Fluorescent probe based on small molecule inhibitor and preparation method and application thereof
阅读说明：本技术 一种基于小分子抑制剂的荧光探针及其制备方法和应用 (Fluorescent probe based on small molecule inhibitor and preparation method and application thereof ) 是由 王宏达 吴强 陈俊玲 蔡明军 于 2020-06-15 设计创作，主要内容包括：本发明涉及一种基于小分子抑制剂的荧光探针及其制备方法和应用,属于荧光成像的小分子标记探针领域。本发明选择具有特定靶点的小分子抑制剂作为研究对象,通过合成手段对小分子抑制剂的合适位点进行衍生修饰,借助柔性PEG链或其它链接分子将其与荧光染料分子相连来构建一种新型的小分子荧光探针,具有体积小、标记特异性高、制备简便等优点,可广泛用于细胞蛋白的分布、定位和组装的研究。本发明的基于小分子抑制剂的荧光探针的制备方法简便、制备时间短、质量易控制,可以广泛应用与对应蛋白的分布、定位与组装研究。本发明的基于小分子抑制剂的荧光探针特别适用于超分辨成像。(The invention relates to a fluorescent probe based on a small molecular inhibitor, a preparation method and application thereof, and belongs to the field of small molecular labeled probes for fluorescence imaging. The invention selects the small molecule inhibitor with specific target as a research object, performs derivative modification on the proper site of the small molecule inhibitor by a synthesis means, and connects the small molecule inhibitor with the fluorescent dye molecule by virtue of the flexible PEG chain or other linking molecules to construct a novel small molecule fluorescent probe, has the advantages of small volume, high labeling specificity, simple and convenient preparation and the like, and can be widely used for research on distribution, positioning and assembly of cell protein. The preparation method of the fluorescent probe based on the small molecular inhibitor is simple and convenient, short in preparation time and easy in quality control, and can be widely applied to research on distribution, positioning and assembly of corresponding proteins. The fluorescent probe based on the small-molecule inhibitor is particularly suitable for super-resolution imaging.)
1. A fluorescent probe based on a small molecule inhibitor is characterized in that the structure of the fluorescent probe is shown as formula 1:
in formula 1:
represents a small molecule inhibitor which is gefitinib, erlotinib, taliquida, GDC-0941, A-674563 or AZD-805;
represents a length-adjustable linker molecule, which is a flexible PEG chain, a saturated carbon chain, or an unsaturated carbon chain;
represents a fluorescent dye molecule, which is Alexa532, Alexa647, Cy3, Cy5, Cy5.5, Cy7, Atto532, Atto488 or TAMRA.
2. The fluorescent probe based on the small molecule inhibitor as claimed in claim 1, wherein the structure is as shown in formula 2 or formula 3:
3. the preparation method of the fluorescent probe based on the small molecule inhibitor as claimed in claim 1 or 2, which is characterized by comprising the following steps:
the method comprises the following steps: carrying out derivative modification on the small molecule inhibitor to obtain a derivative of the small molecule inhibitor;
step two: connecting the derivative of the small molecule inhibitor obtained in the step one with a linking molecule to obtain a product II;
step three: and (5) reacting the product II obtained in the step II with fluorescent dye molecules to obtain the fluorescent probe based on the small molecule inhibitor.
4. The method for preparing a fluorescent probe based on a small molecule inhibitor as claimed in claim 3, wherein the reaction solvent in the first step is N, N-dimethylformamide, methanol, acetonitrile or tetrahydrofuran.
5. The method for preparing a fluorescent probe based on a small molecule inhibitor as claimed in claim 3, wherein the reaction temperature in the first step is 20-120 ℃ and the reaction time is 1-16 hours.
6. The method for preparing a fluorescent probe based on a small molecule inhibitor as claimed in claim 3, wherein the molar ratio of the derivative of the small molecule inhibitor to the linker molecule in the second step is 1: 1.1-1: 3.
7. The method for preparing a fluorescent probe based on a small molecule inhibitor according to claim 3, wherein the reaction solvent in the second step is N, N-dimethylformamide, acetonitrile or tetrahydrofuran, the reaction temperature is 50-120 ℃, and the reaction time is 10-24 hours.
8. The method for preparing the fluorescent probe based on the small molecule inhibitor according to claim 3, wherein the reaction temperature in the third step is room temperature, the reaction solvent is N, N-dimethylformamide, dimethyl sulfoxide, PBS or water, and the reaction time is 2-24 hours.
9. The method for preparing the fluorescent probe based on the small molecule inhibitor as claimed in claim 3, wherein the molar ratio of the product II in the step III to the fluorescent dye molecules is 1: 1.1-1: 2.
10. Use of the small molecule inhibitor-based fluorescent probe of claim 1 or 2 for super-resolution imaging.
Technical Field
The invention belongs to the field of small molecule labeled probes for fluorescence imaging, and particularly relates to a small molecule inhibitor-based fluorescent probe and a preparation method and application thereof.
Background
The characterization of cellular structures and biomolecules with high spatiotemporal resolution is a prerequisite for the study of cell biology at the molecular level. Fluorescence microscopy with good sensitivity and resolution has been the method of investigation of choice to date, but is limited by the diffraction limit of 200 nm of conventional fluorescence microscopy and is not sufficient to resolve most biological structures. In recent years, with the rapid development of super-resolution fluorescence imaging technology, such as a super-resolution microscope (SMLM), a structured light microscope (SIM), a stimulated emission depletion microscope (STED) and the like based on single-molecule positioning, the limit of resolution limit of the conventional fluorescence imaging technology has been greatly broken through, and the technology gradually becomes an important research means in the field of biochemistry.
For high-quality fluorescence imaging, in addition to advanced imaging techniques, precise fluorescence labeling is often required for realization, and therefore, the selection of a fluorescent probe with high efficiency and high specificity is particularly important. Currently, photoactivatable or photoconvertable fluorescent dyes or fluorescent proteins are the most commonly used fluorescent probes for super-resolution imaging techniques. The fluorescent dye has the characteristics of small size, high brightness, easy modification and the like, but the fluorescent dye does not have targeting property, cannot specifically mark a target object, and usually needs a primary antibody or a secondary antibody to obtain high marking specificity. In 2012, the Heilemann topic group labeled gp210 protein by means of antibodies successfully achieved super-resolution imaging of gp210 protein around the nuclear pore complex, and eight-fold symmetry of gp210 protein was observed for the first time (j.cell.sci,2012,125, 570-575). The group of our subjects also achieved dstormm imaging of proteins such as Band3, EGFR, GLUT1 and the like on the surface of cell membranes using various antibody probes, and further studied the assembly rule and distribution characteristics of these proteins on the surface of cell membranes (Nanoscale,2013,5, 11582; sci.rep-UK,2015,5, 9045; pro.natl.acad.sci.u.s.a.,2018,115,7033.). Fluorescent proteins with high specificity are also widely used in the field of super-resolution imaging. In 2008, by combining PALM and single particle tracer techniques, the Schwartz group used fluorescent proteins to label GAG and VSVG proteins in cell membranes, and obtained higher order single cell movement trajectories (NatureMethods,2008,5, 155-. The fluorescent labeling of specific proteins is realized by the reaction of protein residues and specific fluorescent dyes through the introduction of specific protein tags (such as Halo-tag, SNAP-tag and the like) (Nat. chem.2013,5, 132-139; ACS chem.biol.2008,3, 373-382). The nano antibody has smaller volume, and is more favorable for accurately marking target protein. In 2012, the Ewers topic group developed a general and efficient method for labeling nanobodies, which achieved more precise fluorescent labeling of target proteins by combining nanobodies with fluorescent proteins (Nature Methods,2012,9, 582-. Nucleic acid aptamers that have emerged in recent years have received attention as having a relatively small volume due to their high labeling specificity. In 2012, the application of aptamer labeling technology in the super-resolution field was first reported by the Rizzoli group, and the super-resolution imaging of Transferrin (TRF), growth factor receptor (EGFR) was successfully achieved (Nature Methods,2012,9, 938-.
Although antibody, fluorescent protein, nanobody and aptamer labeling have their own advantages, these labeling methods have their own labeling disadvantages. The antibody has large volume, and has the defects of multivalent crosslinking, incomplete labeling, large connection error and the like in the labeling process, so that the antibody labeling often causes a labeling false image and can not reflect the real expression of the protein. Compared with antibodies, the fluorescent protein and the protein label have higher labeling specificity, and can be realized by means of a gene editing technology, so that the real expression and function of the target protein are often damaged in the expression process of the fluorescent protein and the protein label. Although the nano antibody with smaller size has advantages in volume, like fluorescent protein, the expression is controlled by means of gene editing technology, so that the unreal expression of target protein is caused, the real function of the protein is influenced, and meanwhile, the screening of the nano antibody is strict and complicated, so that the wide application of the nano antibody in the field of super-resolution imaging is further limited. The aptamer labeling technology which has emerged in recent years is easier to operate in screening, high specificity is difficult to guarantee because the technology cannot depend on mature animal immune systems, and only limited target proteins can be labeled by using aptamers at present.
The small size, high specificity and no interference on the expression and function of the target protein are the precondition for realizing accurate fluorescence labeling, so that the finding of a labeling method with smaller size, wide application and high labeling specificity is the key for improving the accuracy of super-resolution imaging.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art, and provides a fluorescent probe based on a small molecular inhibitor and a preparation method and application thereof based on the specific recognition of the small molecular inhibitor on a specific target protein.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides a fluorescent probe based on a small molecular inhibitor, which has a structure shown in a formula 1:
in formula 1:
represents a small molecule inhibitor, including but not limited to gefitinib, erlotinib, taliquinad, GDC-0941, A-674563 or AZD-805, etc., preferably gefitinib or taliquinad;
the length-adjustable linking molecules are represented, and include but are not limited to flexible PEG chains, saturated carbon chains, unsaturated carbon chains and the like, preferably flexible PEG chains, wherein the chain length of the PEG chains is preferably 3 glycol molecule chain lengths, and the lengths of the PEG chains can be adjusted;
representative fluorescent dye molecules include, but are not limited to, Alexa532, Alexa647, Cy3, Cy5, Cy5.5, Cy7, Atto532, Atto488, or TAMRA, and the like, preferably Alexa532 or TAMRA.
In the above technical solution, it is further preferable that: the structure of the fluorescent probe based on the small molecule inhibitor is shown as a formula 2 or a formula 3:
the invention also provides a preparation method of the fluorescent probe based on the small molecule inhibitor, which comprises the following steps:
the method comprises the following steps: carrying out derivative modification on the small molecule inhibitor to obtain a derivative of the small molecule inhibitor;
step two: connecting the derivative of the small molecule inhibitor obtained in the step one with a linking molecule to obtain a product II;
step three: and (5) reacting the product II obtained in the step II with fluorescent dye molecules to obtain the fluorescent probe based on the small molecule inhibitor.
In the above technical solution, preferably, the reaction solvent in the step one is N, N-dimethylformamide, methanol, acetonitrile or tetrahydrofuran.
In the above technical scheme, preferably, the reaction temperature in the first step is 20 to 120 ℃, and the reaction time is 1 to 16 hours.
In the above technical solution, preferably, the molar ratio of the derivative of the small molecule inhibitor to the linker molecule in the second step is 1: 1.1-1: 3.
In the above technical scheme, preferably, the reaction solvent in the second step is N, N-dimethylformamide, acetonitrile, or tetrahydrofuran, the reaction temperature is 50 to 120 ℃, and the reaction time is 10 to 24 hours.
In the above technical solution, preferably, the reaction temperature in the third step is room temperature, the reaction solvent is N, N-dimethylformamide, dimethyl sulfoxide, PBS or water, and the reaction time is 2-24 hours.
In the above technical scheme, preferably, the molar ratio of the product two in the step three to the fluorescent dye molecules is 1: 1.1-1: 2.
The invention also provides application of the fluorescent probe based on the small molecule inhibitor in super-resolution imaging.
The invention has the beneficial effects that:
the invention selects the small molecule inhibitor with specific target as the research object, the suitable site of the small molecule inhibitor is derivative modified by the synthesis means, and the small molecule inhibitor is connected with the fluorescent dye molecule by the flexible PEG chain or other linking molecules to construct a novel small molecule fluorescent probe which is used for the specific marking of cell protein, thereby carrying out the corresponding super-resolution imaging research. Compared with the traditional fluorescent protein and antibody probes, the fluorescent probe based on the small molecule inhibitor has the following advantages: firstly, the molecular weight and the volume of the small molecular probe are small, the labeling density of the corresponding protein is higher and accurate, and the antibody cannot accurately label each target due to the self volume; secondly, each small molecule inhibitor is combined with the corresponding protein one to one, so that a more real distribution form of the protein can be represented, and the real expression and function of the target protein are usually influenced by the expression of the common fluorescent protein through a gene editing technology. Thirdly, the chemoselectivity of the synthesis reaction ensures that the small molecule inhibitor binds to the dye molecule 1:1, which more accurately shows the distribution of the target protein than an indeterminate number of dye-linked antibodies; the preparation method of the fluorescent probe based on the small molecular inhibitor is simple and convenient, short in preparation time and easy in quality control, and can be widely applied to research on distribution, positioning and assembly of corresponding proteins. The fluorescent probe based on the small-molecule inhibitor is particularly suitable for super-resolution imaging.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a schematic diagram of the preparation of a small molecule inhibitor-based fluorescent probe of the present invention;
FIG. 2 is an assembly distribution diagram of EGFR on A549 cell membrane marked by Gefitinib small molecule probe Gefitinib-Alexa 532, and the scale is 5 microns;
FIG. 3 is a superimposed graph of co-localization distribution of EGFR on A549 cell membrane marked by Gefitinib small molecule probe Gefitinib-Alexa 532 and antibodyy-Alexa 647 in two-color super-resolution fluorescence imaging, and the scale is 5 microns;
FIG. 4 is a graph showing the distribution of the assembly of the P-glycoprotein on the cell membrane of Tariquidar-TAMRA labeled MCF-10A with a scale of 5 μm.
FIG. 5 is a graph showing the distribution of the assembly of the P-glycoprotein on the cell membrane of the Tariquidar-TAMRA marker 231 of the small molecule probe tariquida, with a scale of 5 μm.
Detailed Description
The invention provides a fluorescent probe based on a small molecular inhibitor, which has a structure shown in a formula 1:
in the formula 1, the reaction mixture is,represents a small molecule inhibitor, including but not limited to gefitinib, erlotinib, tacrine, GDC-0941, A-674563, AZD-805, or the like. Preferably gefitinib or tacrine.
Representative fluorescent dye molecules include, but are not limited to, Alexa532, Alexa647, Cy3, Cy5, Cy5.5, Cy7, Atto532, Atto488, or TAMRA, among others. Alexa532 or TAMRA is preferred.
According to the present invention, most preferably, the structure of the fluorescent probe based on the small molecule inhibitor is represented by formula 2 or formula 3:
the invention also provides a preparation method of the fluorescent probe based on the small molecule inhibitor, which comprises the following steps as shown in figure 1:
the method comprises the following steps: carrying out derivative modification on the small molecule inhibitor to obtain a derivative of the small molecule inhibitor;
step two: connecting the derivative of the small molecule inhibitor obtained in the step one with a linking molecule to obtain a product II;
step three: and (5) reacting the product II obtained in the step II with fluorescent dye molecules to obtain the fluorescent probe based on the small molecule inhibitor.
According to the invention, firstly, modification is carried out on proper sites of the small molecular inhibitor, the modification process is a multi-step reaction, conditions are slightly different among different reactions, preferably, a reaction solvent is N, N-dimethylformamide, acetonitrile, methanol or tetrahydrofuran, the reaction temperature is preferably 20-120 ℃, and the reaction time is 1-16 hours; secondly, connecting the obtained derivative of the small molecular inhibitor with a linking molecule to obtain a product II, preferably, the molar ratio of the derivative of the small molecular inhibitor to the linking molecule is 1: 1.1-1: 3, the reaction solvent is N, N-dimethylformamide, acetonitrile or tetrahydrofuran, the reaction temperature is preferably 50-120 ℃, and the reaction time is 10-24 hours; and finally, reacting the obtained product II with fluorescent dye molecules to obtain the fluorescent probe based on the small molecule inhibitor, wherein the preferable reaction solvent is N, N-dimethylformamide, dimethyl sulfoxide, PBS or water, the reaction temperature is room temperature, the molar ratio of the product II to the fluorescent dye molecules is 1: 1.1-1: 2, and the reaction time is 2-24 hours.
According to the invention, the product II obtained in the step II is reacted with fluorescent dye molecules, and the reaction needs to be determined according to the molecular groups at the two ends of the product. When the molecular terminal of the product II is an amino group, directly carrying out nucleophilic substitution reaction on the product II and the active ester of the fluorescent dye molecule, wherein the substitution reaction is preferably carried out in a reaction solvent, the reaction solvent is preferably N, N-dimethylformamide, dimethyl sulfoxide, PBS or water, an alkali is required to be added in the reaction process, the alkali is preferably triethylamine or diisopropylethylamine, the temperature of the substitution reaction is preferably room temperature and dark, and the reaction time is preferably 2-24 hours;
when the molecular tail end of the product II is an azide group, a click chemistry method can be selected to react with a dye molecule with an alkynyl group to obtain a required small molecular probe, the reaction temperature is preferably room temperature, and the reaction time is preferably 2-24 hours;
when the molecular terminal of the product II is an alkynyl group, a click chemistry method can be selected to react with a dye molecule with an azide group to obtain a required small molecular probe, the reaction temperature is preferably room temperature, and the reaction time is preferably 2-24 hours;
when the molecular end of the product II is a sulfydryl group, the product II can be selected to perform substitution reaction with dye molecules with maleic anhydride groups to obtain a required small molecular probe, the reaction temperature is preferably room temperature, and the reaction time is preferably 2-24 hours;
the present invention is described in further detail below with reference to specific examples, in which the starting materials are all commercially available.