Construction and biological application of novel selenoprotein marking method based on selenol conversion
A four-step selenol conversion method enables selective detection of selenoproteins by alkylation and oxidation, addressing the selectivity issues of existing methods and facilitating reproducible detection.
Patent Information
- Application Number
- CN202411313827.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2024-12-20
AI Technical Summary
Existing methods for detecting selenoproteins are not highly selective due to the low abundance and high homology of selenocysteine (Sec) with cysteine (Cys), making it difficult to explore their recombinant expression and functional effects, especially given the varying expression levels influenced by selenium content.
A four-step method involving alkylation of thiols and selenols, oxidation of thioethers and selenoethers, reduction, and DHA derivatization is developed to selectively detect selenoproteins through selenol conversion.
The method allows for simple, reproducible, and selective detection of selenoproteins, demonstrating high application value and biological applicability.
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Figure CN119161406A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the fields of organic synthesis and molecular medicine, and in particular to the construction and biological application of a new selenoprotein labeling method based on selenol conversion. Background Art
[0002] Selenoprotein, as the most important carrier of selenium in organisms, has many functions including maintaining cellular redox homeostasis and controlling the formation of protein disulfide bond structures. Although there are many types and functions of selenoproteins, they all have one thing in common, that is, they contain selenocysteine (Sec). About half of the functions and effects of the 25 known human selenoproteins have not yet been determined. The main reason for the difficulty in detecting selenoproteins is the low abundance of proteins. At the same time, since the expression level of selenoproteins will change with the content and concentration of selenium in the environment, it is very challenging to explore the recombinant expression and functional effects of selenoproteins. Cysteine (Cysteine, Cys), as a homologous amino acid of Sec, contains a thiol residue and plays a similar or even consistent functional role with Sec in organisms. It is precisely because Cys has many similar properties to Sec and its content in organisms is much higher than Sec that most methods for detecting Sec are not highly selective. Therefore, the present invention discloses for the first time a new type of selenol conversion method for selectively detecting Sec for labeling and detecting selenoproteins. Summary of the invention
[0003] The first purpose of the present invention is to provide a simple, easily reproducible molecular synthesis route for detecting selenoproteins, which has high application value.
[0004] The second purpose of the present invention is to provide a method for detecting selenoproteins, which detects selenoproteins through four steps: alkylation of thiols and selenols, oxidation of thioethers and selenoethers, reduction and elimination of selenoketones, and DHA derivatization. The steps are simple and the detection is convenient. BRIEF DESCRIPTION OF THE DRAWINGS
[0005] Figure 1 are different halogenated hydrocarbons used in the alkylation step of thiol and selenol in the synthetic route provided in Experimental Example 1 of the present invention;
[0006] Figure 2 A is a schematic diagram of the conversion rates of different halogenated hydrocarbons used in the alkylation step of thiol and selenol in the synthetic route provided in Experimental Example 1 of the present invention;
[0007] Figure 2 B is a schematic diagram of the blank control experimental results in the alkylation step of thiol and selenol in the synthetic route provided in Experimental Example 1 of the present invention;
[0008] Figure 3A is a schematic diagram of a specific scheme for simulating the synthetic route provided in Experimental Example 1 of the present invention using cystine;
[0009] Figure 3 B is an HPLC image of using cystine to simulate the synthesis route provided in Experimental Example 1 of the present invention and compared with the results of other groups;
[0010] Figure 4 A is a schematic diagram of the structure of the thiol-containing probe used in the in vivo experiment;
[0011] Figure 4 B is a graph showing the fluorescence intensity of various organs of mice after being treated by the method provided in Experimental Example 1 of the present invention;
[0012] Figure 4 C is the result of the detection of selenium content in various organs of mice after treatment by standard elemental analysis method (ICP-MS)
[0013] Example 1
[0014] The present invention discloses a selenoprotein labeling method based on selenol conversion, and its synthesis route is:
[0015]
[0016] The synthetic routes of the selenocystine mimetic and DHA derivatives of the present invention are as follows:
[0017] The preparation method of the selenocystine mimetic and DHA derivative of the present invention is as follows: Synthesis of compound Boc-Ser: NaOH (4.00 g, 500 mmol) solid is placed in a 500 mL round-bottom flask, 100 mL ultrapure water is added under ice bath, L-serine methyl ester hydrochloride (7.50 g, 48 mmol) is added after stabilization, and 100 mL dioxane is added to form a mixed solvent. Di-tert-butyl dicarbonate (10.90 g, 50 mmol) is dissolved in 20 mL dioxane. Di-tert-butyl dicarbonate solution is added dropwise to the system under ice bath, and the reaction is carried out at 0° C. for 30 min and at room temperature for 2.5 h. After the reaction is completed, dioxane is removed by reduced pressure distillation at 30° C., and the remaining solution is extracted with ethyl acetate. After the organic phases are combined and dried with anhydrous sodium sulfate, the solvent is removed by reduced pressure distillation to obtain a colorless liquid (10.50 g, 91% yield). It is directly used in the next step without purification.
[0018] Synthesis of compound Br-Boc: Compound Boc-Ser (4.38 g, 20 mmol) and triphenylphosphine (6.64 g, 25 mmol) were placed in a 100 mL round-bottom flask, 50 mL THF was added, and the mixture was stirred in an ice bath for 30 min to form a suspension, and then NBS (4.51 g, 25 mmol) was added in batches within 30 min. After TLC detected that the raw material completely disappeared, the solid was filtered, and the filtrate was distilled under reduced pressure to remove the solvent, and then ethyl acetate was added to redissolve the residue, and the compound was separated by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain a white target product (3.96 g, yield 70%). 1 H NMR (400MHz, CDCl3) δ: 5.40 (d, J = 7.9 Hz, 1H), 4.76 (dt, J = 7.8, 3.4 Hz, 1H), 3.80 (s, 4H), 3.71 (dd, J = 10.5, 3.6 Hz, 1H), 1.46 (s, 9H). 13 CNMR(100MHz, CDCl3)δ:169.69,80.53,53.93,52.95,34.07,28.27.
[0019] Synthesis of compound Boc-SeCst-Me: Selenium powder (0.90 g, 11 mmol) was placed in a 25 mL three-necked flask, and 5.0 mL of ultrapure water was added to form a suspension. Sodium borohydride (0.90 g, 24 mmol) dissolved in 25 mL of water was dripped into the three-necked flask under an ice bath. Then 0.90 g of selenium powder was added to form a reddish brown solution. After 30 min, the excess selenium powder was filtered to obtain a Na2Se2 solution. In a 25 mL round-bottom flask, compound Br-Boc (987 mg, 3 mmol) was dissolved in 5 mL of THF. At 0°C, 1.6 mL of Na2Se2 solution was added to the system in an argon atmosphere, and the reaction was carried out at 25°C for 6 h after an ice bath reaction for 30 min. After the reaction, the organic phases were extracted and combined with ethyl acetate, and the organic phases were dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography (petroleum ether: ethyl acetate = 4:1). The column chromatography product was then recrystallized from tetrahydrofuran and n-hexane to obtain a yellow solid (654 mg, yield 72%). 1 H NMR (400MHz, CDCl3) δ: 5.39 (s, 2H), 4.48 (d, J = 75.8Hz, 2H), 3.72 (s, 6H), 3.45–2.86 (m, 4H), 1.41 (s, 18H). 13 C NMR(100MHz, CDCl3)δ:171.27,155.03,80.21,53.69,52.56,32.31,28.30.HRMS(ESI)calculated for[C18 H 32 N2O8Se2Na] + [M+Na] + :m / z=587.0381,found 587.0366.
[0020] Synthesis of compound Boc-DHA-Me-Me: Compound Boc-Ser (4.38 g, 20 mmol) was placed in a 100 mL round-bottom flask and 50 mL DCM was added to dissolve the compound. Triethylamine (11.2 mL, 80 mmol) was slowly added dropwise to the reaction system and stirred continuously for 5 min. Methanesulfonyl chloride (3.08 mL, 40 mmol) was then added dropwise to the reaction system and the reaction was stirred at room temperature for 3 h. 40 mL of saturated sodium bicarbonate solution was added to the system to quench the reaction, the solution was transferred to a separatory funnel, extracted with DCM and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, then concentrated by distillation under reduced pressure, and the crude compound was purified by column chromatography (petroleum ether: ethyl acetate = 20:3) to obtain a yellow oily liquid (3.46 g, yield 86%). 1 H NMR (400MHz, CDCl3) δ: 6.40 (s, 1H), 5.97 (d, J = 1.5Hz, 1H), 4.07 (s, 3H), 1.73 (s, 9H). 13 C NMR(100MHz, CDCl3)δ:164.47,152.57,131.31,105.18,80.70,52.88,28.25.HRMS(ESI)calculated for[C9H 15 NO4Na] + [M+Na] + : m / z=224.0893, found 224.0888.
[0021] Experimental Example 2
[0022] This experimental example provides the conversion results of the reaction between selenocystine mimics and different halogenated hydrocarbons and the blank control experimental results;
[0023] Experimental method: Add sodium borohydride, methanol, and 20mM Boc-SeCst-Me methanol solution to a centrifuge tube, and react in an argon ice bath for 10 minutes. Then add various halogenated hydrocarbons to the system, react at 37°C for 1 hour, and then detect by HPLC. Finally, add 30% hydrogen peroxide, react at 37°C for 1 hour, and detect by HPLC. Then use Boc-SeCst-Me as the substrate, add sodium borohydride and C6 methanol solution, react at 37°C for 1 hour, then add hydrogen peroxide solution, and react at 37°C for 1 hour. The blank components are replaced with the corresponding solvents, and the reaction progress is finally detected by HPLC.
[0024] Experimental results: The experimental results show that the final conversion rate of compound C6 is the highest among all compounds, so we selected compound C6 as the alkylation agent for subsequent experiments. It also shows that the selenol conversion method can produce DHA quickly and efficiently.
[0025] Experimental Example 3
[0026] This experimental example provides HPLC graphs of multiple groups of selenols and thiols after selenol conversion;
[0027] Experimental methods:
[0028] (1) Boc-DHA-Me group: 10 mM Boc-DHA-Me methanol solution;
[0029] (2) Sec+C6 group: Add 10mM Boc-SeCst-Me methanol solution to a round-bottom flask, then add sodium borohydride, and react for 10min in an ice bath. Add 1M methanol solution of compound C6 under argon atmosphere, and react at 37°C for 4h. Finally, add 30% hydrogen peroxide, and after 2h, the reaction progress is detected by HPLC (75% MeOH, 0.4mL / min);
[0030] (3) Cys+C6 group: Add 100 mM methanol solution of cystine analog to a round-bottom flask, then add sodium borohydride, and react for 10 min in an ice bath. Add 1 M methanol solution of compound C6 under argon atmosphere, and react for 4 h at 37°C. Finally, add 30% hydrogen peroxide, stir at 37°C for 2 h, and then detect by HPLC (75% MeOH, 0.4 mL / min);
[0031] (4) Sec+Cys+C6 group: Sodium borohydride was added to a mixture of 200 mM cystine analogs and 20 mM selenocystine analogs, and the mixture was reacted in an ice bath for 10 min. A 1 M methanol solution of compound C6 was added under an argon atmosphere, and the mixture was reacted at 37°C for 4 h. Finally, 30% hydrogen peroxide was added, and HPLC detection was performed after 2 h (75% MeOH, 0.4 mL / min). Experimental results: The above experiments show that under conditions similar to those of biological systems, both Sec and Cys analogs can be labeled with alkylating agents, but only selenoether can be oxidized and eliminated to form Boc-DHA-Me, indicating that the selenol conversion method can be applied at the biological level.
[0032] Experimental Example 4
[0033] This experimental example provides the fluorescence intensity and selenium content results of various organs of mice after being converted to selenol and reacting with probe S3;
[0034] Experimental method: The lysates of five organs of mouse heart, liver, brain, lung and kidney were added to centrifuge tubes respectively, and then sodium borohydride was added and reacted at 0°C for 30 minutes. Then the reaction solution was added to four new centrifuge tubes, numbered as Control group, C6 group, H2O2 group and C6 / H2O2 group. Among them, DMSO solution of C6 was added to the C6 / H2O2 group and incubated at 37°C for 4 hours. Then 100mM hydrogen peroxide solution was added and incubated at 37°C for 2 hours. Then 50mM DMSO solution of probe S3 was added, incubated at 37°C for 2 hours, 1mL acetone was added to the centrifuge tube, and precipitated at -80°C for 3 hours. The protein was centrifuged at low temperature and dissolved in TE buffer. In addition, hydrogen peroxide in the C6 group was replaced by ultrapure water, DMSO was used to replace the solution of compound C6 in the H2O2 group, and the Control group was a group in which neither hydrogen peroxide nor compound C6 existed. The fluorescence intensity of each group of solutions after the reaction was tested by a fluorescence meter, and the experiment was repeated twice. Then, an appropriate amount of protein was drawn from the lysate of each organ into a centrifuge tube, and 1 mL of acetone was added to precipitate at -80°C overnight, and the supernatant was removed. Then, concentrated nitric acid and 30% hydrogen peroxide were added, and ultrasonic treatment was performed for 30 minutes at room temperature overnight. The selenium content was detected by ICP-MS.
[0035] Experimental results: The distribution pattern of selenoprotein in different organs and tissues is relatively reliable. The distribution pattern is: liver > heart > brain > lung > kidney.
Claims
1. A design route for a selenoprotein labeling method based on selenol conversion, the route is:
2. The synthetic route according to claim 1, characterized in that It includes four steps: alkylation of thiols and selenols, oxidation of thioethers and selenoethers, reduction elimination of selenosulfoxides, and derivatization of DHA.
3. Application of the synthetic route as claimed in claim 1 or 2 in selenoprotein labeling detection.
4. A design of a synthetic route, characterized in that: include: The thiols and selenols are converted into DHA derivatives by the four steps as described in claim 1 or 2.