusting alkalinity, extracting with ethyl acetate, and concentrating to obtain a compound 3; 0.329g of Compound 3 was dissolved in 10mL of DMF, and 0.102g of Cy7 was added to the solution to react at 80 ℃ for 12 hours. Purifying by thin layer chromatography, wherein the eluent ratio is ethyl acetate: methanol 10: 1.
example 2
1g of 5-nitro salicylaldehyde was dissolved in 9mL of acetonitrile, and 1.68g of potassium carbonate and 790. mu.L of benzyl bromide were added to the solution, followed by reflux reaction at 85 ℃ for 4 hours. After the reaction is finished, filtering, washing with ethyl acetate, extracting with saturated NaCl, and concentrating to obtain a compound 1; 0.257g of Compound 1 was dissolved in 2mL of methylene chloride, and 92.5. mu.L of aniline, 190. mu.L of TMSCN, 0.0233g I were added2Stirred at room temperature for 5 h. After the reaction is finished, purifying by using a thin layer chromatography, wherein the eluent ratio is n-hexane: ethyl acetate ═ 3:1, obtaining a compound 2 which is a black spot under 254nm illumination and a dark part under 365nm illumination; 0.359g of Compound 2 are taken and dissolved in 5.3mL of ethanol, 10.4mL of concentrated hydrochloric acid, and a total of 0.93g of SnCl is added in portions2·2H2O, reacting for 13 hours at 50 ℃, and using saturated Na after the reaction is finished2CO3Adjusting alkalinity, extracting with ethyl acetate, and concentrating to obtain a compound 3; 0.329g of Compound 3 was dissolved in 13mL of DMF, and 0.109g of Cy7 was added to react at 85 ℃ for 13 hours. Purifying by thin layer chromatography, wherein the eluent ratio is ethyl acetate: methanol 11: 1. finally, the probes were dissolved in Tris buffer (pH 7.4) for further characterization and performance evaluation.
In experimental example 1 and experimental example 2, the probes were dissolved in Tris buffer (pH 7.4) for characterization and performance evaluation.
Experimental example 1
The optical property experiment of the near infrared fluorescent probe of the invention.
The near-infrared fluorescent probes prepared in example 1 were subjected to the following experiments, respectively:
ultraviolet and visible light experiments; as shown in FIG. 1a, the maximum absorption wavelength of the near-infrared fluorescent probe prepared by the invention is 625 nm.
Fluorescence intensity experiment of the exciting light; as shown in FIG. 1b, the near-infrared fluorescent probe prepared by the present invention has an excitation wavelength (EX) of 625nm and an emission wavelength (EM) of 770 nm.
The influence experiment of the fluorescence intensity of the near-infrared fluorescent probe under different pH conditions is carried out; as shown in FIG. 1c, the fluorescence intensity of the near-infrared fluorescent probe prepared by the invention is basically unchanged under different pH conditions.
Experiment of the influence of the fluorescence intensity of the near-infrared fluorescent probe under different interference conditions; as shown in FIG. 1d (1. Fe)3+，2.Cu+，3.Fe2+，4.Ca2+，5.Mn2+，6.K+，7.Na+，8.Al3+，9.Zn2+，10.Mg2+，11.S2O3 2-，12.O2 ·-，13.·OH，14.1O2，15.ClO-，16.H2O2TBHP, 18 NO, 19 GSH, 20 Cys, 21 probe), the near-infrared fluorescent probe prepared by the method is free from the interference of ROS, RNS, metal ions and the like, and the fluorescence intensity is basically unchanged.
Experimental example 2
The invention relates to an experiment for positioning intracellular Golgi apparatus by using near-infrared fluorescent probe.
10nM Golgi Red fluorescent probe (Golgi-Tracker Red), mitochondrial Green fluorescent probe (Mito-Tracker Green), or lysosomal Green fluorescent probe (Lyso-Tracker Green) was added to PC12 cells, and after incubation at 37 ℃ for 15 minutes, 1. mu.M probe was added and incubation at 37 ℃ continued for 15 minutes. Extracellular residual probe was washed away with PBS followed by confocal microscopy. FIGS. 3a and b are images of cells incubated with Golgi red and probe, respectively, and c (overlay) is a superposition of a and b. d and e are images of cells incubated by mitochondria green and the probe, and f is a superimposed image of d and e. g and h are lysosome green, probe incubation cell imaging graphs, and i is a superposition graph of g and h. The golgi overlap factor is 0.92, the mitochondrial localization overlap factor is 0.65, and the lysosomal localization overlap factor is 0.62. The near-infrared fluorescent probe of the invention has the same positioning position with the Golgi body red fluorescent probe in PC12 cells, but has different positioning positions with the mitochondria green-red fluorescent probe and the lysosome green-red fluorescent probe, so the near-infrared fluorescent probe of the invention can position the Golgi body.
Comparative example 1
Different from example 1 is 3-nitrosalicylaldehyde.
The compound was prepared to differ from the structure shown in formula I in that Cy7 was located at the site of the enzyme inhibitor, which would prevent the binding of the enzyme inhibitor to sphingomyelin synthase 1.
Comparative example 2
The difference from example 1 is that the fluorescent molecule is Cy 5.
The prepared compound is different from the structure shown in the formula I in that Cy5 replaces Cy7, the fluorescence intensity of the compound is influenced by the intracellular environment, and the Golgi apparatus cannot be located.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (22)
1. A near-infrared fluorescent probe for positioning a Golgi apparatus is characterized in that: the structural formula is shown as formula I:
2. the method for preparing a near-infrared fluorescent probe according to claim 1, characterized in that:
(1) taking 5-nitro salicylaldehyde as an initial raw material, and carrying out halogenation reaction on the initial raw material and benzyl bromide under the condition of taking potassium carbonate as alkali to obtain a compound 2;
(2) simultaneously carrying out nucleophilic addition reaction on the compound 2 and aniline respectively, and carrying out nucleophilic substitution reaction on the compound 2 and TMSCN to obtain a compound 3;
(3) compound 3 with SnCl2·2H2Carrying out reduction reaction on O to obtain a compound 4;
(4) nucleophilic substitution reaction of the compound 4 and Cy7 is carried out to obtain the structure shown in the formula I.
3. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: the condition of the halogenation reaction in the step (1) is reflux for 2-4h at 75-85 ℃.
4. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (1), the volume of the benzyl bromide corresponding to 1g of 5-nitro salicylaldehyde is 770-790 mu L.
5. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (1), the mass ratio of the 5-nitro salicylaldehyde to the potassium carbonate is 1: 1.6-1.7.
6. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (1), acetonitrile is used as a solvent for 5-nitro salicylaldehyde, and the volume of acetonitrile corresponding to 1g of 5-nitro salicylaldehyde is 7-9 mL.
7. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (1), after the reaction, filtering, washing with ethyl acetate, extracting with saturated NaCl, and concentrating to obtain a compound 2.
8. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (2), the reaction conditions are room temperature.
9. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (2), the dichloromethane is used as the solvent for the compound 2, and the volume of the dichloromethane corresponding to 1g of the compound 2 is 7-8 mL.
10. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in step (2), the volume of aniline corresponding to 1g of compound 2 is 350-.
11. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (2), the catalyst for the reaction is I2Compounds 2 and I2The mass ratio of (1): 9-11.
12. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (2), after the reaction is finished, purifying by using a thin layer chromatography, wherein the eluent ratio is n-hexane: ethyl acetate 2-3: 1 to give compound 3.
13. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: in the step (2), the reaction condition of the step (2) is room temperature, and the reaction time is 4-6 h.
14. The method for preparing a near-infrared fluorescent probe according to claim 2, characterized in that: the reaction condition of the step (3) is that the temperature is 40-50 ℃ and the reaction time is 11-13 h.
15. The method of claim 2, wherein: in the step (3), the solvent of the compound 3 is a mixed solution of ethanol and concentrated hydrochloric acid, the volume of ethanol corresponding to 1g of the compound 3 is 13-15mL, and the volume of concentrated hydrochloric acid corresponding to 1g of the compound 3 is 27-29 mL.
16. The method of claim 2, wherein: compound (I)3 with SnCl2·2H2The mass ratio of O is 2.4-2.6.
17. The method of claim 2, wherein: saturated Na is used after the reaction in the step (3) is finished2CO3Adjusting the alkalinity, extracting with ethyl acetate, and concentrating to obtain the compound 4.
18. The method of claim 2, wherein: the solvent of the compound 4 in the step (4) is DMF, and the volume of the solvent corresponding to 1g of the compound 4 is 30-40 mL.
19. The method of claim 2, wherein: the mass ratio of the compound 4 to the Cy7 is 3-4: 1.
20. the method of claim 2, wherein: the reaction temperature in the step (4) is 70-90 ℃, and the reaction time is 11-13 h.
21. The method of claim 2, wherein: purifying by using thin layer chromatography after the reaction in the step (4), wherein the eluent ratio is ethyl acetate: methanol 9-11: 1.
22. use of the near-infrared fluorescent probe of claim 1 for detection of localized intracellular Golgi.
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| CN105112048A (en) * | 2015-08-20 | 2015-12-02 | 上海交通大学 | Novel intracellular Golgi-complex fluorescence molecular probe and application thereof |
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| CN101993382A (en) * | 2009-08-21 | 2011-03-30 | 复旦大学 | Aromatic amine derivative or analog and application thereof |
| CN105112048A (en) * | 2015-08-20 | 2015-12-02 | 上海交通大学 | Novel intracellular Golgi-complex fluorescence molecular probe and application thereof |
| CN109053802A (en) * | 2018-09-01 | 2018-12-21 | 青岛科技大学 | A kind of Ratio-type near infrared fluorescent probe and its synthetic method and application |
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