Polypeptide editing method based on methionine desulfurization strategy

Abstract

The invention relates to a polypeptide editing method based on a methionine desulfurization strategy, and the method comprises the following steps: 1) a reaction formula 1 is shown in the specification, (2) as shown in a reaction formula 2, the target sulfonium 1, electron-deficient olefin 2, a photocatalyst and N-heterocyclic carbene-borane are dissolved in the solvent, and a blue light irradiation reaction is performed at room temperature to obtain a modified product 3, and (3) as shown in the reaction formula 2, the target sulfonium 1, the electron-deficient olefin 2, the photocatalyst and the N-heterocyclic carbene-borane are dissolved in the solvent. According to the method, the desulfurization modification of methionine is realized by utilizing the homocleavage substitution reaction of sulfonium, on one hand, the modification strategy provides more choices for a biological coupling mode; on the other hand, the polypeptide obtained through modification has the advantages in the aspect of structural stability compared with a modified product in the prior art.

Description

Technical Field

[0001] This application belongs to the field of peptide or protein modification, specifically a peptide editing method based on a methionine desulfurization strategy. Background Technology

[0002] Biomolecules such as peptides and proteins are essential components of living organisms and participate in various fundamental life activities. Therefore, research on the structure and function of biomolecules such as peptides and proteins has always been a hot topic in life sciences. Through chemical covalent modification strategies, biomolecules can be endowed with different structural and functional properties, greatly promoting related research and leading to numerous successful applications in cutting-edge fields such as chemical biology, biomaterials, and biomedicine.

[0003] Genetic codon amplification technology allows for the precise insertion of non-natural amino acids into target locations within proteins, followed by bioorthogonal reactions to regulate the structure and function of these proteins—a common research strategy in chemical biology. However, this strategy also faces challenges such as high experimental technical barriers and potential impact on target protein expression levels. In contrast, covalent modification of specific types or sites of amino acid residues in natural proteins (peptides) via chemical reactions can achieve their regulation. This strategy is simple to implement, does not affect protein expression, and serves as a valuable complement to the aforementioned non-natural amino acid insertion strategy.

[0004] Traditional bioconjugation relies on the nucleophilic properties of amino acid residues to react with suitable electrophilic reagents, thereby achieving selective modification of natural proteins (peptides). Currently, the most mature bioconjugation methods are based on cysteine ​​and lysine. In recent years, with the development of novel chemical reactions, especially redox reactions, bioconjugation methods targeting aromatic amino acid residues have been reported, with successful examples including those targeting tyrosine, tryptophan, and histidine.

[0005] Methionine, another sulfur-containing natural amino acid, plays a crucial role not only as a starting amino acid in protein synthesis but also, similar to cysteine, is closely related to intracellular redox responses. Furthermore, methionine has a relatively low natural abundance among natural amino acids, which facilitates better reaction selectivity. These unique biological functions and relatively low natural abundance make chemical biology research on methionine an important research topic. Developing methionine-selective bioconjugation methods can not only provide important research tools for this research but also offer novel approaches to the structural and functional modification of biomolecules.

[0006]

[0007] In 2017, Christopher J. Chang's research group developed a methionine-selective bioconjugation strategy using oxocyclopropane substrates, which can efficiently convert the thioether structure of methionine into a thionimide structure. This strategy was applied to the preparation of antibody-drug conjugates and methionine-based proteomics research. In 2018, Matthew J. Gaunt et al. achieved a methionine-selective S-alkylation strategy using high-valent iodine reagents, providing a novel chemical method for the selective modification of proteins (see Equation (1) above). As mentioned above, both methods couple the modifying group to the sulfur atom site of methionine. In contrast, in 2020, David WCMacMillan's research group achieved selective modification of the methyl site of methionine using a suitable photocatalyst and applied this method to the covalent modification of a series of proteins (see Equation (2) above).

[0008] In summary, current modification strategies for methionine mainly focus on coupling modifying groups to the sulfur or methyl groups, which shows certain limitations in terms of the innovation and diversity of chemical structures. Summary of the Invention

[0009] Technical issues

[0010] Based on the aforementioned research significance, research foundation, and existing limitations, the inventors aim to develop a novel bioconjugation method based on methionine, overcoming the limitations of current bioconjugation methods based on methionine in terms of the innovation and diversity of chemical structures.

[0011] Technical solution

[0012] This invention aims to provide a peptide editing method based on a methionine desulfurization strategy. This method represents a novel peptide editing strategy, offering a new technical means for bioconjugation and showing great promise for peptide modification. This invention also aims to provide peptides edited using the above method.

[0013] On one hand, the present invention provides a peptide editing method based on a methionine desulfurization strategy, the method comprising the following steps:

[0014] (1) Preparation of thionium:

[0015]

[0016] As shown in reaction formula 1 above, the substrate (Met-X)1' and S1, which have a methyl thioether structure, are dissolved in a solvent, acid is added, and the reaction is stirred at room temperature to obtain the target thionium 1.

[0017] The substrate (Met-X)1' having a methyl sulfide structure is selected from:

[0018]

[0019] Wherein, case (i) represents unmodified or modified methionine;

[0020] Case (ii) represents a polypeptide or protein containing methionine, wherein the amino acid side chains of the polypeptide or protein do not contain alkenyl groups, and

[0021] The position of methionine in the polypeptide or protein is not limited;

[0022] (2) Desulfurization reaction:

[0023]

[0024] As shown in reaction formula 2 above, the target thionium 1, electron-deficient olefin 2, photocatalyst, and azacarbene-borane are dissolved in a solvent, and the reaction is carried out under blue light irradiation at room temperature to obtain the modified product 3.

[0025] or

[0026] The method includes the following steps:

[0027] (1') Preparation of thionium:

[0028]

[0029] As shown in reaction formula 1' above, the polypeptide or protein 4' containing methionine and S1 are dissolved in a solvent, acid is added, and the reaction is stirred at room temperature to obtain the target thionium 4.

[0030] The methionine-containing polypeptide or protein 4' further has at least one acrylated amino acid, the position of methionine in the polypeptide or protein is not limited, and the position of the acrylated amino acid in the polypeptide or protein 4' is any position other than methionine.

[0031] (2') Desulfurization reaction:

[0032]

[0033] As shown in reaction formula 2' above, the target thionium 4, photocatalyst, and azircone-borane are dissolved in a solvent and irradiated with blue light at room temperature to obtain a cyclic peptide structure 5 linked by a valeryl linking group.

[0034] In a specific embodiment, in step (1), the substrate 1' having the methyl thioether structure is selected from 1a' to 1m': their corresponding amino acid sequences are SEQ ID No.:1-13, respectively.

[0035]

[0036] In a specific embodiment, in step (1'), the N-terminal amino acid of the polypeptide or protein 4' containing methionine is acrylated.

[0037] In a specific embodiment, in step (1'), the polypeptide or protein 4' containing methionine contains acrylated lysine.

[0038] In a specific embodiment, in step (1'), the polypeptide or protein 4' containing methionine is selected from 4a'-4h': the corresponding amino acid sequences are SEQ ID No.:14-21 respectively:

[0039]

[0040] In a specific embodiment, in step (1) or (1'), an acid is used for catalysis, and the acid can also inhibit the side reaction of alkylation of lysine and other side chains. The acid is selected from formic acid, acetic acid, trifluoroacetic acid, preferably formic acid, and the concentration of the acid is 1% to 100% (volume ratio), preferably 5%. The solvent is selected from water, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 1,4-dioxane or combinations thereof; preferably a mixed solvent of acetonitrile and water with a volume ratio of 1:1, and the reaction time is 6-12 hours.

[0041] In a specific embodiment, in step (2), EWG in the electron-deficient olefin 2 structure represents an electron-withdrawing group, such as a group containing carbonyl, sulfonyl, phosphono, nitro, cyano, trifluoromethyl, and electron-deficient heterocyclic groups and their derivative groups.

[0042] In a specific embodiment, the electron-deficient olefin is selected from the following 2a-2r:

[0043]

[0044] In a specific embodiment, in step (2) or (2'), the azacarbene-borane is selected from the following:

[0045]

[0046] In a specific embodiment, in step (2) or (2'), the solvent is selected from: water, formic acid, acetic acid, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, methanol or a combination thereof; preferably dimethyl sulfoxide.

[0047] In a specific embodiment, in step (2) or (2'), the photocatalyst is selected from: [Ir(dtbbpy)(ppy)2]PF6, Ir[dF(CF3)ppy]2(dtbbpy)PF6, fac-Ir(ppy)3, fac-Ir(dF-ppy)3, [Ru(bpy)3][PF6]2, [Ru(bpz)3][PF6]2, [Ru(phen)3][PF6]2, [Ru(bpm)3][PF6]2, [Ru(bpz)3][PF6]2, 4CzIPN, Eosin Y, Fluorescein, preferably fac-Ir(ppy)3.

[0048] In a specific implementation, in step (2) or (2'), the wavelength of the blue light is 254nm-600nm, preferably 420nm-440nm.

[0049] In a specific implementation, in step (2) or (2'), the blue light irradiation time is 15-60 minutes.

[0050] In a specific embodiment, the method further includes a step of semi-preparative HPLC purification and freeze-drying of the intermediate products of steps (1) and (1') or the final products of steps (2) and (2').

[0051] On the other hand, the present invention provides polypeptides or proteins edited by the above method.

[0052] In a specific embodiment, the polypeptide or protein is selected from the following:

[0053]

[0054]

[0055]

[0056]

[0057] In another aspect, the present invention provides the use of polypeptides having the following structure in the preparation of antibacterial reagents.

[0058]

[0059] In a specific embodiment, the antibacterial agent has antibacterial activity against methicillin-resistant Staphylococcus aureus USA300, vancomycin-resistant Enterococcus faecalis O649, Gram-negative Escherichia coli AB1157, Acinetobacter baumannii (Aba.), and Pseudomonas aeruginosa PAO1.

[0060] Beneficial effects

[0061] The inventors of this application utilize the homolytic substitution reaction of thionium to achieve the desulfurization modification of methionine. This modification strategy differs significantly from existing strategies, offering substantial innovation in the diversity of chemical structures and providing a novel method for the covalent modification of methionine, thus offering more options for bioconjugation. Furthermore, the peptides modified in this application exhibit superior structural stability compared to modified products from existing technologies. Detailed Implementation

[0062] The specific operation process of the method of this application will be described in detail below through embodiments to enable those skilled in the art to better understand the present invention. However, the provision of these embodiments is not intended to limit the scope of this application.

[0063] the term:

[0064] In this application, the letters used in the polypeptide sequence represent amino acids as commonly understood in the art. Additionally, as will be readily understood by those skilled in the art, the intermediate and final products generated in the methods of this application contain... Used to indicate the portion of methionine with the side chain removed.

[0065] In the following examples, the starting peptides used are as follows:

[0066]

[0067]

[0068] It should be noted that for the above acrylamide peptides, N-terminal acrylamide refers to the NH2 group of the N-terminal amino acid being acrylamided; while intra-chain amino acid acrylamide refers to the side chain group of that amino acid being acrylamided.

[0069] The electron-deficient olefin structures used are as follows:

[0070]

[0071] Example 1:

[0072]

[0073] The first step involved dissolving peptide 1a' (1.0 equivalent) and S1 (5.0 equivalent) in a mixed solvent of acetonitrile / water (1:1, volume ratio), adding 5% formic acid, and stirring at room temperature for 6-12 hours. After the reaction was completed by HPLC monitoring, the target thionium 1a was obtained by semi-preparative HPLC purification and freeze-drying.

[0074] In the second step, the thionium 1a (0.02 mmol, 1.0 equivalent), electron-deficient olefin 2a (1.1 equivalent), photocatalyst fac-Ir(ppy)3 (0.01 equivalent), and azacarbene-borane (NHC-BH3-1, 1.1 equivalent) obtained in the previous step were dissolved in dimethyl sulfoxide (0.4 mL). The mixture was irradiated with blue light at room temperature for 15-60 minutes. After washing the reaction system three times with diethyl ether, the mixture was analyzed by LC-MS. After purification by semi-preparative HPLC and lyophilization, the modified peptide 3aa was obtained.

[0075] The target product 3aa was obtained in 85% by HPLC and 73% by separation. ESI-MS: Ca was calculated. 39 H 54 N8O 11 Sm / z = 842.3633, measured 843.36 [M+H] + .

[0076] 1 ¹H NMR (800MHz, deuterium oxide) δ 7.76 (ddt, J = 7.2, 2.2, 1.3Hz, 2H), 7.73–7.68 (m, 1H), 7.56 (dddd, J = 8.5, 6.3, 2.5, 1.3Hz, 2H), 7.53–7.46 (m, 1H), 7.38 (dq, J = 8.2, 1.3Hz, 1H), 7.17 (q, J = 2.1Hz, 1H), 7.12 (ddt, J = 8.1, 6.9, 1.2Hz, 1H), 7.03 (ddt, J = 8.0, 7.0, 1.1Hz, 1H), 4.53–4.44 (m, 2H), 4.28 (ddd, J = 8.9, 4.3, 2.0Hz, 1H), 4.22–4. 18(m,1H),4.16–4.12(m,1H),4.09(ddd,J=8.6,4.3,1.6Hz,1H),3.21–3.12(m, 3H),3.08(dd,J=14.8,7.7Hz,1H),2.88(td,J=7.8,3.1Hz,2H),2.55–2.44(m,2 H),1.86(d,J=2.1Hz,3H),1.82(tdd,J=11.3,5.5,3.1Hz,1H),1.68(dt,J=18.7 ,6.0Hz,2H),1.61–1.49(m,6H),1.42–1.19(m,5H),1.15(dq,J=6.8,2.2Hz,3H).

[0077] 13C NMR (126MHz, deuterium oxide) δ 175.03, 174.24, 173.92, 173.55, 173.37, 171.94, 171.43, 136.12, 134.75, 129.79, 127.71, 127.06, 124.14, 121.99, 119.37, 118.33, 111.93, 108.93, 66.99, 59.41, 54.82, 53.39, 52.46, 50.16, 39.16, 35.57, 30.08, 26.87, 26.17, 23.58, 22.04, 21.88, 21.55, 19.06.

[0078] Example 2:

[0079] Replace 2a with 2b in Example 1, and the rest of the operation is the same.

[0080]

[0081] The yield of the target product 3ab was 89%. ESI-MS: C2 was calculated. 40 H 54 N8O 13 Sm / z = 886.3531, measured 887.43 [M+H] + .

[0082] Example 3:

[0083] Replace 2a with 2c in Example 1, and the rest of the operation is the same.

[0084]

[0085] The yield of the target product 3ac was 81%. ESI-MS: C2 was calculated. 43 H 57 N9O 12 Sm / z = 923.3847, measured 924.39 [M+H] + .

[0086] Example 4:

[0087] Replace 2a with 2d in Example 1, and the rest of the operation is the same.

[0088]

[0089] The yield of the target product 3ad was 78% (monosubstituted:disubstituted 16:1), ESI-MS: calculated C 37 H 59 N8O 12P m / z = 838.3990, measured 839.40 [M+H] + .

[0090] Example 5:

[0091] Replace 2a with 2e in Example 1, and the rest of the operation is the same.

[0092]

[0093] The yield of the target product 3ae in the reaction was 87%. ESI-MS calculated C2. 40 H 60 N8O 13 m / z = 860.4280, measured value 861.43 [M+H] + .

[0094] Example 6:

[0095] Replace 2a with 2f in Example 1, and the rest of the operation is the same.

[0096]

[0097] The yield of the target product 3af was 81% (monosubstituted:disubstituted 7:1), ESI-MS: calculated C 41 H 56 N8O 11 m / z = 836.4069, measured 837.40 [M+H] + .

[0098] Example 7:

[0099] Replace 2a with 2g in Example 1, and the rest of the operation is the same.

[0100]

[0101] The yield of the target product 3ag was 78%. ESI-MS calculated C2. 38 H 53 N9O9m / z = 779.3966, measured 780.40 [M+H] + .

[0102] Example 8:

[0103] Replace 2a with 2h in Example 1, and the rest of the operation is the same.

[0104]

[0105] The yield of the target product 3ah was 80% (monosubstituted:disubstituted 7:1), ESI-MS: calculated C38 H 53 N9O9m / z = 779.3966, measured 780.40 [M+H] + .

[0106] Example 9:

[0107] Replace 2a with 2i in Example 1, and the rest of the operation is the same.

[0108]

[0109] The yield of the target product 3ai was 80% (monosubstituted:disubstituted 5:1), ESI-MS: calculated C 43 H 68 N8O 15 m / z = 936.4804, measured 937.49 [M+H] + .

[0110] Example 10:

[0111] Replace 2a with 2j in Example 1, and the rest of the operation is the same.

[0112]

[0113] The yield of the target product 3aj in the reaction was 68%. ESI-MS calculated Cj. 43 H 52 F 16 N8O 11 m / z = 1160.3500, measured value 1161.35 [M+H] + .

[0114] Example 11:

[0115] Replace 2a with 2k in Example 1, and the rest of the operation is the same.

[0116]

[0117] The yield of the target product 3ak was 74% (monosubstituted:disubstituted 4:1), ESI-MS calculated C 48 H 70 N 10 O 18 m / z = 1074.4870, measured 1075.49 [M+H] + .

[0118] Example 12:

[0119] Replace 2a with 2l in Example 1, and the rest of the operation is the same.

[0120]

[0121] The yield of the target product 3al was 77% (monosubstituted:disubstituted 3:1), ESI-MS: calculated C 48 H 69 N9O 19 m / z = 1075.4710, measured 1076.46 [M+H] + .

[0122] Example 13:

[0123] Replace 2a with 2m in Example 1, and the rest of the operation is the same.

[0124]

[0125] The yield of the target product 3am was 79% (monosubstituted:disubstituted 2.5:1). ESI-MS calculated C0. 42 H 62 N 10 O 15 m / z = 948.4553, measured 949.47 [M+H] + .

[0126] Example 14:

[0127] Replace 2a with 2n in Example 1, and the rest of the operation is the same.

[0128]

[0129] The yield of the target product 3an was 82% (monosubstituted:disubstituted 6:1), ESI-MS: calculated C 52 H 73 N 13 O 14 m / z = 1103.5400, measured 1104.55 [M+H] + .

[0130] Example 15:

[0131] Replace 2a with 2o in Example 1, and the rest of the operation is the same.

[0132]

[0133] The yield of the target product 3ao in the reaction was 57%. ESI-MS calculated C0. 61 H 96 N 10 O 14 Sm / z = 1224.6828, measured 1225.66 [M+H] + .

[0134] Example 16:

[0135] Replace 2a with 2p in Example 1, and the rest of the operation is the same.

[0136]

[0137] The yield of the target product 3ap was 76% (monosubstituted:disubstituted 5:1), ESI-MS: calculated C 73 H 113 N 13 O 18 m / z = 1459.8327, measured 1460.83 [M+H] + .

[0138] Example 17:

[0139] Replace 2a with 2q in Example 1, and the rest of the operation is the same.

[0140]

[0141] The yield of the target product 3aq was 81% (monosubstituted:disubstituted 17.5:1). ESI-MS calculated C1... 77 H 101 N9O 24 m / z = 1535.6959, measured 1536.69 [M+H] + .

[0142] Example 18:

[0143] Replace 2a with 2r in Example 1, and the rest of the operation is the same.

[0144]

[0145] The yield of the target product 3ar was 77% (monosubstituted:disubstituted 2:1), ESI-MS: calculated C 92 H 142 N 18 O 22 m / z = 1851.0546, measured value 1852.03 [M+H] + .

[0146] Example 19:

[0147] Replace 1a' with 1b' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0148]

[0149] The yield of the target product 3be was 73%. ESI-MS calculated C2. 36 H 52 N8O 11 m / z = 772.3756, measured 773.40 [M+H] + .

[0150] Example 20:

[0151] Replace 1a' with 1c' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0152]

[0153] The yield of the target product 3ce was 81%, the isolated yield was 73%, and the calculated Cg by ESI-MS was [not specified]. 41 H 60 N 10 O 13 m / z = 900.4341, measured value 901.46 [M+H] + .

[0154] 1 ¹H NMR (500 MHz, deuterium oxide) δ 8.56 (d, J = 1.4 Hz, 1H), 7.32–7.22 (m, 5H), 7.19 (dd, J = 7.0, 1.6 Hz, 2H), 4.63–4.53 (m, 2H), 4.32 (t, J = 5.7 Hz, 1H), 4.26–4.03 (m, 8H), 3.80–3.66 (m, 2H), 3.35 (dd, J = 8.0, 2.5 Hz, 1H), 3.27–3.19 (m, 1H), 3.17–3.02 (m, 2H), 2.93 (dd, J = 1.4 Hz, 1H), δ 8.56 (d, J = 1.4 Hz, 1H), 7.32–7.22 (m, 5H), 7.19 (dd, J = 7.0, 1.6 Hz, 2H), δ 2.93 (dd, J = 1.4 Hz, 1 ... 13.9,9.0Hz,1H),2.14(dt,J=14.0,8.4Hz,2H),2.04(dtd,J=15.3,7.7,3.0Hz,1H),1.97(d,J=2.2Hz,4H),1.87(dq,J=13.5, 7.0Hz, 1H), 1.79 (p, J=7.4, 7.0Hz, 1H), 1.26 (d, J=7.3Hz, 3H), 1.18 (tdd, J=7.1, 4.3, 1.6Hz, 8H), 0.89 (dd, J=6.8, 4.2Hz, 4H).

[0155] 13C NMR (126MHz, deuterium oxide) δ 177.52, 174.53, 173.81, 172.80, 171.23, 170.58, 136.31, 133.47, 129.19, 128.81, 128.64, 127.29, 117.26, 62.58, 61.19, 57.45, 57.28, 55.19, 53.93, 53.09, 52.02, 49.90, 36.90, 33.10, 32.68, 30.80, 29.65, 26.54, 26.31, 21.81, 16.39, 16.28, 16.16, 13.46.

[0156] Example 21:

[0157] Replace 1a' with 1d' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0158]

[0159] The yield of the target product 3de was 81%, the separation yield was 77%, and the calculated C0 by ESI-MS was [not specified]. 43 H 63 N 11 O 14 m / z = 957.4556, measured 958.48 [M+H] + .

[0160] Example 22:

[0161] Replace 1a' with 1e' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0162]

[0163] The yield of the target product 3ee in the reaction was 81%. ESI-MS: calculated C 45 H 66 N 12 O 15 m / z = 1014.4771, measured 1015.50 [M+H] + .

[0164] Example 23:

[0165] Replace 1a' with 1f' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0166]

[0167] The yield of the target product 3fe was 61%. ESI-MS calculated C2. 36 H 52 N6O 13 m / z = 776.3592, measured 777.37 [M+H] + .

[0168] Example 24:

[0169] Replace 1a' with 1g' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0170]

[0171] The yield of the target product 3ge in the reaction was 86%. ESI-MS calculated Cg. 35 H 47 N5O 11 m / z = 713.3272, measured 714.33 [M+H] + .

[0172] Example 25:

[0173] Replace 1a' with 1h' in Example 1, replace 2a (1.1 equivalent) with 2f (2.0 equivalent), and the rest of the operations are the same.

[0174]

[0175] The yield of the target product 3hf was 89% (monosubstituted:disubstituted 11:1), ESI-MS: calculated C 43 H 61 N9O 10 m / z = 863.4541, measured 864.46 [M+H] + .

[0176] Example 26:

[0177] Replace 1a' with 1i' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0178]

[0179] The yield of the target product 3ie was 79%. ESI-MS calculated Ci. 39 H 58 N 10 O 12 m / z = 858.4236, measured 859.43 [M+H] + .

[0180] Example 27:

[0181] Replace 1a' with 1j' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0182]

[0183] The yield of the target product 3je was 80%. ESI-MS calculated C2. 39 H 60 N8O 11 m / z = 816.4382, measured value 817.45 [M+H] + .

[0184] Example 28:

[0185] Replace 1a' with 1k' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0186]

[0187] The yield of the target product 3 ke was 75% in the reaction. ESI-MS calculated C2. 56 H 90 N 12 O 16 m / z = 1186.6598, measured 1187.67 [M+H] + .

[0188] Example 29:

[0189] Replace 1a' with 1l' in Example 1, replace 2a (1.1 equivalent) with 2e (5.0 equivalent), and the rest of the operations are the same.

[0190]

[0191] The yield of the target product 3e was 91%, and ESI-MS calculated C1. 68 H 104 N 20 O 23 m / z = 1568.7583, measured 1569.68 [M+H] + .

[0192] Example 30:

[0193] Replace 1a' with 1m' and 2a with 2o in Example 1, and the rest of the operations are the same.

[0194]

[0195] The yield of the target product in the reaction was 43% over 3 months. ESI-MS calculated C0.05. 76 H 140 N 14 O 13 Sm / z = 1489.0445, measured 1490.05 [M+H] + .

[0196] Example 31:

[0197] Replace 1a' in Example 1 with N-terminal acryloylated polypeptide 4a'. Step 2 does not require 2a. The rest of the operation is the same.

[0198]

[0199] The yield of the target product 5a was 66%, the isolated yield was 57%, and the calculated C by ESI-MS was... 36 H 53 N9O9 m / z = 755.3966, measured 756.39 [M+H] + .

[0200] 1 ¹H NMR (800MHz, deuterium oxide) δ 7.32–7.29 (m, 2H), 7.24 (tt, J = 6.6, 1.4 Hz, 1H), 7.23–7.20 (m, 2H), 4.97 (s, 1H), 4.32 (dd, J = 8.7, 6.1 Hz, 1H), 4.29–4.23 (m, 1H), 4.10 (dd, J = 8.3, 4.2 Hz, 1H), 3.84–3.71 (m, 2H), 3.06 (dd, J = 13.5, 7.7 Hz, 1H), 3.00– 2.92(m,1H),2.87(dd,J=16.0,10.3Hz,1H),2.68(dd,J=16.0,4.9Hz,1H),2.32(ddd,J=12.8,6.2,1.9Hz,1H),2.29– 2.17(m,1H),2.08–1.78(m,3H),1.72–1.62(m,1H),1.60–1.48(m,2H),0.88(d,J=6.2Hz,3H),0.80(d,J=6.0Hz,2H).

[0201] 13C10 NMR (126 MHz, deuterium oxide) δ 177.91, 176.74, 176.70, 174.87, 174.57, 173.29, 171.99, 171.63, 136.20, 129.25, 128.90, 127.37, 61.69, 56.24, 54.43, 53.73, 52.26, 48.32, 36.00, 33.94, 31.25, 30.84, 29.50, 24.57, 24.46, 22.51, 20.03. ESI-MS: Calculated C10 NMR 36 H 53 N9O9 m / z = 755.3966, measured 756.39 [M+H] + .

[0202] Example 32:

[0203] Replace 1a' in Example 1 with N-terminal acryloylated polypeptide 4b'. Step 2 does not require 2a. The rest of the operation is the same.

[0204]

[0205] The yield of the target product 5b in the reaction was 53%. ESI-MS calculated C5b. 48 H 76 N 12 O 12 m / z = 1012.5706, measured 1013.57 [M+H] + .

[0206] Example 33:

[0207] Replace 1a' in Example 1 with lysine-acrylated polypeptide 4c'. Step 2 does not require 2a. The rest of the operation is the same.

[0208]

[0209] The yield of the target product 5c in the reaction was 61%. ESI-MS calculated C5c. 50 H 78 N 12 O 13 m / z = 1054.5811, measured 1055.59 [M+H] + .

[0210] Example 34:

[0211] Replace 1a' in Example 1 with lysine-side-chain acrylated polypeptide 4d'. Step 2 does not require 2a. The rest of the operation is the same.

[0212]

[0213] The yield of the target product in the reaction was 76% after 5 days. ESI-MS calculated C5. 47 H 72 N 12 O 12 m / z = 996.5393, measured 997.54 [M+H] + .

[0214] Example 35:

[0215] Replace 1a' in Example 1 with N-terminal acryloylated polypeptide 4e'. Step 2 does not require 2a. The rest of the operation is the same.

[0216]

[0217] The yield of the target product 5e in the reaction was 51%. ESI-MS calculated C5e. 60 H 90 N 20 O 19 m / z = 1394.6691, measured 1395.89 [M+H] + .

[0218] Example 36:

[0219] Replace 1a' in Example 1 with 4f', which is alycylated polypeptide with a lysine side chain. Step 2 does not require 2a. The rest of the operation is the same.

[0220]

[0221] The yield of the target product 5f was 51%. ESI-MS calculated C5. 65 H 103 N 19 O 19 m / z = 1453.7678, measured 1454.75 [M+H] + .

[0222] Example 37:

[0223] Replace 1a' in Example 1 with N-terminal acryloylated cyclic peptide 4g'. Step 2 does not require 2a. The rest of the operation is the same.

[0224]

[0225] The yield of the target product (5g) was 33%. ESI-MS calculated C0.05. 48 H 72 N 12 O 13 S²m / z = 1088.4783, measured value 1089.49 [M+H]+ .

[0226] Example 38:

[0227] Replace 1a' in Example 1 with 4h', a cyclic peptide with lysine side chain acrylylation. Step 2 does not require 2a. The rest of the operation is the same.

[0228]

[0229] The yield of the target product after 5 hours of reaction was 57%. ESI-MS calculated C2. 54 H 83 N 19 O 18 S²m / z = 1285.6163, measured value 1286.88 [M+H] + .

[0230] Test Example 1:

[0231] Antimicrobial peptides are a class of polypeptides with broad-spectrum antimicrobial activity. Due to their unique antimicrobial mechanisms and broad antimicrobial spectrum, they hold promise as a novel therapeutic approach to overcome bacterial resistance. However, the poor in vivo stability and metabolic properties of polypeptide drugs significantly limit their application. This application utilizes a bioconjugation strategy to modify antimicrobial peptides, introducing functional modules to enhance both antimicrobial activity and metabolic properties, thereby improving drug-likeness.

[0232] In Example 30 above, a modified peptide 3mo was synthesized using 1m' as the starting peptide. In this test example, the antibacterial activities of peptide 1m' and modified peptide 3mo against methicillin-resistant Staphylococcus aureus USA300, vancomycin-resistant Enterococcus faecium O649, Gram-negative Escherichia coli AB1157, Acinetobacter baumannii (Aba.), and Pseudomonas aeruginosa PAO1 were further tested as follows:

[0233] Strains: methicillin-resistant Staphylococcus aureus USA300, vancomycin-resistant Enterococcus faecalis O649, Gram-negative Escherichia coli AB1157, Acinetobacter baumannii (Aba.), and Pseudomonas aeruginosa PAO1.

[0234] Culture medium: CAMHB medium (acid-hydrolyzed casein 17.5 g / L, beef meal 3 g / L, soluble starch 1.5 g / L, calcium ions 20-25 g / L, magnesium ions 10-12.5 g / L).

[0235] Test Methods: In vitro antimicrobial activity MIC determination was performed using Mueller-Hinton II broth. MIC values ​​of all antimicrobial agents were measured by micro-dilution with the broth. Typically, compounds were dissolved in dimethyl sulfoxide to a stock solution of 2.56 mg / mL. All samples were diluted with culture medium to an initial concentration of 128 μg / mL. Further 1:2 serial dilutions were performed by adding culture medium to achieve concentrations from 64 μg / mL to 0.0625 μg / mL or lower. 150 μL of each dilution was distributed in 96-well plates, along with a sterile control (bacteria-free), a growth control (containing culture medium and dimethyl sulfoxide, but without the compound), and a positive control antimicrobial peptide PL-13 (Ac-FKKLKKLFSKLWNWK-NH2, SEQ ID No.:22). Except for the sterile control, each well was inoculated with 5 μL of bacterial suspension (approximately 10 CFU / well). The 96-well plates were incubated at 37°C for 24 hours. The MIC values ​​for these compounds are defined as the minimum concentration required to completely inhibit bacterial growth. All MIC values ​​are interpreted according to recommendations from the Clinical and Laboratory Standards Association.

[0236] The results are shown in Table 1 below.

[0237] Table 1

[0238]

[0239] As shown in Table 1 above, compared to the unmodified peptide 1m', the sulfonamide-containing dipeptide-modified antimicrobial peptide 3mo exhibits enhanced killing effects against various bacterial species, including methicillin-resistant Staphylococcus aureus USA300, vancomycin-resistant Enterococcus faecalis O649, Gram-negative Escherichia coli AB1157, Acinetobacter baumannii (Aba.), and Pseudomonas aeruginosa PAO1. Those skilled in the art can also use the methods of this application to structurally modify other antimicrobial peptides.

[0240] In summary, the method of this application has broad application prospects in peptide modification, and those skilled in the art can flexibly adopt the method of this application to modify peptides as needed.

Claims

1. A peptide editing method based on a methionine desulfurization strategy, the method comprising the following steps: (1) Preparation of thionium: As shown in reaction formula 1 above, the substrate (Met-X)1' and S1, which have a methyl thioether structure, are dissolved in a solvent, acid is added, and the reaction is stirred at room temperature to obtain the target thionium 1. The substrate (Met-X)1' having a methyl sulfide structure is selected from: Wherein, case (i) represents unmodified or modified methionine; Case (ii) represents a polypeptide or protein containing methionine, wherein the amino acid side chains of the polypeptide or protein do not contain alkenyl groups, and The position of methionine in the polypeptide or protein is not limited; (2) Desulfurization reaction: As shown in reaction formula 2 above, the target thionium 1, electron-deficient olefin 2, photocatalyst, and azacarbene-borane are dissolved in a solvent, and the reaction is carried out under blue light irradiation at room temperature to obtain the modified product 3. or The method includes the following steps: (1') Preparation of thionium: As shown in reaction formula 1' above, the polypeptide or protein 4' containing methionine and S1 are dissolved in a solvent, acid is added, and the reaction is stirred at room temperature to obtain the target thionium 4. The methionine-containing polypeptide or protein 4' further has at least one acrylated amino acid, the position of methionine in the polypeptide or protein is not limited, and the position of the acrylated amino acid in the polypeptide or protein 4' is any position other than methionine. (2') Desulfurization reaction: As shown in reaction formula 2' above, the target thionium 4, photocatalyst, and azircone-borane are dissolved in a solvent and irradiated with blue light at room temperature to obtain a cyclic peptide structure 5 linked by a valeryl linking group.

2. The method according to claim 1, wherein, In step (1), the substrate 1' having the methyl thioether structure is selected from 1a' to 1m': their corresponding amino acid sequences are SEQ ID No.: 1-13, 3. The method according to claim 1, wherein, In step (1'), The N-terminal amino acid of the 4' end of the methionine-containing polypeptide or protein is acrylated; or The polypeptide or protein containing methionine 4' contains acrylated lysine. Specifically, the polypeptide or protein 4' containing methionine is selected from 4a'-4h': and their corresponding amino acid sequences are SEQ ID No.:14-21 respectively:

4. The method according to claim 1, wherein, In step (1) or (1'), the acid is selected from formic acid, acetic acid, trifluoroacetic acid, preferably formic acid, and the concentration of the acid is 1% to 100 vol%, preferably 5 vol%. The solvent is selected from water, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 1,4-dioxane, or combinations thereof; preferably, it is a mixture of acetonitrile and water in a volume ratio of 1:

1. The reaction time is 6-12 hours.

5. The method according to claim 1, wherein, In step (2), EWG in the electron-deficient olefin 2 structure represents an electron-withdrawing group, such as a group containing carbonyl, sulfonyl, phosphono, nitro, cyano, trifluoromethyl, or an electron-deficient heterocyclic group, and its derivative groups. Specifically, the electron-deficient olefin is selected from the following 2a-2r: and / or The azacarbene-borane is selected from the following: and / or The solvent is selected from: water, formic acid, acetic acid, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, methanol, or combinations thereof, preferably dimethyl sulfoxide; and / or The photocatalyst is selected from: [Ir(dtbbpy)(ppy)2]PF6, Ir[dF(CF3)ppy]2(dtbbpy)PF6, fac-Ir(ppy)3, fac-Ir(dF-ppy)3, [Ru(bpy)3][PF6]2, [Ru(bpz)3][PF6]2, [Ru(phen)3][PF6]2, [Ru(bpm)3][PF6]2, [Ru(bpz)3][PF6]2, 4CzIPN, Eosin Y, Fluorescein, preferably fac-Ir(ppy)3; and / or The wavelength of the blue light is 254nm-600nm, preferably 420nm-440nm; and / or The blue light irradiation time is 15-60 minutes.

6. The method according to claim 1, wherein, The method further includes a step of semi-preparative HPLC purification and freeze-drying of the intermediate products of steps (1) and (1') or the final products of steps (2) and (2').

7. A polypeptide or protein edited by any one of claims 1-6.

8. The polypeptide or protein according to claim 7, wherein the polypeptide or protein is selected from the following:

9. The uses of polypeptides with the following structures in the preparation of antibacterial reagents.

10. The use according to claim 9, wherein, The antibacterial reagent has antibacterial activity against methicillin-resistant Staphylococcus aureus USA300, vancomycin-resistant Enterococcus faecalis O649, Gram-negative Escherichia coli AB1157, Acinetobacter baumannii (Aba.), and Pseudomonas aeruginosa PAO1.

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