Biotinylation modified amino acid probe, preparation method and application thereof, and method and application for identifying amino acid binding protein

Abstract

The invention relates to a biotinylation modified amino acid probe, a preparation method and application thereof, and a method and application for identifying amino acid binding protein. The amino acid probe is amino acid with biotin modification at an amino terminal or a carboxyl terminal; or, the amino acid probe is amino acid with an azide group at the carboxyl end, the amino acid probe reacts with alkynyl-containing biotin under the catalysis of copper ions through a click chemical reaction in a cell to generate biotin-modified amino acid, an azide-modified amino acid probe library is supplemented, and the amino acid probe is combined with immunoprecipitation and mass spectrometry technologies, so that the biotin-modified amino acid is obtained. Proteins capable of being directly combined with amino acids can be identified, an important tool is provided for follow-up research of amino acid sensing proteins, and the method has important application value in identification of amino acid binding proteins, research of amino acid sensing mechanisms and functions and determination of drug targets or screening of lead compounds.

Description

Technical Field

[0001] The present invention relates to the field of amino acid sensing technology, in particular to the field of amino acid probe technology, and specifically to a biotinylated amino acid probe and a preparation method and application thereof, and a method and application for identifying amino acid binding proteins. Background Art

[0002] The construction and application of amino acid probes is an active research field, especially biosensors developed based on fluorescence resonance energy transfer (FRET) technology. These probes can detect amino acid concentrations inside and outside cells in real time and dynamically, which is crucial for understanding the role of amino acids in physiological and pathological processes.

[0003] The construction principle of existing amino acid fluorescent probes: Amino acid probes usually consist of two parts: one is a protein that can bind to specific amino acids (such as amino acid binding protein), and the other is a fluorescent protein (such as green fluorescent protein GFP, yellow fluorescent protein YFP, etc.). When amino acids bind to binding proteins, they cause changes in the conformation of fluorescent proteins, thereby changing the intensity or wavelength of the fluorescent signal. This change can be used to quantitatively analyze the concentration of amino acids in samples.

[0004] Amino acid fluorescent probes are widely used in many fields: they are used to study the role and metabolic process of amino acids in cells; in drug development, they are used for high-throughput screening of compounds that can affect amino acid metabolism; in disease research, they are used to study diseases related to amino acid metabolism, such as cancer, diabetes, etc.; in nutrition, they are used to evaluate the amino acid content in different foods or supplements.

[0005] At present, some amino acid probes have been developed, and biotin-labeled nucleic acid probes and small molecule probes have appeared in the prior art. Biotin probes are molecular tools that use the high affinity binding properties between biotin and avidin or streptavidin. Such probes are widely used in biochemical and molecular biological research, especially in the detection, localization and quantitative analysis of nucleic acids and proteins. The following are some key applications and characteristics of biotin probes.

[0006] Molecular hybridization technology: Biotin-labeled nucleic acid probes can be used to detect complementary nucleic acid sequences. By hybridizing with nucleic acid molecules fixed on the filter membrane and then detecting using an avidin-enzyme complex, high-sensitivity and specific detection of specific DNA or RNA sequences can be achieved.

[0007] In situ hybridization: Biotin-labeled nucleic acid probes can be used for in situ hybridization of tissue sections or cells to directly detect specific DNA sequences in tissue sections, single cells or chromosomes fixed on slides.

[0008] Gene localization and sequence analysis: Biotin probes can be used for gene cloning, gene localization and nucleic acid sequence analysis, such as chemical degradation method or terminal termination method for nucleic acid sequence analysis.

[0009] Purification of proteins and nucleic acids: Biotin-labeled oligonucleotides or antibodies can be used for affinity purification through avidin-agarose columns to enrich specific DNA fragments or proteins.

[0010] Immunodetection: Biotin-labeled antibodies can be used in techniques such as immunohistochemistry, immunoblotting, and immunofluorescence microscopy to detect and localize the target protein by binding to avidin-labeled detection reagents.

[0011] Photosensitive biotin probes: These probes combine a photosensitive group with biotin and can undergo cross-linking reactions with nearby molecules under light conditions. They are used to study protein-protein interactions, cell and tissue analysis, and protein purification and capture.

[0012] Fluorescent Labeling: Fluorescent biotin derivatives can be used for fluorescence detection and quantification of biotin-binding proteins, and the results can be observed by fluorescence microscopy, providing information about the localization of the molecule.

[0013] Small molecule fluorescent probes: In drug analysis and precision medicine, small molecule fluorescent probes are used to analyze drug-target interactions and promote the precision and personalization of drug development, disease diagnosis, and treatment monitoring.

[0014] Biotin probes have the advantages of high sensitivity, high specificity, high resolution and easy operation, which make biotin probes an indispensable tool in biomedical research.

[0015] Currently known amino acid probes are mainly limited to the detection of amino acid concentrations in cells or in vivo, such as the amino acid probes that have been used, such as cysteine ​​probes, glutamate and aspartate probes, arginine probes and tryptophan probes. Although these amino acid probes can achieve specific, high-responsive and ultra-sensitive monitoring of the metabolism of corresponding amino acids in living cells, tissues, living bodies and clinical samples. However, for example, how to capture proteins that can directly sense changes in amino acid abundance without bias and high throughput still faces challenges.

[0016] The significance of amino acid sensing research is multifaceted. It involves how organisms perceive and respond to changes in amino acid levels, and how these changes affect metabolic processes and health status. It is of great significance for understanding the metabolic regulation mechanism of organisms, developing new treatments, and improving biomanufacturing efficiency. At present, in the field of amino acid sensing research, there is still a lack of probes for global analysis of amino acid sensing proteins. There is an urgent need to develop amino acid probes combined with active proteomics to explore new drug targets and lead compounds. Summary of the invention

[0017] The purpose of the present invention is to address at least one of the deficiencies in the prior art and to provide a biotinylated amino acid probe and a preparation method and application thereof, as well as a method and application for identifying amino acid binding proteins, so as to solve the problem of the lack of amino acid probes for global analysis of amino acid sensing proteins in the related art.

[0018] To achieve the above object, the technical solution adopted by the present invention is:

[0019] The first aspect of the present invention is to provide a biotinylated amino acid probe, wherein the amino acid probe is an amino acid modified with biotin at the amino or carboxyl terminus; or, the amino acid probe is an amino acid with an azide group at the carboxyl terminus, which reacts with alkyne-containing biotin under the catalysis of copper ions through a click chemistry reaction in cells to generate a biotin-modified amino acid.

[0020] Furthermore, the structure of the amino acid with biotin modification at the amino or carboxyl terminus is selected from at least one of the following:

[0021]

[0022] Furthermore, the structure of the amino acid with an azide group at the carboxyl end is selected from at least one of the following:

[0023]

[0024] The second aspect of the present invention is to provide a method for preparing a biotinylated amino acid probe, the method comprising the following steps: reacting a compound of formula 1 with a compound of formula 2, formula 3 or formula 4 in a first solvent system, and removing the first solvent after the reaction; reacting the reaction product in a second solvent system, and removing the second solvent after the reaction, separating and purifying to obtain a biotinylated amino acid probe of formula I, formula II or formula III, respectively;

[0025] Wherein, the first solvent system is dichloromethane and N, N-diisopropylethylamine; the second solvent system is dichloromethane and trifluoroacetic acid;

[0026] The structures of the compounds of Formula 1, Formula 2, Formula 3 and Formula 4 are shown below:

[0027]

[0028] The structures shown in Formula I, Formula II and Formula III are as follows:

[0029]

[0030] Furthermore, in the first solvent system, the mass ratio of the compound of formula 1 to the compound of formula 2, formula 3 or formula 4 is 1:0.5-2, the amount of the compound of formula 1 and dichloromethane and N,N-diisopropylethylamine is (40-150) mg: (5-10) mL: (40-100) μL, and the reaction conditions are: reaction at room temperature for 6-24 hours.

[0031] Furthermore, in the first solvent system, the mass ratio of the compound of formula 1 to the compound of formula 2, formula 3 or formula 4 is 1:1 to 1.3.

[0032] Furthermore, in the first solvent system, the amount of the compound of formula 1, dichloromethane and N,N-diisopropylethylamine is (50-100) mg: (5-10) mL: (45-92) μL, and the reaction conditions are: reaction at room temperature for 8-24 hours.

[0033] Furthermore, in the second solvent system, the volume ratio of dichloromethane to trifluoroacetic acid is 0.5-2:1, and the reaction conditions are room temperature for 2-4 hours.

[0034] Furthermore, the volume ratio of dichloromethane to trifluoroacetic acid is 1:1.

[0035] In a specific embodiment, the compound Biotin-Val-NH 2 The specific synthesis steps include: the compound of formula 1 (100 mg, 0.35 mmol, 1 eq) is dissolved in 10 mL of dichloromethane, and the compound of formula 2 (121 mg, 0.39 mmol, 1.1 eq) and 92 μL of N, N-diisopropylethylamine (DIPEA) are then dissolved in the above system and reacted at room temperature for 8 hours. After the reaction is completed, the solvent is removed by rotary evaporation, washed twice with dichloromethane and saturated sodium bicarbonate solution, and the organic phase is dried and filtered and then rotary evaporated to remove the solvent. Then 0.5 mL of dichloromethane and 0.5 mL of trifluoroacetic acid are added and reacted at room temperature for 3 hours. After the reaction is completed, the solvent is removed by rotary evaporation, and after the sodium bicarbonate solution is neutralized, it is extracted multiple times with a mixed solvent of chloroform and isopropanol, the organic phase is rotary evaporated to remove the solvent, and then separated and purified with flash to finally obtain Biotin-Val-NH 2 ;

[0036] Amino acid probe Biotin-Leu-NH2 The specific synthesis steps include: the compound of formula 1 (50mg, 0.17mmol, 1eq) is dissolved in 5mL of dichloromethane, and the compound of formula 3 (63mg, 0.19mmol, 1.1eq) and 45μL N, N-diisopropylethylamine (DIPEA) are then dissolved in the above system, and the reaction is carried out at room temperature overnight. After the reaction is completed, the solvent is removed by rotary evaporation, and the organic phase is washed twice with dichloromethane and saturated sodium bicarbonate solution. The organic phase is dried and filtered and then the solvent is removed by rotary evaporation. Then 0.5mL of dichloromethane and 0.5mL of trifluoroacetic acid are added and reacted at room temperature for 2h. After the reaction is completed, the solvent is removed by rotary evaporation, and after the sodium bicarbonate solution is neutralized, it is extracted several times with a mixed solvent of chloroform and isopropanol, the organic phase is rotary evaporated to remove the solvent, and then separated and purified with flash, and finally a white solid compound Biotin-Leu-NH is obtained. 2 ;

[0037] Amino acid probe Biotin-Ile-NH 2 The specific synthesis steps include: the compound of formula 1 (100 mg, 0.35 mmol, 1 eq) is dissolved in 5 mL of dichloromethane, and the compound of formula 4 (128 mg, 0.39 mmol, 1.1 eq) and 92 μL of N, N-diisopropylethylamine (DIPEA) are then dissolved in the above system and reacted at room temperature overnight. After the reaction is completed, the solvent is removed by rotary evaporation, washed twice with dichloromethane and saturated sodium bicarbonate solution, and the organic phase is dried and filtered and then rotary evaporated to remove the solvent. Then 0.5 mL of dichloromethane and 0.5 mL of trifluoroacetic acid are added and reacted at room temperature for 4 hours. After the reaction is completed, the solvent is removed by rotary evaporation, and after the sodium bicarbonate solution is neutralized, it is extracted several times with a mixed solvent of chloroform and isopropanol, the organic phase is rotary evaporated to remove the solvent, and then separated and purified with flash to finally obtain a white solid compound Biotin-Ile-NH 2 .

[0038] The third aspect of the present invention is to provide a method for identifying amino acid binding proteins, the method using any biotinylated amino acid probe as described in the first aspect;

[0039] Wherein, when the amino acid probe is an amino acid modified with biotin at the amino or carboxyl terminus, the protein that can directly bind to the amino acid is identified by an in vitro biotin pull-down experiment combined with mass spectrometry detection;

[0040] When the amino acid probe is an amino acid with an azide group at the carboxyl end, the protein bound to the amino acid is obtained by immunoprecipitation technology through the high affinity binding characteristics between biotin and streptavidin, and the protein that can directly bind to the amino acid is identified by combining mass spectrometry detection.

[0041] Furthermore, when the amino acid probe is an amino acid modified with biotin at the amino terminus or carboxyl terminus, the method specifically includes the steps of: subjecting the cells to corresponding amino acid starvation treatment, lysing the cells with a lysis solution, reacting the biotinylated amino acid probe incubated on streptavidin agarose beads with the cell lysis solution overnight, and obtaining a protein bound to the amino acid through an in vitro pulldown experiment; obtaining peptides from the obtained protein through proteolysis, and identifying the protein that can directly bind to the amino acid through a mass spectrometry detection method.

[0042] Furthermore, when the amino acid probe is an amino acid with an azide group at the carboxyl end, the method specifically includes the steps of: subjecting the cells to corresponding amino acid starvation treatment, treating them with an azide-modified amino acid probe and UV cross-linking, lysing the cells with a lysis solution, subjecting the lysis solution to a click chemistry reaction with alkyne-containing biotin, and enriching the reaction product with streptavidin agarose beads to obtain amino acid-bound proteins; obtaining peptides by proteolysis of the obtained proteins, and identifying proteins that can directly bind to amino acids by mass spectrometry detection.

[0043] The fourth aspect of the present invention is to provide an application of a biotinylated amino acid probe as described in any one of the first aspects or a method as described in any one of the third aspects, wherein the application is selected from one of the following applications: application in identifying amino acid binding proteins, application in studying amino acid sensing mechanisms and their functions, application in determining drug targets or screening lead compounds.

[0044] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0045] The present invention uses direct chemical synthesis to modify the amino or hydroxyl ends of 21 kinds of amino acids with biotin to synthesize biotin-modified amino acid probes, and then can identify proteins that can directly bind to amino acids through in vitro biotin pull-down experiments combined with mass spectrometry detection; and through chemical synthesis, the carboxyl end of the amino acid is modified with an azide group, and the azide group and alkyne-containing biotin are reacted in the cell under the catalysis of copper ions through a click chemistry reaction to generate biotin-modified amino acids, and the high-affinity binding properties between biotin and streptavidin are used to obtain proteins bound to amino acids using immunoprecipitation technology, and combined with mass spectrometry technology, proteins that can directly bind to amino acids are identified. The present invention provides a very comprehensive and effective tool for systematically identifying the sensor proteins of various amino acids by formulating biotinylated amino acid probes, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0047] Figure 1 It is a diagram of the experimental steps and experimental results of using a biotin-modified amino acid probe to identify proteins bound to amino acids in one embodiment of the present invention;

[0048] Figure 2 Schematic diagram of the mass spectrometry analysis experimental steps using azide-modified valine and leucine probes in one embodiment of the present invention;

[0049] Figure 3 This is a WB result diagram of valine and leucine binding proteins after treatment with azide-modified valine and leucine probes in one embodiment of the present invention;

[0050] Figure 4 This is a mass spectrometry result of a valine click chemistry reaction after treatment with an azide-modified valine probe and binding to a protein in one embodiment of the present invention;

[0051] Figure 5 This is a graph showing the mass spectrometry results of a leucine click chemistry reaction after an azide-modified leucine probe is treated with the binding protein in one embodiment of the present invention. DETAILED DESCRIPTION

[0052] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0053] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0054] The biotin-alkyne group of the present invention was purchased from APExBIO. Other biotin-alkyne groups not specifically marked with a manufacturer in the examples can be obtained through conventional commercial purchases.

[0055] The present invention is described below by way of example.

[0056] Example 1-Synthesis of biotinylated amino acid probe

[0057] This example relates to the synthesis of a preferred biotinylated amino acid probe.

[0058] The following is a detailed description of the amino acid probe Biotin-Val-NH 2 , amino acid probe Biotin-Leu-NH 2 and amino acid probe Biotin-Ile-NH 2 The synthetic route and steps.

[0059] (I) Amino acid probe Biotin-Val-NH 2 Synthesis

[0060] 1. Amino acid probe Biotin-Val-NH 2 The synthetic route is as follows:

[0061]

[0062] 2. Amino acid probe Biotin-Val-NH 2 The specific synthesis steps include: compound 1 (100 mg, 0.35 mmol, 1 eq) is dissolved in 10 mL of dichloromethane, and compound 2 (121 mg, 0.39 mmol, 1.1 eq) and 92 μL of N, N-diisopropylethylamine (DIPEA) are then dissolved in the above system and reacted at room temperature for 8 hours. After the reaction is completed, the solvent is removed by rotary evaporation, and the organic phase is washed twice with dichloromethane and saturated sodium bicarbonate solution. The organic phase is dried and filtered, and the solvent is removed by rotary evaporation. Then 0.5 mL of dichloromethane and 0.5 mL of trifluoroacetic acid are added and reacted at room temperature for 3 hours. After the reaction is completed, the solvent is removed by rotary evaporation, and after the sodium bicarbonate solution is neutralized, it is extracted several times with a mixed solvent of chloroform and isopropanol, the organic phase is rotary evaporated to remove the solvent, and then separated and purified by flash, and finally a white solid compound Biotin-Val-NH is obtained. 2 .

[0063] 3. The NMR data are as follows:

[0064] 1 H NMR (400 MHz, MeOD-d 4)δ4.50 (dd, J=7.8, 4.6Hz, 1H), 4.31 (dd, J=7.8, 4.5Hz, 1H), 3.61 (d, J=5.9Hz, 1H), 3.37-3.36 (brs, 2H), 3.27-3.17 (m, 2H), 2.93 (dd, J=12.8, 4 .96Hz, 1H), 2.71 (d, J=12.7Hz, 1H), 2.24-2.12 (m, 3H), 1.77-1.54 (m, 4H), 1.48-1.40 (m, 2H), 1.04 (dd, J=6.7, 5.3Hz, 6H); HRMS (ESI)calcd.for C 17 H 31 N 5 O 3 S[M+H] + 386.527, found:386.5.

[0065] (ii) Amino acid probe Biotin-Leu-NH 2 Synthesis

[0066] 1. Amino acid probe Biotin-Leu-NH 2 The synthetic route is as follows:

[0067]

[0068] 2. Amino acid probe Biotin-Leu-NH 2 The specific synthesis steps include: compound 1 (50 mg, 0.17 mmol, 1 eq) is dissolved in 5 mL of dichloromethane, and compound 3 (63 mg, 0.19 mmol, 1.1 eq) and 45 μL N, N-diisopropylethylamine (DIPEA) are then dissolved in the above system and reacted at room temperature overnight. After the reaction is completed, the solvent is removed by rotary evaporation, washed twice with dichloromethane and saturated sodium bicarbonate solution, and the organic phase is dried and filtered and then rotary evaporated to remove the solvent. Then 0.5 mL of dichloromethane and 0.5 mL of trifluoroacetic acid are added to react at room temperature for 2 hours. After the reaction is completed, the solvent is removed by rotary evaporation, and after the sodium bicarbonate solution is neutralized, it is extracted several times with a mixed solvent of chloroform and isopropanol, the organic phase is rotary evaporated to remove the solvent, and then separated and purified by flash, and finally a white solid compound Biotin-Leu-NH is obtained. 2 .

[0069] 3. The NMR data are as follows:

[0070] 1 H NMR (400 MHz, MeOD-d 4)δ4.49 (dd, J=7.8, 4.9Hz, 1H), 4.30 (dd, J=7.9, 4.5Hz, 1H), 3.36-3.34 (m, 1H), 3.23-3.19 (m, 1H), 2.93 (dd, J=12.7, 5.0Hz, 1H), 2.71 (d, J=12.7Hz, 1H), 2.22 (t, 2H), 1.77-1.51 (m, 6H), 1.48-1.34 (m, 3H), 0.95 (dd, J=8.6, 6.6Hz, 6H).

[0071] (III) Amino acid probe Biotin-Ile-NH 2 Synthesis

[0072] 1. Amino acid probe Biotin-Ile-NH 2 The synthetic route is as follows:

[0073]

[0074] 2. Amino acid probe Biotin-Ile-NH 2 The specific synthesis steps include: compound 1 (100 mg, 0.35 mmol, 1 eq) is dissolved in 5 mL of dichloromethane, and compound 4 (128 mg, 0.39 mmol, 1.1 eq) and 92 μL N, N-diisopropylethylamine (DIPEA) are then dissolved in the above system and reacted at room temperature overnight. After the reaction is completed, the solvent is removed by rotary evaporation, washed twice with dichloromethane and saturated sodium bicarbonate solution, and the organic phase is dried and filtered and then rotary evaporated to remove the solvent. Then 0.5 mL of dichloromethane and 0.5 mL of trifluoroacetic acid are added and reacted at room temperature for 4 hours. After the reaction is completed, the solvent is removed by rotary evaporation, and after the sodium bicarbonate solution is neutralized, it is extracted several times with a mixed solvent of chloroform and isopropanol, the organic phase is rotary evaporated to remove the solvent, and then separated and purified by flash, and finally a white solid compound Biotin-Ile-NH is obtained. 2 .

[0075] 3. The NMR data are as follows:

[0076] 1 H NMR (400 MHz, MeOD-d 4)δ4.49 (dd, J=7.8, 5.1Hz, 1H), 4.30 (dd, J=7.9, 4.4Hz, 1H), 3.60 (q, J=7.0 Hz, 1H), 3.23-3.19 (m, 1H), 3.12 (d, J=5.9Hz, 1H), 2.92 (dd, J=12.7, 4.9Hz, 1H), 2.71 (d, J=12.7Hz, 1H), 2.21 (t, J=7.4Hz, 2H), 1.77-1.57 (m, 5H), 1.55 -1.40(m, 3H), 1.20-1.11(m, 2H), 0.93(d, J=7.2, 6H).HRMS(ESI)calcd.for C 18 H 33 N 5 O 3 S[M+H] + 400.554, found:400.4.

[0077] In this embodiment, the amino or hydroxyl ends of 21 amino acids, including alanine (Ala); arginine (Arg); aspartic acid (Asp); glutamic acid (Glu); phenylalanine (Phe); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); valine (Val); cysteine ​​(Cys); proline (Pro); asparagine (Asn); glutamine (Gln); and selenocysteine ​​(Sec), were modified with biotin at the amino or hydroxyl ends to obtain the corresponding amino acid probes, including: Biotin-Val-NH 2 , Biotin-Leu-NH 2 , Biotin-Ile-NH 2 , Biotin-Arg-OH, Biotin-His-OH, Biotin-Lys-OH, Biotin-Asp-OH, Biotin-Glu-OH, Biotin-Ser-OH, Biotin-Thr-OH, Biotin-Asn-OH, Biotin-Gln-OH, Biotin-Cys-OH, Biotin -Sec-OH, Biotin-Gly-OH, Biotin-Pro-OH, Biotin-Ala-OH, Biotin-Met-OH, Biotin-Phe-OH, Biotin-Trp-OH, Biotin-Tyr-OH, Biotin-Val-OH, Biotin-Leu-OH, Biotin-Ile-OH.

[0078] The structural formula corresponding to the above amino acid probe is shown below.

[0079]

[0080] Example 2-Amino acid probe with azide group

[0081] In this example, an azide group is added to the carboxyl end of an amino acid by chemical synthesis, and the azide group is reacted with alkyne-containing biotin by a click chemistry reaction in cells under the catalysis of copper ions to generate a biotin-modified amino acid.

[0082] The amino acid probe with an azide group includes: N 3 -Val-NH 2 , N 3 -Leu-NH 2 , N 3 -Ile-NH 2 , N 3 -Arg-NH 2 , N 3 -His-NH 2 , N 3 -Asp-NH 2 , N 3 -Glu-NH 2 , N 3 -Ser-NH 2 , N 3 -Thr-NH 2 , N 3 -Asn-NH 2 , N 3 -Cys-NH 2 , N 3 -Sec-NH 2 , N 3 -Gly-NH 2 , N 3 -Pro-NH 2 , N 3 -Ala-NH 2 , N 3 -Phe-NH 2 , N 3 -Trp-NH 2 , N 3 -Tyr-NH 2 , N 2 -Val-NH 2 , N 2 -Leu-NH 2 , N 3 -Lys-NH 2 , N3 -Gln-NH 2 , N 3 -Met-NH 2 , N 2 -Ile-NH 2 .

[0083] The structural formula corresponding to the above amino acid probe with an azide group is shown below:

[0084]

[0085]

[0086] Example 3 - Application of biotinylated amino acid probes

[0087] In this example, the biotin-modified amino acid probe synthesized in Example 1 was selected as the amino acid probe Biotin-Val-NH 2 、Biotin-Leu-NH 2 and Biotin-Ile-NH 2 , and selected the colorectal cancer cell line HCT116 (purchased from the cell bank of the Chinese Academy of Sciences) for the experiment, which specifically includes the following steps:

[0088] Before collecting cell samples, amino acid starvation treatment is performed on the amino acids of the corresponding probes (for example, the biotinylated valine group is bound to the culture medium lacking valine for 6 hours in advance). The cells are lysed with a lysis buffer, and the biotinylated amino acid probe incubated on streptavidin agarose beads is reacted with the HCT116 cell lysate overnight. Proteins binding to different amino acids (leucine, isoleucine, and valine) are obtained through in vitro pull-down experiments. The protein obtained by immunoprecipitation is hydrolyzed to obtain peptides. After the peptides are desalted, the sample molecules are converted into charged particles, and the mass spectra of the ions are recorded by a mass spectrometer. The obtained mass spectrometry data are processed and analyzed by corresponding software to identify and quantify the peptides in the sample and search the library to determine the protein. By comparing with the biotin control group, the binding proteins of different amino acids are analyzed. The specific steps are as follows Figure 1 As shown in Part A of Figure 1 As shown in Part B.

[0089] Through statistical analysis, it was found that there are 185 valine binding proteins, 749 leucine binding proteins, and 204 isoleucine binding proteins. Further analysis of the Venn diagram intersection revealed that there are common and specific binding proteins for different amino acids. Specifically, there are 7 common binding proteins for valine, leucine, and isoleucine, 77 proteins that specifically bind to valine, 614 proteins that specifically bind to leucine, and 158 proteins that specifically bind to isoleucine.

[0090] From the above, it can be seen that the use of biotin-modified amino acid probes combined with immunoprecipitation technology can obtain proteins bound to amino acids. Combined with mass spectrometry technology, it is possible to identify proteins directly bound to amino acids and analyze the binding proteins of different amino acids to find out whether there are common and specific binding proteins.

[0091] Example 4 - Application of Azide-Modified Valine and Leucine Probes

[0092] This example uses the amino acid probes with azide groups synthesized in Example 2, and selects azide-modified valine and leucine probes N 2 -Val-NH 2 、N 2 -Leu-NH 2 Human embryonic kidney HEK293T cell line (purchased from the Chinese Academy of Sciences Cell Bank) was used for the experiment. The experimental steps are as follows: Figure 2 As shown, the specific steps include:

[0093] HEK293T cells were starved for 50 minutes with the corresponding probe amino acids (such as leucine or valine). Specifically, the original culture medium was discarded, washed with PBS, starved for 50 minutes with DMEM culture medium containing 0mM leucine or valine, and then treated with azide-modified amino acid probes for 10 minutes. After UV crosslinking for 10 minutes, the cells were lysed with lysate, and the lysate was subjected to click chemistry reaction with biotin with alkynyl groups. The products after the reaction were enriched with streptavidin agarose beads, and the enriched samples were hydrolyzed to obtain peptides. After the peptides were desalted, the sample molecules were converted into charged particles, and the mass spectra of the ions were recorded by the mass spectrometer. The obtained mass spectrometry data were processed and analyzed by the corresponding software to identify and quantify the peptides in the sample and search the library to determine the protein. By comparing with the biotin control group, the binding proteins of different amino acids were analyzed.

[0094] Through analysis, it was found that Figure 3 As shown, SDS-PAGE gel and Coomassie brilliant blue staining were used to confirm the use of N 2 -Val-NH 2 、N 2 -Leu-NH 2The probe can be enriched with proteins, and these proteins can be competitively eluted by exogenously added valine or leucine; e.g. Figure 4 and Figure 5 As shown in the figure, 132 valine-binding proteins and 310 leucine-binding proteins were obtained using azide-modified valine and azide-modified leucine probes, respectively. KEGG gene pathway enrichment analysis was performed on these proteins, and it was found that valine-binding proteins were enriched in pathways such as metabolism, ribosome and lipid metabolism, and leucine-binding proteins were enriched in pathways such as RNA transport, endocytosis and aminoacyl-tRNA synthesis.

[0095] In summary, the present invention utilizes chemical synthesis technology to directly connect biotin modification to the amino end or hydroxyl end of an amino acid, and connects biotin modification to the carboxyl end of an amino acid through a click chemistry reaction in a cell, provides a biotin-modified amino acid probe and an azide-modified amino acid probe library, and utilizes the amino acid probe in combination with immunoprecipitation and mass spectrometry technology to identify a series of amino acid binding proteins, which not only supplements the amino acid azide probe library, but also provides an important tool for subsequent experiments on biotin addition after click chemistry reaction with biotin-alkyne in cells and exploration of amino acid sensing proteins, and also lays a foundation for subsequent research on amino acid sensing mechanisms and functions, and has important application significance and research value in applications such as identifying amino acid binding proteins, studying amino acid sensing mechanisms and functions, and determining drug targets or screening lead compounds.

[0096] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A biotinylated amino acid probe, characterized in that: The amino acid probe is an amino acid modified with biotin at the amino or carboxyl end; or, the amino acid probe is an amino acid with an azide group at the carboxyl end, which reacts with alkyne-containing biotin under the catalysis of copper ions through a click chemistry reaction in cells to generate a biotin-modified amino acid.

2. The amino acid probe according to claim 1, characterized in that The structure of the amino acid with biotin modification at the amino or carboxyl terminus is selected from at least one of the following:

3. The amino acid probe according to claim 1, characterized in that The structure of the amino acid with an azide group at the carboxyl end is selected from at least one of the following:

4. A method for preparing a biotinylated amino acid probe, characterized in that: The method comprises the following steps: reacting the compound of formula 1 with the compound of formula 2, formula 3 or formula 4 respectively in a first solvent system, and removing the first solvent after the reaction; reacting the reaction product in a second solvent system, and removing the second solvent after the reaction, separating and purifying to obtain the biotinylated amino acid probes shown in formula I, formula II or formula III respectively; Wherein, the first solvent system is dichloromethane and N, N-diisopropylethylamine; the second solvent system is dichloromethane and trifluoroacetic acid; The structures of the compounds of Formula 1, Formula 2, Formula 3 and Formula 4 are shown below: The structures shown in Formula I, Formula II and Formula III are as follows:

5. The preparation method according to claim 4, characterized in that: In the first solvent system, the mass ratio of the compound of formula 1 to the compound of formula 2, formula 3 or formula 4 is 1:0.5-2, the amount of the compound of formula 1 and dichloromethane and N,N-diisopropylethylamine is (40-150) mg: (5-10) mL: (40-100) μL, and the reaction conditions are: reaction at room temperature for 6-24 hours.

6. The preparation method according to claim 4, characterized in that: In the second solvent system, the volume ratio of dichloromethane to trifluoroacetic acid is 0.5-2:1, and the reaction conditions are room temperature for 2-4 hours.

7. A method for identifying amino acid binding proteins, characterized in that: The method uses the biotinylated amino acid probe as described in any one of claims 1 to 3; Wherein, when the amino acid probe is an amino acid modified with biotin at the amino or carboxyl terminus, the protein that can directly bind to the amino acid is identified by an in vitro biotin pull-down experiment combined with mass spectrometry detection; When the amino acid probe is an amino acid with an azide group at the carboxyl end, the protein bound to the amino acid is obtained by immunoprecipitation technology through the high affinity binding characteristics between biotin and streptavidin, and the protein that can directly bind to the amino acid is identified by combining mass spectrometry detection.

8. The method according to claim 7, characterized in that When the amino acid probe is an amino acid modified with biotin at the amino or carboxyl terminus, the method specifically comprises the steps of: subjecting cells to corresponding amino acid starvation treatment, lysing the cells with a lysis solution, reacting the biotinylated amino acid probe incubated on streptavidin agarose beads with the cell lysis solution overnight, obtaining a protein bound to the amino acid by an in vitro pull-down experiment; obtaining peptides from the obtained protein by proteolysis, and identifying the protein that can directly bind to the amino acid by a mass spectrometry detection method.

9. The method according to claim 7, characterized in that: When the amino acid probe is an amino acid with an azide group at the carboxyl end, the method specifically includes the steps of: subjecting cells to corresponding amino acid starvation treatment, treating with an azide-modified amino acid probe and UV cross-linking, lysing the cells with a lysis solution, subjecting the lysis solution to a click chemistry reaction with an alkynyl-containing biotin, and enriching the reaction product with streptavidin agarose beads to obtain amino acid-bound proteins; obtaining peptides from the obtained proteins by proteolysis, and identifying proteins that can directly bind to amino acids by mass spectrometry detection.

10. A use of a biotinylated amino acid probe as claimed in any one of claims 1 to 3, or a method as claimed in any one of claims 7 to 9, characterized in that: The application is selected from one of the following applications: application in identifying amino acid binding proteins, application in studying amino acid sensing mechanisms and functions, and application in determining drug targets or screening lead compounds.

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