Selenohydration reaction of alkynyl amide compounds and its application in selenium-containing polypeptide and protein modification

Abstract

This invention discloses a selenylamide-based hydrogenation reaction of acetylacetamide compounds and its application in the modification of selenium-containing peptides and proteins, belonging to the fields of organic chemistry and chemical biology. The reaction involves the reaction of a selenium-containing compound with an acetylacetamide compound in a solvent with the addition of an additive. The applications of this reaction include: 1) selective modification and labeling of selenocysteine ​​in peptides and proteins; 2) cyclization reactions of selenium-containing peptides and selenoproteins; and 3) sequential modification of selenium-containing peptides in the presence of cysteine ​​by precise pH control combined with the hydrogen sulfide reaction of acetylacetamide. This reaction has advantages such as mild conditions, simple operation, fast reaction rate, high yield, good stability, and good selectivity. It provides a new and robust approach for the selective modification and labeling of selenium groups in bioconjugations, peptides, and / or proteins, and also provides a new method for the construction of cyclic peptides, offering a powerful tool for the development of selenium-containing peptide drugs.

Description

Technical Field

[0001] This invention relates to the fields of organic chemistry and chemical biology, and in particular to the selenization reaction of acetylacetamide compounds and their application in the modification of selenium-containing peptides and proteins. Background Technology

[0002] Both peptides and proteins are highly active biological macromolecules, forming the basic organic components of cells and playing irreplaceable roles in various life activities within the body, serving as the material basis of life. Peptide drugs have attracted widespread attention in the pharmaceutical community due to their advantages such as strong drug-like properties, high targeting selectivity, good specificity, high stability, low toxicity and side effects, and low residue in the body. They have become one of the hottest areas in drug development and are currently the most active and fastest-growing segment of the field, belonging to one of the most promising drug types.

[0003] Although some naturally occurring or synthetic peptides possess high activity, these active peptides often cannot be directly developed into clinical drugs for therapeutic purposes due to defects such as poor stability and membrane permeability, difficulty in oral administration, and short in vivo half-life. Therefore, chemical modification methods (such as side chain functional group modification or cyclization strategies) are necessary to improve the properties of these active peptides before they can be developed into drugs. At the same time, selective modification of peptides and proteins is also a necessary means to understand protein structure and its biological function.

[0004] With the development of science and technology, selenocysteine ​​(abbreviated as Sec or U) has gradually become known to people. It differs from cysteine ​​(abbreviated as Cys or C) by only one atom and is known as the "twenty-first amino acid". At present, there are 25 known selenoproteins in the human body, but very few selenoproteins have been comprehensively studied and reported on in terms of their related biological functions.

[0005] Structurally and chemically, cysteine ​​(Cys) and selenocysteine ​​(Sec) are quite similar, both exhibiting strong nucleophilicity and relatively weak redox potential. However, the abundance of cysteine ​​(Cys) proteins in organisms is far higher than that of selenocysteine ​​proteins, making selective modification of Sec a challenging task. Despite these subtle differences, such as the lower pKa of Sec's side chain SeH (pKa 5.24) compared to Cys's side chain SH (pKa 8.3) and the lower redox potential of Sec relative to Cys (approximately -220 mV, approximately -388 mV), scientists have developed methods for specifically modifying Sec, as documented in Nat. Rev. Chem. 2024, 8 (3), 211-229. However, the currently reported methods for modifying selenopeptides and selenoproteins are limited, and some methods have shortcomings, such as complex reaction conditions, poor selectivity, and the need to use expensive photocatalysts, making it difficult to apply them to the selective modification of selenoproteins in organisms.

[0006] In addition, Chinese patent CN115108953B discloses the hydrogen sulfide reaction of acetylacetamide and its method for selective modification of polypeptide cysteine. However, as mentioned above, the content of proteins containing cysteine ​​in the body is much higher than that of selenium-containing proteins, and Se and S have different physicochemical properties. The inventors replaced -SH with -SeH in the method but failed to obtain satisfactory results.

[0007] Therefore, in order to better study the structure and biological function of selenoproteins, and to accelerate the research and development of selenopeptides and selenoprotein drugs, it is essential to develop a simple and efficient method for selectively modifying selenopeptides and selenoproteins. Summary of the Invention

[0008] One of the objectives of this invention is to provide a selenium hydrogenation reaction of acetylamides to solve the above-mentioned problems.

[0009] To achieve the above objectives, the technical solution adopted in this invention is as follows: a selenium hydrogenation reaction of acetylamides, wherein a selenium-containing compound (A) reacts with an acetylamide compound (B) in a solvent with the addition of an additive to obtain product (C), and the reaction is shown in the following general formula (1):

[0010]

[0011] (1),

[0012] Among them, R 1 Selected from C1 to C 22 Alkyl, C4-C 10One or more of the following: aromatic ring group, substituted aromatic ring group, heterocyclic aryl group, alkenyl group, alkynyl group, protected α-selenocysteine ​​residue, β-selenocysteine ​​residue, γ-selenocysteine ​​residue, and alkyl group of polypeptide chain.

[0013] R 2 It is selected from one or more of alkyl, hydroxyethyl, propargyl, indomethacin-containing groups, biotin-containing groups, and coumarin-containing fluorescent groups.

[0014] EWG is selected from one or more of the following: C1-C5 alkylsulfonyl, C1-C5 alkylyl, C6-C10 arylsulfonyl, and C6-C10 arylyl.

[0015] As a preferred technical solution, the additive is selected from one or more of phosphate buffer (PB), N,N-diisopropylethylamine (DIEA), triethylamine (NEt3), sodium carbonate, potassium carbonate, sodium bicarbonate, 4-dimethylaminopyridine (DMAP), cesium carbonate (Cs2CO3), lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, imidazole, N-methylimidazolium, pyridine, sodium ethoxide (EtONa), lithium ethoxide (EtOLi), DBU, TCEP, DTT, Bu3P, and ascorbic acid salts (such as NaSAc), with sodium carbonate being preferred.

[0016] As a preferred technical solution, the solvent is selected from one or more of dichloromethane (DCM), dichloroethane (DCE), trichloroethane, dimethyl sulfoxide (DMSO), methanol, acetonitrile, N,N-dimethylformamide (DMF), tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, ethyl acetate, methanol, isopropanol, n-butanol, and buffer solutions (such as phosphate buffer (pH = 5-8)). The preferred solvent is isopropanol, or a mixture of acetonitrile and phosphate buffer at pH = 6.8.

[0017] A second objective of this invention is to provide the use of the above-mentioned acetylamide compound for the hydrogen selenization reaction, specifically for the selective modification of selenopeptides. The method involves using the acetylamide compound (B) to perform a hydrogen selenization reaction with selenools in selenopeptides, wherein the reaction is carried out in a weakly acidic system with pH 5-7; preferably, it is used for the selective modification and labeling of side-chain selenools in peptides and proteins.

[0018] As a preferred technical solution, the weakly acidic reaction system is a mixed system composed of a slightly acidic buffer solution and an organic solvent;

[0019] The volume ratio of the slightly acidic buffer solution to the organic solvent is 1 to 50:1, preferably 5 to 40:1, and more preferably 10 to 30:1;

[0020] The slightly acidic buffer solution is selected from PBS buffer and Tris buffer; the organic solvent is selected from acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; and / or

[0021] The molar ratio of the acetylamid compound having general structural formula (B) to the selenium-containing peptide or protein having general structural formula (A) is 1-10:1, preferably 1.1-4:1, and more preferably 1.2-2:1.

[0022] As a further preferred technical solution, the reaction steps are as follows: a acetylacetamide compound having the general structural formula (B) and a polypeptide or protein containing selenocysteine ​​having the general structural formula (A) are dissolved in a weakly acidic reaction system, mixed evenly, and then reacted. The reaction is carried out by stirring at 0–50 °C for 1–120 min, preferably at 25–40 °C for 5–50 min. After the reaction is completed, the alkenyl selenide compound having the general structural formula (C) is obtained by column chromatography.

[0023] The third objective of this invention is to provide another application of the selenization reaction of the above-mentioned acetylamides, namely, the selective cyclization modification of selenopeptides. Specifically, the acetylamide compound (B) in reaction formula (I) is linked to the selenopeptide (i.e., a polypeptide containing selenocysteine) via an amide bond / ester bond / ether bond. Then, under weakly alkaline conditions, intramolecular selenization of selenools is carried out for cyclization. Preferably, this method is used for the selective cyclization modification of side-chain selenools in peptides and proteins, and includes the following reaction formulas (2) and (3): that is, selenopeptides D or F containing acetylamides (which can be attached to a solid support or are free) undergo a cyclization reaction in a weakly alkaline reaction system, ultimately obtaining alkenyl selenide compounds with structural formulas (E) or (G).

[0024] (2)

[0026] (3)

[0028] Among them, R 4 One or more of the following: H, Fmoc, Fmoc-protected polypeptide chain alkyl, Boc, Boc-protected polypeptide chain alkyl, Cbz, protected polypeptide chain alkyl, Ac, Ac-protected polypeptide chain alkyl;

[0029] AA n These are amino acids in the peptide chain that have been modified with acetylacetamide compounds;

[0030] EWG is selected from one or more of C1~C5 alkylsulfonyl, C1~C5 alkylyl, C6~C10 arylsulfonyl, and C6~C10 arylyl.

[0031] The additives are selected from one or more of the following: phosphate buffer (PB), N,N-diisopropylethylamine (DIEA), triethylamine (NEt3), sodium carbonate, potassium carbonate, sodium bicarbonate, 4-dimethylaminopyridine (DMAP), cesium carbonate (Cs2CO3), lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, imidazole, N-methylimidazolium, pyridine, sodium ethoxide (EtONa), lithium ethoxide (EtOLi), DBU, TCEP, DTT, Bu3P, and ascorbic acid salts (such as NaSAc).

[0032] The solvent is selected from one or more of the following: dichloromethane (DCM), dichloroethane (DCE), trichloroethane, dimethyl sulfoxide (DMSO), methanol, acetonitrile, N,N-dimethylformamide (DMF), tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, ethyl acetate, methanol, isopropanol, n-butanol, and buffer solutions (such as phosphate buffer (pH = 5-8)).

[0033] As a preferred technical solution, the weakly alkaline reaction system is a mixed system composed of an organic solvent and a slightly alkaline buffer solution;

[0034] The volume ratio of the organic solvent to the slightly alkaline buffer solution is 1 to 50:1, preferably 1 to 30:1, and more preferably 1 to 10:1;

[0035] The alkaline buffer solution is selected from PB buffer and Tris buffer; the organic solvent is selected from acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

[0036] Preferably, the reaction steps are as follows: dissolve the selenized polypeptide or protein D or F (which can be attached to a solid support or be free) containing the acetylamid compound (B) in a weakly alkaline reaction system, mix thoroughly, and then react (stirring at 0-50 °C for 1-120 min, preferably at 20-40 °C for 5-50 min); after the reaction is completed, obtain the alkenyl selenide cyclic compound with the general structural formula E or G by column chromatography, recrystallization, or semi-preparative separation.

[0037] The fourth objective of this invention is to provide another use for the selenization reaction of the above-mentioned acetylamides, specifically for the cyclization modification of diselenomeric polypeptides. The method includes the following reaction formula (4): an acetylamide compound having reaction formula I reacts with a polypeptide H containing two selenocysteine ​​residues under weakly alkaline conditions to achieve the cyclization of the polypeptide H containing two selenocysteine ​​residues (Sec) via the selenization reaction of selenools (including in a solid or liquid phase); preferably, it is used for the cyclization modification of side-chain selenools in peptides and proteins. Finally, an alkenyl selenide compound having structural formula J is obtained.

[0038] (4)

[0040] Among them, R 5 It is one or more of the following: H, Fmoc, Fmoc-protected polypeptide residues, Boc, Boc-protected polypeptide chain residues, Cbz, protected polypeptide chain residues, Ac, Ac-protected polypeptide chain residues.

[0041] R 6 -OH, -NH2, C1-C5 alkoxy groups, C6-C 10 One or more of the following: phenolic group, amino acid residue, carboxyl-protected (such as tert-butyl ester, amide, etc.) amino acid residue (peptide group), and solid-phase support linked by ester or amide bonds;

[0042] The additive is selected from one or more of the following: phosphate buffer (PB), N,N-diisopropylethylamine (DIEA), triethylamine (NEt3), sodium carbonate, potassium carbonate, sodium bicarbonate, 4-dimethylaminopyridine (DMAP), cesium carbonate (Cs2CO3), lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, imidazole, N-methylimidazolium, pyridine, sodium ethoxide (EtONa), lithium ethoxide (EtOLi), DBU, TCEP, DTT, Bu3P, and ascorbic acid salts (such as NaSAc).

[0043] The solvent is selected from one or more of the following: dichloromethane (DCM), dichloroethane (DCE), trichloroethane, dimethyl sulfoxide (DMSO), methanol, acetonitrile, N,N-dimethylformamide (DMF), tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, ethyl acetate, methanol, isopropanol, n-butanol, and buffer solutions (such as phosphate buffer (pH=5-8)).

[0044] As a preferred technical solution, the weakly alkaline reaction system is a mixed system composed of an organic solvent and a slightly alkaline buffer solution;

[0045] The volume ratio of the organic solvent to the slightly alkaline buffer solution is 1 to 50:1, preferably 1 to 30:1, and more preferably 1 to 10:1;

[0046] Preferably, the alkaline buffer solution is selected from PB buffer and Tris buffer; the organic solvent is selected from acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

[0047] As a preferred technical solution, the reaction steps are as follows: a polypeptide H containing two selenocysteines (which can be attached to a solid support or be free) and an acetylacetamide compound with the general reaction formula I are dissolved in a weakly alkaline reaction system, mixed evenly, and then reacted. Specifically, the reaction is carried out by stirring at 0–50°C for 1–360 min, preferably at 20–40°C for 5–300 min. After the reaction is completed, the cyclic compound with the general structural formula J is obtained by column chromatography, recrystallization, or semi-preparative separation.

[0048] The fifth objective of this invention is to provide a final application of the selenization reaction of the aforementioned acetylamides, wherein the application is to use the pH of the reaction system in conjunction with the sulfidation reaction of acetylamides for the sequential modification of selenopolypeptides in the presence of cysteine ​​by adjusting the pH of the reaction system. The specific method is as follows: including the following general reaction formula (5), the acetylamides (B1) in general reaction formula (1) are first used in a weakly acidic solvent 1 to selectively modify the selenopolypeptide (K) containing cysteine ​​under the combined action of additives. After the reaction, an alkenyl selenide compound with the structural formula L is obtained. Then, in a weakly alkaline solvent 2, the acetylamides (B2) in general reaction formula (1) is added, and the sulfidation reaction of acetylamides is used to further modify cysteine, and finally, a compound with the structural formula M containing both alkenyl selenide and alkenyl sulfide, which is sequentially modified by selenocysteine, is obtained.

[0049] (5)

[0051] Among them, R 4 One or more of the following: H, Fmoc, Fmoc-protected polypeptide chain alkyl, Boc, Boc-protected polypeptide chain alkyl, Cbz, protected polypeptide chain alkyl, Ac, Ac-protected polypeptide chain alkyl;

[0052] AA is an amino acid in a peptide chain;

[0053] EWG is selected from one or more of C1~C5 alkylsulfonyl, C1~C5 alkylyl, C6~C10 arylsulfonyl, and C6~C10 arylyl.

[0054] The molar ratio of the acetylamid compound BI or B2 having the general structural formula (B) to the selenium-containing peptide or protein having the general structural formula (K) is 1-10:1, preferably 1.1-4:1, and more preferably 1.2-2:1.

[0055] The additive is selected from one or more of the following: phosphate buffer, N,N-diisopropylethylamine, triethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, 4-dimethylaminopyridine, cesium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, imidazole, N-methylimidazolium, pyridine, sodium ethoxide, lithium ethoxide, DBU, TCEP, DTT, Bu3P, and ascorbic acid salts (such as NaSAc).

[0056] Solvent 1 and solvent 2 are selected from one or more of the following: dichloromethane, dichloroethane, trichloroethane, dimethyl sulfoxide, methanol, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, ethyl acetate, methanol, isopropanol, n-butanol, dilute hydrochloric acid, aqueous sodium hydroxide solution, and buffer solution.

[0057] Preferably, the slightly acidic / slightly alkaline buffer solution is selected from PB buffer and Tris buffer; the organic solvent is selected from acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

[0058] Compared with the prior art, the advantages of the present invention are as follows:

[0059] (1) The trifluoromethanesulfonyl acetylide compounds preferred in this invention not only have the advantages of ordinary acetylide amides, but can also selectively react with selenocysteine ​​under specific conditions to selectively modify and label the peptides without affecting other active groups. They have the advantages of high reaction selectivity, fast reaction rate, no other side reactions, simple and mild conditions, easy operation, wide substrate applicability and high atom economy. The fast reaction rate of this invention means fast response, which is of positive significance for its application in medical imaging examinations.

[0060] (2) The trifluoromethanesulfonyl acetylide compounds preferred in this invention, when combined with selenool or selenium powder to form alkenyl selenide structures, exhibit good stability and remain relatively stable under both acidic and alkaline conditions. Furthermore, their reactions with selenium groups are all trans-additions, demonstrating excellent regioselectivity.

[0061] (3) The present invention can precisely modify the order of cysteine ​​and selenocysteine ​​in a polypeptide by controlling the acidity and alkalinity (pH) of the reaction system.

[0062] In other words, the reaction of this invention has the advantages of mild conditions, simple operation, fast reaction rate, high yield, and good selectivity. The alkenyl selenide compounds formed by the reaction have good stability under acidic, alkaline, and oxidative conditions, and can remain stable at room temperature for several months. This provides a new and robust approach for the selective modification and labeling of selenium groups in bioconjugations, peptides, and / or proteins, and also provides a new method for the construction of cyclic peptides, offering a powerful tool for the development of selenium-modified peptide drugs. Detailed Implementation

[0063] To explain the technical content, objectives, and effects of the present invention in detail, the following specific embodiments are provided to further illustrate the content of the present invention. However, the content of the present invention is far more than the following examples.

[0064] Examples 1-2 below describe the selenization reaction of acetylacetamide.

[0065] Example 1

[0066]

[0067] In a 4 mL reaction flask equipped with a magnetic stir bar, 0.12 mmol of selenophenol A-1, 0.1 mmol of acetylene amide B-1, 0.24 mmol of sodium carbonate and 1 mL of isopropanol were added. The mixture was stirred at room temperature. The reaction was monitored by thin-layer chromatography (TLC). The reaction ended after 5 min. Compound C-1 was obtained by column chromatography with a yield of 99%.

[0068] NMR data for compound C-1: 1 H NMR (400 MHz, Methylene Chloride-d2) δ 7.72 (d,J = 8.2 Hz, 2H), 7.53 – 7.47 (m, 2H), 7.40 – 7.35 (m, 2H), 7.33 – 7.27 (m,3H), 6.40 (d, J = 7.2 Hz, 1H), 6.28 (d, J = 7.3 Hz, 1H), 3.03 (s, 3H), 2.45(s, 3H). 13 C NMR (100 MHz, Methylene Chloride-d2) δ 144.6, 134.3, 132.2, 131.4, 130.2, 129.6, 129.6, 127.8, 127.7, 116.2, 36.4, 21.7.

[0069] Example 2

[0070]

[0071] In a 4 mL reaction flask equipped with a magnetic stir bar, 0.12 mmol of selenophenol A-1, 0.1 mmol of acetylene amide B-2, 0.24 mmol of sodium carbonate and 1 mL of isopropanol were added. The mixture was stirred at room temperature. The reaction was monitored by thin-layer chromatography (TLC). The reaction ended after 5 min. Compound C-2 was obtained by column chromatography with a yield of 90%.

[0072] NMR data for compound C-2: 1 H NMR (400 MHz, Methanol-d4) δ 7.53 – 7.48 (m,2H), 7.35 – 7.27 (m, 3H), 6.59 (d, J = 7.1 Hz, 1H), 6.35 (d, J = 7.1 Hz, 1H), 3.15 (s, 3H), 2.96 (s, 3H). 13 C NMR (100 MHz, Methanol-d4) δ 133.0, 132.0, 130.5, 130.3, 128.5, 117.1, 36.4, 36.4.

[0073] Examples 3-15 below illustrate the application of selenylamide hydrogenation reaction in the selective modification of selenized peptides and proteins.

[0074] Example 3

[0075]

[0076] In a 10 mL reaction flask equipped with a magnetic stir bar, selenopeptide A-3 (0.02 mmol), acetylene amide B-1 (0.05 mmol), reducing agent DTT 2.5 eq., and 4 mL of a mixture of phosphate buffer (0.2 M, pH 7.6) and acetonitrile (volume ratio 5:3) were added. The mixture was stirred at 30 °C. The reaction was monitored by thin-layer chromatography (TLC). The reaction was completed in 1.5 h. Compound C-3 was obtained by column chromatography with a yield of 93%.

[0077] NMR data for compound C-3: 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 7.3 Hz, 1H), 6.20 (d, J = 7.2 Hz, 1H), 6.00 (d, J = 7.2 Hz, 1H), 5.20 (d, J = 7.3 Hz, 1H), 4.93 – 4.82 (m, 1H), 4.25– 4.11 (m, 1H), 3.69 (s, 3H), 3.18 – 3.02 (m, 2H), 2.89 (s, 3H), 2.39 (s,3H), 1.40 (s, 9H), 1.32 (d, J = 7.1 Hz, 3H); 13 C10 NMR (100 MHz, CDCl3) δ 172.6, 170.5, 155.4, 144.0, 133.9, 129.8, 128.9, 127.5, 116.0, 80.1, 52.7, 52.5, 50.1, 35.9, 29.0, 28.3, 21.5, 18.2; Low-resolution MS (ESI-TOF) m / z: calcd for C10 22 H 33 N3NaO7SSe + [M+Na] + : 586.1097, found 586.37.

[0078] Example 4

[0079]

[0080] In a 25 mL reaction flask equipped with a magnetic stir bar, selenopeptide A-3 (0.02 mmol), acetylene amide B-3 (0.025 mmol), reducing agent DTT 2.5 eq., and 10 mL of a mixture of phosphate buffer (0.2 M, pH 6.8) and acetonitrile (volume ratio 4:1) were added. The mixture was stirred at room temperature and the reaction was monitored by thin-layer chromatography (TLC). The reaction was completed after 50 min. Compound C-4 was obtained by column chromatography with a yield of 94%.

[0081] NMR data for compound C-4: 1H NMR (400 MHz, CD2Cl2) δ 7.25 (s, 1H), 6.67 (d, J= 6.6 Hz, 1H), 6.27 (d, J = 6.6 Hz, 1H), 5.41 – 5.34 (m, 1H), 5.31 – 5.29 (m,1H), 4.93 – 4.85 (m, 1H), 4.17 – 4.09 (m, 1H), 3.81 – 3.68 (m, 5H), 3.63 –3.55 (m, 1H), 3.39 – 3.10 (m, 3H), 1.42 (s, 9H), 1.30 (d, J = 7.1 Hz, 3H); 13 CNMR (100 MHz, CD2Cl2) δ 173.3, 171.0, 156.0, 128.6, 125.5, 120.5 (q, J = 323Hz), 80.5, 60.8, 53.2, 53.2, 53.2, 50.5, 29.5, 28.5, 18.4.

[0082] Example 5

[0083]

[0084] In a 10 mL reaction flask equipped with a magnetic stir bar, selenopeptide A-4 (0.02 mmol), acetylamide B-3 (0.03 mmol), reducing agent DTT 2.5 eq., and 4 mL of phosphate buffer (0.2 M, pH 6.8) were added. The mixture was stirred at room temperature and monitored by high performance liquid chromatography (HPLC). The reaction ended in 5 min with a conversion rate of 99%. Compound C-5 was obtained by semi-preparative separation and purification with a yield of 69%.

[0085] NMR data for compound C-5: 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.56 (d,J = 7.9 Hz, 1H), 8.25 (s, 1H), 8.12 – 7.95 (m, 3H), 7.80 (s, 2H), 7.39 (s,1H), 7.28 – 7.24 (m, 4H), 7.23 – 7.17 (m, 1H), 7.08 (s, 1H), 7.04 (d, J = 6.6Hz, 1H), 6.42 (d, J = 6.6 Hz, 1H), 5.61 – 5.53 (m, 1H), 5.02 – 4.93 (m, 1H),4.68 – 4.47 (m, 2H), 4.23 – 4.13 (m, 1H), 3.86 (s, 1H), 3.80 – 3.75 (m, 2H),3.63 – 3.48 (m, 5H), 3.18 – 3.04 (m, 2H), 2.98 – 2.90 (m, 1H), 2.87 – 2.79(m, 1H), 2.79 – 2.71 (m, 2H), 1.75 – 1.62 (m, 1H), 1.57 – 1.45 (m, 3H), 1.37– 1.24 (m, 2H), 1.15 (d, J = 6.3 Hz, 3H); 13 C NMR (100 MHz, DMSO-d6) δ 173.5,170.7, 170.0, 168.3, 166.9, 137.3, 129.2, 128.6, 128.2, 126.4, 123.1, 119.6(q, J = 322 Hz), 66.0, 57.9, 57.8, 54.1, 53.4, 52.1, 51.6, 42.1, 38.7, 37.4,31.4, 28.3, 26.7, 22.3, 19.6。

[0086] MS (ESI-TOF) m / z: calcd for C 29 H 46 F3N8O9SSe + [M+H] + : 819.2220, found819.2221。

[0087] The relevant materials were analyzed by HPLC. The detection conditions were as follows: a Jupiter® 5μm C18 300 Å column, 250×4.6 mm; mobile phase A was an acetonitrile solution containing 0.039 vol.% trifluoroacetic acid (TFA) and 10 vol.% water; mobile phase B was an aqueous solution containing 0.045 vol% trifluoroacetic acid (TFA); the gradient elution program was to increase the proportion of mobile phase A from 10% to 100% within 30 minutes; the flow rate was 1.0 mL / min; and the detection wavelength was 214 nm.

[0088] Results: The retention time of raw material A-4 was 12.357 min, the retention time of acetylene amide B-3 was 13.980 min, the retention time of the reaction solution for preparing C-5 was 12.083 min, and the retention time of purified C-5 was 12.087 min.

[0089] Example 6

[0090]

[0091] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-5 (0.005 mmol), acetylamide B-3 (0.0065 mmol), reducing agent DTT 2.5 eq., and 1 mL of phosphate buffer (0.2 M, pH 6.8) were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction ended in 10 min with a conversion rate of 99%. Compound C-6 was obtained by semi-preparative separation and purification, with a yield of 76%. MS (ESI-TOF) m / z: calcd for C 46 H 67 F3N 11 O 12 SSe + [M+H] + :1134.3804, found 1134.3801.

[0092] The relevant materials were analyzed by HPLC, and the detection conditions were the same as in Example 5.

[0093] Results: The retention time of raw material A-5 was 12.273 min, the retention time of acetylene amide B-3 was 13.980 min, the retention time of the reaction solution for preparing C-6 was 14.153 min, and the retention time of purified C-6 was 14.063 min.

[0094] Example 7

[0095] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-6 (0.005 mmol), acetylamide B-3 (0.0065 mmol), reducing agent DTT 2.5 eq., and 1 mL of phosphate buffer (0.2 M, pH 6.8) were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction ended in 10 min with a conversion rate of 99%. Compound C-7 was obtained by semi-preparative separation and purification, with a yield of 72%. MS (ESI-TOF) m / z: calcd for C 30 H 48 F3N 10 O 12 S2Se + [M+H] + :941.2006, found 941.2001.

[0096] The relevant materials were analyzed by HPLC, and the detection conditions were the same as in Example 5.

[0097] Results: The retention time of raw material A-6 was 5.983 min, the retention time of acetylene amide B-3 was 13.980 min, the retention time of the reaction solution for preparing C-7 was 10.920 min, and the retention time of purified C-7 was 10.777 min.

[0098] Example 8

[0099]

[0100] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-7 (0.005 mmol), acetylamide B-3 (0.0065 mmol), reducing agent DTT 2.5 eq., and 1 mL of phosphate buffer (0.2 M, pH 6.8) were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction ended in 10 min with a conversion rate of 99%. Compound C-8 was obtained by semi-preparative separation and purification, with a yield of 77%. MS (ESI-TOF) m / z: calcd for C 39 H 60 F3N 14 O 14 SSe + [M+H] + :1117.3246, found 1117.3240.

[0101] The relevant materials were analyzed by HPLC, and the detection conditions were the same as in Example 5.

[0102] Results: The retention time of raw material A-7 was 8.300 min, the retention time of acetylene amide B-3 was 13.980 min, the retention time of the reaction solution for preparing C-8 was 11.960 min, and the retention time of purified C-8 was 11.927 min.

[0103] Example 9

[0104]

[0105] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-8 (0.005 mmol), acetylamide B-3 (0.0065 mmol), reducing agent DTT 5 eq., and 1 mL of phosphate buffer (0.2 M, pH 6.8) were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction was completed in 10 min. Compound C-9 was obtained by semi-preparative separation and purification, along with a C-9 dimer (with thiol groups forming disulfide bonds). This dimer could be converted back to C-9 by adding the reducing agent tributylphosphine. The overall yield of C-9 was 82%. High-resolution MS (ESI-TOF) m / z of the C-9 dimer: calcd for C 92 H 147 F6N 26 O 26 S4Se2 + [M+H] + : 2433.8092, found 2433.8044. Low-resolution MS (ESI-TOF) m / z for C-9: calcd for C 46 H 75 F3N 13 O 13 S2Se + [M+H] + : 1218.4161, found 1218.20.

[0106] The relevant materials were analyzed by HPLC, and the detection conditions were the same as in Example 5.

[0107] Results: The retention time of raw material A-8 was 14.127 min, the retention time of acetylene amide B-3 was 13.980 min, the retention time of the reaction solution for preparing C-9 was 12.210 min, the retention time of the thiol group obtained during the purification of C-9 oxidized to a disulfide bond compound was 13.460 min, and the retention time of the disulfide bond compound obtained after purification of C-9 with the addition of reducing agent Bu3P was 12.003 min.

[0108] Example 10

[0109]

[0110] In a 4 mL reaction flask equipped with a magnetic stir bar, selenopeptide A-9 (0.002 mmol), acetylamide B-3 (0.005 mmol), reducing agent DTT (0.005 mmol), and 1 mL of phosphate buffer (0.2 M, pH 6.8) were added. The mixture was stirred at room temperature and monitored by high performance liquid chromatography (HPLC). The reaction ended after 5 min. Compound C-10 (with the same structure as C-5) was obtained by semi-preparative separation and purification, with a yield of 85%.

[0111] Example 11

[0112]

[0113] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-10 (0.002 mmol), indomethacin-modified acetyleneamide B-4 (0.005 mmol), reducing agent DTT (0.005 mmol), and 1 mL of a mixture of phosphate buffer (0.2 M, pH 6.8) and acetonitrile (1:1) were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction was completed in 1.5 h. Compound C-11 was obtained by semi-preparative separation and purification, with a yield of 51%. MS (ESI-TOF) m / z:calcd for C 48 H 60 ClF3N9O 12 SSe + [M+H] + : 1158.2882, found 1158.2878.

[0114] The relevant materials were analyzed by HPLC, and the detection conditions were the same as in Example 5.

[0115] Results: The retention time of raw material A-10 was 8.410 min, the retention time of acetylene amide B-4 was 28.493 min, the retention time of the reaction solution for preparing C-11 was 21.620 min, and the retention time of purified C-11 was 21.697 min.

[0116] Example 12

[0117]

[0118] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-11 (0.002 mmol), coumarin-modified acetyleneamide B-5 (0.005 mmol), reducing agent DTT (0.005 mmol), and 2 mL of a mixture of phosphate buffer (0.2 M, pH 6.8) and acetonitrile (1:1) were added. The mixture was stirred at room temperature, and the reaction was monitored by high-performance liquid chromatography (HPLC). The reaction was completed in 3 h. Compound C-12 was obtained by semi-preparative separation and purification, with a yield of 54%. MS (ESI-TOF) m / z: calcd for C 39 H 51 F3N9O 11 SSe + [M+H] + : 990.2541, found 990.2539.

[0119] The relevant materials were analyzed by HPLC, and the detection conditions were the same as in Example 5.

[0120] Results: The retention time of raw material A-11 was 8.410 min, the retention time of acetylene amide B-5 was 21.360 min, the retention time of the reaction solution for preparing C-12 was 15.877 min, and the retention time of purified C-12 was 15.873 min.

[0121] Example 13

[0122]

[0123] In a 4 mL reaction flask equipped with a magnetic stir bar, selenopeptide A-12 (0.002 mmol), polyethylene glycol-containing acetylacetamide B-6 (0.005 mmol), reducing agent DTT (0.01 mmol), and 4 mL of phosphate buffer (0.2 M, pH 6.8) were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction was completed in 0.5 h. Compound C-13 was obtained by semi-preparative separation and purification, with a yield of 82%. The MS of the product followed a normal distribution. When n = 11, the MS (ESI-TOF) m / z: calcd: 3303.7027, found 3303.1. When n = 12, the MS (ESI-TOF) m / z: calcd: 3391.7551, found 3391.3.

[0124] The relevant materials were analyzed by HPLC, and the detection conditions were the same as in Example 5.

[0125] Results: The retention time of raw material A-12 was 8.410 min, the retention time of acetylene amide B-6 was 17.800 min, the retention time of the reaction solution for preparing C-13 was 16.263 min, and the retention time of purified C-13 was 16.390 min.

[0126] Examples 14-18 below illustrate the application of selenylamide hydrogenation in the cyclization of selenized peptides and proteins.

[0127] Example 14

[0128]

[0129] In a 25 mL reaction flask equipped with a magnetic stir bar, selenopeptide A-13 (0.045 mmol), reducing agent tris(2-carboxyethyl)phosphine (TCEP) (0.135 mmol), sodium ascorbate (NaSAC, 0.27 mmol), and 15 mL of a 1:3 mixture of phosphate buffer (0.2 M, pH 6.8) and acetonitrile were added. The mixture was stirred at room temperature and monitored by high performance liquid chromatography (HPLC). The reaction was completed in 3 h. Compound C-14 was obtained by semi-preparative separation and purification with a yield of 82%.

[0130] NMR data for compound C-14: 1 H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 6.5 Hz,1H), 8.18 – 8.10 (m, 1H), 7.96 – 7.86 (m, 5H), 7.71 (dd, J = 7.4, 2.7 Hz,2H), 7.41 (td, J = 7.5, 3.2 Hz, 2H), 7.34 – 7.16 (m, 8H), 6.38 (d, J = 6.4Hz, 1H), 4.43 – 3.78 (m, 13H), 3.16 – 2.90 (m, 3H), 2.86 – 2.76 (m, 1H), 1.68– 1.57 (m, 1H), 1.55 – 1.40 (m, 2H), 1.10 (d, J = 7.2 Hz, 3H), 0.83 (d, J =6.2 Hz, 3H), 0.79 (d, J = 6.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 172.4,172.3, 171.1, 170.8, 169.5, 156.3, 143.8, 140.8, 137.3, 131.3, 129.2, 128.2,127.7, 127.2, 126.5, 125.4, 123.6, 120.2, 119.7 (q, J = 325.0), 66.0, 62.5,55.6, 55.2, 51.6, 49.6, 49.2, 46.6, 40.9, 40.3, 37.1, 28.3, 24.0, 23.2, 21.4,17.4. MS (ESI-TOF) m / z: calcd for C 43 H 49 F3N6NaO 10 SSe + [M+Na] + : 1001.2240, found1001.2253.

[0131] The relevant materials were detected by HPLC. The detection conditions were the same as those in Example 5, except that the gradient elution program increased the proportion of mobile phase A from 50% to 100% within 30 minutes.

[0132] Results: The retention time of the selenopolypeptide acid before esterification of raw material A-13 was 15.167 min, the retention time of raw material A-13 was 21.333 min, the retention time of the reaction solution during the preparation of C-14 was 16.227 min, and the retention time of purified C-14 was 16.230 min.

[0133] Example 15

[0134]

[0135] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-14 (0.005 mmol), reducing agent tris(2-carboxyethyl)phosphine (TCEP) (0.015 mmol), sodium ascorbate (NaSAC, 0.03 mmol), and 1 mL of a 1:3 mixture of phosphate buffer (0.2 M, pH 6.8) and acetonitrile were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction was completed after 20 min. Compound C-15 was obtained by semi-preparative separation and purification, with a yield of 72%. MS (ESI-TOF) m / z: calcd for C 40 H 44 F3N5NaO9SSe+ [M+Na] + : 930.1869, found 930.1874.

[0136] The relevant materials were analyzed by HPLC under the same conditions as in Example 14. The retention time of the selenopolypeptide acid before esterification of raw material A-14 was 15.940 min, the retention time of raw material A-14 was 22.517 min, the retention time of C-15 in the reaction solution during the preparation of C-15 was 17.863 min, and the retention time of purified C-15 was 17.983 min.

[0137] Example 16

[0138]

[0139] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-15 (0.005 mmol), reducing agent tris(2-carboxyethyl)phosphine (TCEP) (0.015 mmol), sodium ascorbate (NaSAC, 0.03 mmol), and 1 mL of a 1:3 mixture of phosphate buffer (0.2 M, pH 6.8) and acetonitrile were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction was completed after 55 min. Compound C-16 was obtained by semi-preparative separation and purification, with a yield of 78%. MS (ESI-TOF) m / z: calcd for C 42 H 54 F3N7NaO 11 SSe + [M+Na] + : 1024.2612, found 1024.2623.

[0140] The relevant materials were analyzed by HPLC under the same conditions as in Example 14. The retention time of the selenopolypeptide acid before esterification of raw material A-15 was 13.350 min, the retention time of raw material A-15 was 19.370 min, the retention time of C-16 in the reaction solution during the preparation of C-16 was 14.240 min, and the retention time of purified C-16 was 14.370 min.

[0141] Example 17

[0142]

[0143] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-16 (0.005 mmol), reducing agent tris(2-carboxyethyl)phosphine (TCEP) (0.015 mmol), sodium ascorbate (NaSAC, 0.03 mmol), and 1 mL of a 1:3 mixture of phosphate buffer (0.2 M, pH 6.8) and acetonitrile were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction was completed in 1 h. Compound C-17 was obtained by semi-preparative separation and purification, with a yield of 67%. MS (ESI-TOF) m / z: calcd for C 65 H 88 F3N 11 NaO 15 SSe + [M+Na] + : 1454.5192, found 1454.5216.

[0144] The relevant materials were analyzed by HPLC under the same conditions as in Example 14. The retention time of the selenopolypeptide acid before esterification of raw material A-16 was 20.037 min, the retention time of raw material A-16 was 25.177 min, the retention time of the reaction solution for preparing C-17 was 20.867 min, and the retention time of purified C-17 was 21.050 min.

[0145] Example 18

[0146]

[0147] In a 4 mL reaction flask equipped with a magnetic stirrer, selenopeptide A-17 (0.005 mmol), reducing agent tris(2-carboxyethyl)phosphine (TCEP) (0.015 mmol), sodium ascorbate (NaSAC, 0.03 mmol), and 1 mL of a 1:3 mixture of phosphate buffer (0.2 M, pH 6.8) and acetonitrile were added. The mixture was stirred at room temperature and monitored by high-performance liquid chromatography (HPLC). The reaction was completed after 2 h. Compound C-18 was obtained by semi-preparative separation and purification, with a yield of 70%. MS (ESI-TOF) m / z: calcd for C 57 H 73 F3N 10 NaO 14 SSe + [M+Na] + : 1313.4038, found 1313.4073.

[0148] The relevant materials were analyzed by HPLC under the same conditions as in Example 14. The retention time of the selenopolypeptide acid before esterification of raw material A-17 was 15.037 min, the retention time of raw material A-17 was 20.871 min, the retention time of the reaction solution for preparing C-18 was 15.083 min, and the retention time of purified C-18 was 15.233 min.

[0149] Example 19 below illustrates the application of the selenium hydrogenation reaction of acetylacetamide in the modification of selenium-substituted polypeptides and protein sequences in the presence of cysteine ​​(Cys).

[0150] Example 19

[0151]

[0152] First, in a 4 mL reaction flask equipped with a magnetic stir bar, selenocysteine-containing selenopeptide A-18 (0.005 mmol), reducing agent tris(2-carboxyethyl)phosphine (TCEP) (0.015 mmol), sodium ascorbate (NaSAC, 0.03 mmol), and 2 mL of phosphate buffer (0.2 M, pH 5.0) were added. Then, 2 eq of biotin-modified acetyleneamide B-4 were added. The reaction was monitored by high-performance liquid chromatography (HPLC). The reaction was completed in 25 min, yielding intermediate C-19, a selenocysteine-selectively modified acetyleneamide B-4. MS (ESI-TOF) m / z: calcd for C 108 H 168 F3N 36 O 26 S4Se + [M+H] + :2650.0925, found 2650.0931. Subsequently, sodium hydroxide was added to the reaction system to adjust the pH to 8, followed by the addition of 5 eq. of acetylide B-3. The reaction was monitored by high-performance liquid chromatography (HPLC), and the reaction ended after 50 min. Semi-preparative separation and purification yielded compound C-20, sequentially modified by two acetylide amides, in 25% yield. MS (ESI-TOF) m / z: calcd for C 112 H 170 F6N 37 O 29 S5Se + [M+H] + : 2851.0633, found 2851.0640.

[0153] The relevant materials were analyzed by HPLC under the same conditions as in Example 5. The retention time of raw material A-18 was 7.60 min, the retention time of intermediate C-19 modified by the first acetylene amide B-4 was 9.61 min, and the retention time of product C-20 modified by the second acetylene amide B-3 was 10.20 min.

[0154] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The selenization reaction of acetylamides, characterized in that, The reaction involves the reaction of a selenium-containing compound (A) with an acetylamid compound (B) in a solvent with the addition of an additive to obtain product (C), as shown in the general formula (1) below: （1）， Among them, R 1 Selected from C1 to C 22 Alkyl, C4-C 10 One or more of the following: aromatic ring group, substituted aromatic ring group, heterocyclic aryl group, alkenyl group, alkynyl group, protected α-selenocysteine ​​residue, β-selenocysteine ​​residue, γ-selenocysteine ​​residue, and alkyl group of polypeptide chain. R 2 It is selected from one or more of alkyl, hydroxyethyl, propargyl, indomethacin-containing groups, biotin-containing groups, and coumarin-containing fluorescent groups. EWG is selected from one or more of the following: C1-C5 alkylsulfonyl, C1-C5 alkylyl, C6-C10 arylsulfonyl, and C6-C10 arylyl.

2. The selenium hydrogenation reaction of the acetylamidoid compound according to claim 1, characterized in that, The additive is selected from one or more of the following: phosphate buffer, N,N-diisopropylethylamine, triethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, 4-dimethylaminopyridine, cesium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, imidazole, N-methylimidazolium, pyridine, sodium ethoxide, lithium ethoxide, DBU, TCEP, DTT, Bu3P, and ascorbate, with sodium carbonate being preferred.

3. The selenium hydrogenation reaction of the acetylamides according to claim 1, characterized in that, The solvent is selected from one or more of dichloromethane, dichloroethane, trichloroethane, dimethyl sulfoxide, methanol, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, ethyl acetate, methanol, isopropanol, n-butanol, and buffer solutions; preferably, the solvent is isopropanol, or a mixture of acetonitrile and a phosphate buffer solution with pH=6.

8.

4. The use of the selenium hydrogenation reaction of the acetylamides as described in claim 1, characterized in that, The application is for the selective modification of selenopeptides, specifically by using an acetylamid compound (B) to perform a selenization reaction with selenools in the selenopeptide, wherein the reaction is carried out in a weakly acidic system; preferably, it is used for the selective modification and labeling of side-chain selenools in peptides and proteins.

5. The use according to claim 4, characterized in that, The weakly acidic reaction system is a mixture of a slightly acidic buffer solution and an organic solvent; The volume ratio of the slightly acidic buffer solution to the organic solvent is 1 to 50:1, preferably 5 to 40:1, and more preferably 10 to 30:1; The slightly acidic buffer solution is selected from PB buffer and Tris buffer; the organic solvent is selected from acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; and / or The molar ratio of the acetylamid compound having general structural formula (B) to the selenium-containing peptide or protein having general structural formula (A) is 1-10:1, preferably 1.1-4:1, and more preferably 1.2-2:

1.

6. The use according to claim 5, characterized in that, The specific steps of the reaction are as follows: a acetylacetamide compound having the general structural formula (B) and a polypeptide or protein containing selenocysteine ​​having the general structural formula (A) are dissolved in a weakly acidic reaction system, mixed evenly, and then reacted. The reaction is carried out by stirring at 0-50°C for 1-120 min, preferably at 25-40°C for 5-50 min. After the reaction is completed, the alkenyl selenide compound having the general structural formula (C) is obtained by column chromatography.

7. The use of the selenylamide compound of claim 1 in the hydrogenation reaction of selenium, characterized in that, The application is for the selective cyclization modification of selenopolypeptides. Specifically, the method involves linking the acetylamid compound (B) in reaction formula (1) to the selenopolypeptide (i.e., a polypeptide containing selenocysteine) via an amide bond / ester bond / ether bond, and then, under weakly alkaline conditions, performing intramolecular cyclization of the selenool via hydrogenation. Preferably, the method is used for the selective cyclization modification of side-chain selenools in peptides and proteins, and includes the following reaction formulas (2) and (3): that is, selenopolypeptides D or F (which can be attached to a solid support or are free) containing acetylamid compounds undergo cyclization in a weakly alkaline reaction system, ultimately obtaining alkenyl selenide compounds with the structural formula (E) or (G). （2） （3） Among them, R 4 One or more of the following: H, Fmoc, Fmoc-protected polypeptide chain alkyl, Boc, Boc-protected polypeptide chain alkyl, Cbz, protected polypeptide chain alkyl, Ac, Ac-protected polypeptide chain alkyl; AA n These are amino acids in the peptide chain that have been modified with acetylacetamide compounds; EWG is selected from one or more of C1~C5 alkylsulfonyl, C1~C5 alkylyl, C6~C10 arylsulfonyl, and C6~C10 arylyl. The additives are selected from one or more of the following: phosphate buffer, N,N-diisopropylethylamine, triethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, 4-dimethylaminopyridine, cesium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, imidazole, N-methylimidazolium, pyridine, sodium ethoxide, lithium ethoxide, DBU, TCEP, DTT, Bu3P, and ascorbate. The solvent is selected from one or more of the following: dichloromethane, dichloroethane, trichloroethane, dimethyl sulfoxide, methanol, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, ethyl acetate, methanol, isopropanol, n-butanol, and buffer solutions. Preferably, the weakly alkaline reaction system is a mixture of an organic solvent and a slightly alkaline buffer solution; The volume ratio of the organic solvent to the slightly alkaline buffer solution is 1 to 50:1, preferably 1 to 30:1, and more preferably 1 to 10:1; The alkaline buffer solution is selected from PB buffer and Tris buffer; the organic solvent is selected from acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

8. The use of the selenium hydrogenation reaction of the acetylamides as described in claim 1, characterized in that, The application is for the cyclization modification of diselenyl peptides. The specific method includes the following reaction formula (4): an acetylacetamide compound having structural formula I reacts with a peptide H containing two selenocysteine ​​residues under weakly alkaline conditions to achieve the cyclization of peptide H containing two selenocysteine ​​residues through the selenization of selenools; preferably, it is used for the cyclization modification of side-chain selenools in peptides and proteins. Finally, an alkenyl selenide compound having structural formula J is obtained. （4） Among them, R 5 It is one or more of the following: H, Fmoc, Fmoc-protected polypeptide residues, Boc, Boc-protected polypeptide chain residues, Cbz, protected polypeptide chain residues, Ac, Ac-protected polypeptide chain residues. R 6 -OH, -NH2, C1-C5 alkoxy groups, C6-C 10 One or more of the following: phenolic group, amino acid residue, carboxyl-protected amino acid residue, and solid-phase support linked by ester or amide bonds; The additive is selected from one or more of the following: phosphate buffer, N,N-diisopropylethylamine, triethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, 4-dimethylaminopyridine, cesium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, imidazole, N-methylimidazolium, pyridine, sodium ethoxide, lithium ethoxide, DBU, TCEP, DTT, Bu3P, and ascorbate. The solvent is selected from one or more of the following: dichloromethane, dichloroethane, trichloroethane, dimethyl sulfoxide, methanol, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, ethyl acetate, methanol, isopropanol, n-butanol, and buffer solutions. The weakly alkaline reaction system is a mixture of an organic solvent and a slightly alkaline buffer solution; The volume ratio of the organic solvent to the slightly alkaline buffer solution is 1 to 50:1, preferably 1 to 30:1, and more preferably 1 to 10:1; Preferably, the alkaline buffer solution is selected from PB buffer and Tris buffer; the organic solvent is selected from acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

9. The use according to claim 8, characterized in that, The reaction involves dissolving a polypeptide H containing two selenocysteines and an acetylacetamide compound with general structural formula I in a weakly alkaline reaction system, mixing them thoroughly, and then reacting. Specifically, the reaction is carried out by stirring at 0–50°C for 1–360 min, preferably at 20–40°C for 5–300 min. After the reaction is completed, the cyclic compound with general structural formula J is obtained by column chromatography, recrystallization, or semi-preparative separation.

10. The use of the selenium hydrogenation reaction of the acetylamides as described in claim 1, characterized in that, The intended use is to modify selenomeric peptides in the presence of cysteine ​​by adjusting the pH of the reaction system and combining the hydrogen sulfide reaction of acetylamide. The specific method is as follows: including the following general reaction formula (5), the acetylamide compound (B1) in the general reaction formula (1) is first used in a weakly acidic solvent 1 to selectively modify the selenomeric peptide (K) containing cysteine ​​under the combined action of additives. After the reaction, an alkenyl selenide compound with the general structural formula L is obtained. Then, in a weakly alkaline solvent 2, the acetylamide compound (B2) in the general reaction formula (1) is added, and the hydrogen sulfide reaction of acetylamide is used to further modify cysteine. Finally, a compound with the general structural formula M containing both alkenyl selenide and alkenyl sulfide, which is sequentially modified by selenocysteine, is obtained. （5） Among them, R 4 One or more of the following: H, Fmoc, Fmoc-protected polypeptide chain alkyl, Boc, Boc-protected polypeptide chain alkyl, Cbz, protected polypeptide chain alkyl, Ac, Ac-protected polypeptide chain alkyl; AA is an amino acid in a peptide chain; EWG is selected from one or more of C1~C5 alkylsulfonyl, C1~C5 alkylyl, C6~C10 arylsulfonyl, and C6~C10 arylyl. The molar ratio of the acetylamid compound BI or B2 having the general structural formula (B) to the selenium-containing peptide or protein having the general structural formula (K) is 1-10:1, preferably 1.1-5:

1. The additive is selected from one or more of the following: phosphate buffer, N,N-diisopropylethylamine, triethylamine, sodium carbonate, potassium carbonate, sodium bicarbonate, 4-dimethylaminopyridine, cesium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, imidazole, N-methylimidazolium, pyridine, sodium ethoxide, lithium ethoxide, DBU, TCEP, DTT, Bu3P, and ascorbate. Solvent 1 and solvent 2 are selected from one or more of the following: dichloromethane, dichloroethane, trichloroethane, dimethyl sulfoxide, methanol, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, n-pentane, cyclohexane, diethyl ether, petroleum ether, ethyl acetate, methanol, isopropanol, n-butanol, dilute hydrochloric acid, aqueous sodium hydroxide solution, and buffer solution. Preferably, the acidic / alkaline buffer solution is selected from PB buffer and Tris buffer; the organic solvent is selected from acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

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