CN108761083B - Biotin-labeled ebselen probe, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of protein detection, and particularly relates to a biotin-labeled ebselen probe, and a preparation method and application thereof. The inventionThe structural formula of the probe is as follows:

the ebselen biotin probe prepared by the invention has a stable structure, is simple to synthesize, can rapidly mark thiol small molecules and ebselen protein capable of covalent targeting, can enrich the targeted protein through biotin, and can identify the type of the protein by using mass spectrometry. The probe of the invention has profound influence on the research of the action mechanism of ebselen.

Description

Biotin-labeled ebselen probe, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of protein detection, and particularly relates to a biotin-labeled ebselen probe, and a preparation method and application thereof.

Background

Ebselen (Ebselen) is a small molecule antioxidant of organic selenium compound, and can scavenge excessive peroxide in the body and block chain reaction of generating free radicals, thereby treating various diseases such as cardiovascular disease, arthritis, stroke, atherosclerosis and cancer. In addition, ebselen is also well recognized as acting as a mimic of glutathione peroxidase activity (GPx), which catalyzes a number of important reactions to protect cellular components from free radical and oxidative damage. It has also been shown that ebselen is an excellent substrate for mammalian thioredoxin (TrxR) and acts as a dehydroascorbic acid (DHA) reductase mimetic. However, the molecular mechanism of ebselen action is still unclear, and it is urgently necessary to solve the explanation of the cellular mechanism of ebselen.

Over the years, the research on proteins capable of covalent targeting with ebselen has been based on the discovery that a single protein or class of proteins to be studied has been targeted, and only 19 potential protein targets are now covalently targeted by ebselen. Very few studies have been made on proteins that can be covalently targeted to ebselen in the proteome range. The designed and synthesized ebselen biotinylation probe plays an important role in exploring the protein covalently targeted by ebselen, but the designed and synthesized ebselen analog biotinylation probe cannot explore the protein covalently targeted by ebselen more accurately. Therefore, a simple-to-synthesize and sensitive-to-detect biotinylated ebselen probe is needed.

Disclosure of Invention

The invention mainly aims to provide a biotin-labeled ebselen probe. The probe can mark proteins which can be targeted by ebselen in the range of cell or tissue proteome, and has profound influence on the research of the action mechanism of ebselen.

In order to achieve the purpose, the invention adopts the following technical scheme:

one of the purposes of the invention is to provide a biotin-labeled ebselen probe, which has the structural formula shown as follows:

the second object of the present invention is to provide a method for preparing the biotin-labeled ebselen probe, which comprises the following steps:

(1) reacting o-iodobenzoyl chloride with p-phenylenediamine, washing with water, separating liquid, and using CH for water phase₂Cl₂After two times of extraction, organic phases are combined, dried and separated by a chromatographic column to obtain a compound I, wherein the structural formula is as follows:

(2) dissolving and mixing tert-butoxycarbonyl 6-aminocaproic acid, EDCI and HOBT, adding triethylamine, uniformly stirring under an ice bath condition, adding a compound I, and reacting for 24 hours to obtain a compound II, wherein the structural formula is as follows:

(3) dissolving and mixing cuprous iodide and 1, 10-phenanthroline, stirring uniformly, adding a compound II, selenium powder and potassium carbonate, heating for reacting for 24 hours, pouring a reaction solution into a NaCl solution after the reaction is finished, stirring uniformly at room temperature, filtering, washing with water, drying, and separating by using a chromatographic column to obtain a compound III, wherein the structural formula is as follows:

(4) dissolving the compound III, adding trifluoroacetic acid under ice bath condition, and stirring overnight at room temperature to obtain a compound IV, wherein the structural formula is as follows:

(5) dissolving Biotin, N-hydroxysuccinimide and EDCI in DMF, stirring uniformly, reacting for 24 hours, pouring the mixed solution into cold water, and obtaining a white solid, namely Biotin-NHS.

(6) Dissolving and mixing the compound IV and Biotin-NHS, adding triethylamine, uniformly stirring, and reacting for 24 hours to obtain the compound.

The specific reaction process is shown in FIG. 1.

Preferably, in the step (1), the volume mass ratio of the o-iodobenzoyl chloride to the p-phenylenediamine is 1-1.5: 2-3; the eluent for chromatographic column separation is CH₂Cl₂And methanol in a volume ratio of 35-45: 1, and preparing the composition.

Preferably, in the step (2), the mass ratio of the tert-butoxycarbonyl 6-aminocaproic acid to EDCI & HCl to the HOBT to the compound I is as follows: 1-1.5: 0.6-1: 0.9-1.4:1.3-2.

Preferably, the specific steps of step (3) are as follows: the reaction is carried out under the protection of nitrogen, cuprous iodide and 1, 10-phenanthroline are dissolved in DMF, the mixture is stirred uniformly, a compound II, selenium powder and potassium carbonate are added, the mixture is heated at 110 ℃ for reaction for 24 hours, after the reaction is finished, the reaction solution is poured into NaCl solution, the mixture is stirred uniformly at room temperature, and after the filtration, the washing and the drying, the separation by a chromatographic column is carried outTo obtain a compound III; the mass ratio of cuprous iodide to 1, 10-phenanthroline to compound II to selenium powder to potassium carbonate is 0.13-0.16: 0.13-0.16: 2-2.5: 0.35-0.4: 0.8-1.0; the eluent for chromatographic column separation is CH₂Cl₂And methanol in a volume ratio of 20-25: 1, and preparing the composition.

Preferably, in the step (4), the mass-to-volume ratio of the compound III to the trifluoroacetic acid is 1-2: 5-10.

Preferably, in the step (5), the mass ratio of biotin, N-hydroxysuccinimide and EDCI is: 1.2-1.5: 0.7-0.9: 1.2-1.4.

Preferably, the mass ratio of the compound IV to the Biotin-NHS in the step (6) is 0.8-1: 0.7-0.9.

The third purpose of the invention is to provide the biotin-labeled ebselen probe and/or the application of the preparation method in detecting ebselen covalent targeting protein.

The fourth object of the present invention is to provide a method for detecting ebselen covalent targeting protein, comprising the steps of:

(1) cracking cells by adopting an ultrasonic disruption method to obtain a protein solution with the concentration of 2 mg/mL; the biotin-labeled ebselen probe of claim 1 is added to a protein solution and reacted at 25 ℃ for 1 hour.

(2) Carrying out Western blot experiment on the protein solution reacted in the step (1), and observing the marked protein on a membrane;

(3) using CH for the protein solution reacted in the step (1)₃OH/CHCl₃Precipitating, and then washing with cold methanol; ultrasonic redissolving, enriching by using Streptavidin beads, and carrying out enzyme digestion by using trypsin to obtain a peptide segment;

(4) carrying out dimethylation marking on the peptide segment in the step (3), and carrying out alkaline classification by using a high performance liquid chromatography;

(5) and merging the classified peptide fragments, performing data acquisition by using LC-MS, and performing library searching analysis on the data.

Compared with the prior art, the invention has the following advantages:

1. the preparation method of the biotin-labeled ebselen probe is simple, the labeling speed is high, and long-time incubation is not needed in the detection process;

2. the structure of the ebselen probe marked by the biotin is relatively accurate, and the obtained target protein is closer to that of the ebselen medicine;

3. the biotin-labeled ebselen probe can visually observe the labeling condition through a western bolt, and can enrich the labeled protein.

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 exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a diagram showing the reaction process of the Biotin-Ebselen probe.

FIG. 2 is a western blot of different concentrations of the invention after the action of the Biotin-Ebselen probe and the HeLa cytoproteome.

FIG. 3 is a western blot of the Biotin-Ebselen probe of the invention and HeLa cell proteome after different times of action.

FIG. 4 is a western blot of the competition of the Biotin-Ebselen probe of the invention with DMSO and Ebselen, respectively, under conditions targeted to the HeLa cell proteome.

FIG. 5 is a flow chart of the work of the Biotin-Ebselen probe of the present invention in the field of proteomics to probe Ebselen covalently targeted proteins. Panel a is a flow chart of DMSO and Biotin-Ebselen probe-action proteins; b is a flow chart of the Biotin-Ebselen probe and Ebselen competitive protein.

FIG. 6 is a Venn diagram showing the results of exploring the amount of Ebselen covalently targeting proteins using a Biotin-Ebselen probe obtained from two repeated experiments, taking the HeLa proteome as an example.

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 exemplary embodiments according to the invention. 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 the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example a method for preparing a Biotin-labeled Eboselin Probe

The preparation method of the biotin-labeled ebselen probe comprises the following steps:

(1) o-iodobenzoyl chloride (1.33g) and p-phenylenediamine (1.08g) were dissolved in CH, respectively₂Cl₂10mL each, triethylamine (1.04mL) was added to the p-phenylenediamine solution, the o-iodobenzoyl chloride solution was added dropwise to the p-phenylenediamine solution in an ice bath, the reaction was carried out overnight at room temperature, the reaction solution was washed with water, and after separation, the aqueous phase was treated with CH₂Cl₂Extracting twice, mixing organic phases, drying, spin-drying, and separating with chromatographic column (eluent: V)_{Petroleum ether}：V_{Methylene dichloride}40: 1) the solid obtained is compound I.

ESI-MS:calculated for[M+H]⁺＝338.9988,found338.9969.

(2) tert-Butoxycarbonyl 6-aminocaproic acid (1.02g), EDCI & HCl (0.93g) and HOBT (0.65g) were dissolved in CH₂Cl₂(30mL), triethylamine (0.74mL) is added, stirring is carried out for half an hour under the ice bath condition, then compound I (1.35g) is added, reaction is carried out for 24 hours, precipitated solid is obtained, filtration is carried out, and the solid is washed by a solvent, thus obtaining compound II.

ESI-MS:calculated for[M+H]⁺＝574.1173,found 574.1155.

(3) The reaction is carried out under the protection of nitrogen, cuprous iodide (114mg) and 1, 10-phenanthroline (108mg) are dissolved in DMF (5mL) and stirred for 15min, then compound II (1.65g), selenium powder (284mg) and potassium carbonate (621mg) are added, the mixture is heated at 110 ℃ for reaction for 24 hours, after the reaction is finished, the reaction solution is poured into NaCl solution and stirred for 3 hours at room temperature, the mixture is filtered, the solid is washed with water, and the solid is separated by a chromatographic column after being dried (V)_{Methylene dichloride}：V_Methanol25: 1) to obtain a compound III;

ESI-MS:calculated for[M+H]⁺＝526.1216,found 526.1233.

(4) compound III (1g) was dissolved in CH₂Cl₂/CH₃Adding trifluoroacetic acid (5mL) into OH (v/v ═ 10:1) (15mL) under an ice bath condition, and stirring at room temperature overnight to obtain a test compound IV; stopping the reaction, spin-drying, adding saturated NaHCO₃(10mL), quenching reaction, separating liquid, washing an organic phase by using saturated sodium chloride, drying and spin-drying to obtain a solid, namely a compound IV;

(5) biotin (1.47g), N-hydroxysuccinimide (0.76g) and EDCI (1.38g) were dissolved in DMF (10mL), stirred at room temperature, and after 24 hours the reaction was terminated, the reaction solution was poured into cold water to obtain a white solid, which was filtered, washed with methanol several times, and dried to give Biotin-NHS.

(6) Compound IV (0.8g) and Biotin-NHS (0.75g) were dissolved in DMF (10mL), triethylamine (0.61mL) was added, and the mixture was stirred at room temperature for 24 hours to obtain Biotin-Ebselen. When the reaction was stopped, the reaction solution was poured into cold water, filtered, and the obtained solid was washed with methanol to obtain compound Biotin-Ebselen.

Nuclear magnetic and mass spectrum characterization:

¹1H NMR(400MHz,DMSO)δ10.00(s,1H),8.08(d,J＝7.8Hz,1H),7.89(d,J＝7.5Hz,1H),7.77(s,1H),7.66(d,J＝8.6Hz,2H),7.60–7.40(m,3H),6.41(d,J＝27.8Hz,2H),4.29(s,1H),4.12(s,1H),3.20–2.94(m,3H),2.81(dd,J＝11.5,4.4Hz,1H),2.57(d,J＝12.9Hz,1H),2.40–2.24(m,2H),2.04(t,J＝6.4Hz,2H),1.59(d,J＝6.5Hz,3H),1.46(dd,J＝26.1,5.5Hz,5H),1.26(d,J＝29.1Hz,5H).¹³C NMR(400MHz,DMSO)δ172.27,171.69,165.38,163.18,139.35,137.6,134.89,132.60,128.91,128.35,126.69,126.29,125.67,120.00,61.50,59.65,55.92,38.76,36.80,35.69,29.49,28.69,28.50,26.59,25.72,25.33.

ESI-MS:calculated for[M+H]⁺＝630.1648,found 630.1637.

test examples

(one) different concentrations of probe Biotion-Ebselen on HeLa cell proteome labeled western blot experiment.

A series of western blot experiments, such as SDS-PAGE, were performed after the reaction of 100. mu.L of a 2mg/mL HeLa cell proteome solution with 0, 10, 25, 50, and 100. mu.M of the probe Biotion-Ebselen prepared in the examples of the present invention at 25 ℃ for one hour, followed by membrane rotation. And (3) incubating by using HRP-labeled Streptavidin as an antibody, and finally performing development imaging under a gel imaging system. The results of the experiment are shown in FIG. 2, and the amount of the protein that can be labeled increases with the increase in the concentration of the probe.

(II) probe Biotion-Ebselen is used for carrying out western blot experiment on different time periods of the HeLa cell proteome.

mu.M of the probe Biotion-Ebselen prepared in the examples of the present invention was reacted with 600. mu.M of a 2mg/mL HeLa cell proteome solution, 100. mu.L was taken out at 10, 30, 60, 90, 120min and the reaction was terminated by adding a loading buffer, followed by incubation with HRP-labeled Streptavidin as an antibody in a series of western felt procedures, and finally visualized under a gel imaging system. The results of the experiment are shown in FIG. 3, and the probe is basically labeled after 10min of action with the proteome solution.

(II) the Biotin-Ebselen probe of the present invention was used in western blot experiments in which DMSO and Ebselen were used in competition with each other under conditions in which HeLa cell proteome was used as a target.

Proteomic samples were divided into four groups: the group was exposed to DMSO, the group two proteome samples were exposed to probe, and after 1 hour of exposure, the reaction was quenched by adding a sample buffer. And (3) respectively acting the three-group proteome sample and the four-group proteome sample by DMSO and ebselen for 0.5 hour, simultaneously acting the two-group proteome sample by the probe for 1 hour, and adding a sample loading buffer solution to quench the reaction. After that, a series of operations of western felt was performed, and incubation with HRP-labeled Streptavidin as an antibody was performed, and finally, imaging was performed under a gel imaging system. The experimental result is shown in fig. 4, wherein the pore channels from left to right in the figure are respectively group one to group four, and as can be seen from fig. 4, the probe of the present invention can label the ebselen-targeted protein.

FIG. 5 is a flow chart of the probe Biotion-Ebselen in the example of the present invention to probe Ebselen covalently targeted proteins in the field of proteome. and a, finding out proteins which can be marked by the probe and excluding non-specific proteins through the final quantitative result in the experiments of the group I and the group II. And b, the graph shows the experiments of the group three and the group four, and the final quantitative result confirms that the protein which can be covalently targeted by the ebselen and can be marked by the probe. The two processes comprise the steps of probe and protein action, enrichment, enzyme digestion, dimethylation, alkaline classification, LC-MS detection, library searching and the like.

FIG. 6 is a Venn diagram showing the results of probing the amount of Ebselen to covalently target proteins with a Biotin-Ebselen probe obtained by two repeated experiments, taking the HeLa proteome as an example. Here the result sets the quantitative R values to be R respectively_Probe/DMSONot less than 10 and R_{Probe/Competitor}Not less than 1.5. Proteins satisfying both R values are identified as proteins to which ebselen can be covalently targeted. The first biological identification of 625 proteins which can be covalently targeted by ebselen; the second bioassay identified 838 proteins that ebselen could covalently target. The total number of proteins identified in the two biological experiments, i.e. the proteins which are finally thought to be covalently targeted by ebselen, is 462.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (8)

1. A preparation method of biotin-labeled ebselen probe is characterized in that the structural formula of the biotin-labeled ebselen probe is as follows:

the preparation method comprises the following steps:

(1) reacting o-iodobenzoyl chloride with p-phenylenediamine, washing with water, separating liquid, and using CH for water phase₂Cl₂After two times of extraction, organic phases are combined, dried and separated by a chromatographic column to obtain a compound I, wherein the structural formula is as follows:

(2) dissolving and mixing tert-butoxycarbonyl 6-aminocaproic acid, EDCI and HOBT, adding triethylamine, uniformly stirring under an ice bath condition, adding a compound I, and reacting for 24 hours to obtain a compound II, wherein the structural formula is as follows:

(3) dissolving and mixing cuprous iodide and 1, 10-phenanthroline, stirring uniformly, adding a compound II, selenium powder and potassium carbonate, heating for reacting for 24 hours, pouring a reaction solution into a NaCl solution after the reaction is finished, stirring uniformly at room temperature, filtering, washing with water, drying, and separating by using a chromatographic column to obtain a compound III, wherein the structural formula is as follows:

(4) dissolving the compound III, adding trifluoroacetic acid under ice bath condition, and stirring overnight at room temperature to obtain a compound IV, wherein the structural formula is as follows:

(5) dissolving Biotin, N-hydroxysuccinimide and EDCI in DMF, stirring uniformly, reacting for 24 hours, pouring the mixed solution into cold water to obtain a white solid, namely Biotin-NHS;

(6) dissolving and mixing the compound IV and Botin-NHS, adding triethylamine, stirring uniformly, and reacting for 24 hours to obtain the compound.

2. The preparation method according to claim 1, wherein the o-iodobenzoyl chloride and p-phenylenediamine in the step (1) have a volume mass ratio of 1-1.5: 2-3; the eluent for chromatographic column separation is CH₂Cl₂And methanol in a volume ratio of 35-45: 1, and preparing the composition.

3. The process according to claim 1, wherein the mass ratio of t-butoxycarbonyl 6-aminocaproic acid, EDCI HCl, HOBT and Compound I in step (2) is: 1-1.5: 0.6-1: 0.9-1.4:1.3-2.

4. The preparation method according to claim 1, wherein the step (3) comprises the following steps: the reaction is carried out under the protection of nitrogen, cuprous iodide and 1, 10-phenanthroline are dissolved in DMF, the mixture is uniformly stirred, a compound II, selenium powder and potassium carbonate are added, the mixture is heated at 110 ℃ for reaction for 24 hours, after the reaction is finished, the reaction solution is poured into NaCl solution, the mixture is uniformly stirred at room temperature, and after filtration, washing and drying, chromatographic column separation is carried out to obtain a compound III; the mass ratio of cuprous iodide to 1, 10-phenanthroline to compound II to selenium powder to potassium carbonate is 0.13-0.16: 0.13-0.16: 2-2.5: 0.35-0.4: 0.8-1.0; the eluent for chromatographic column separation is CH₂Cl₂And methanol in a volume ratio of 20-25: 1, and preparing the composition.

5. The preparation method according to claim 1, wherein in the step (4), the mass-to-volume ratio of the compound III to the trifluoroacetic acid is 1-2: 5-10.

6. The preparation method according to claim 1, wherein the mass ratio of biotin, N-hydroxysuccinimide, and EDCI in step (5) is: 1.2-1.5: 0.7-0.9: 1.2-1.4.

7. The method according to claim 1, wherein the mass ratio of the compound IV to the Biotin-NHS in the step (6) is 0.8-1: 0.7-0.9.

8. A detection method for detecting ebselen covalent targeting protein is characterized by comprising the following steps:

(1) cracking cells by adopting an ultrasonic disruption method to obtain a protein solution with the concentration of 2 mg/mL; adding the biotin-labeled ebselen probe prepared by the method according to any one of claims 1 to 7 to a protein solution, reacting at 25 ℃ for 1 hour,

(2) carrying out Western blot experiment on the protein solution reacted in the step (1), and observing the marked protein on a membrane;

(3) using CH for the protein solution reacted in the step (1)₃OH/CHCl₃Precipitating, and then washing with cold methanol; ultrasonic redissolving, enriching by using Streptavidin beads, and carrying out enzyme digestion by using trypsin to obtain a peptide segment;

(4) carrying out dimethylation marking on the peptide segment in the step (3), and carrying out alkaline classification by using a high performance liquid chromatography;

(5) and merging the classified peptide fragments, performing data acquisition by using LC-MS, and performing library searching analysis on the data.

Images