Method for n-terminal histidine specific modification of polypeptides or proteins

Abstract

The application discloses a method for specifically modifying N-terminal histidine of polypeptide or protein, which comprises: reacting polypeptide or protein with N-terminal histidine with o-benzoquinone to generate polypeptide or protein product containing oxazine; the polypeptide or protein with N-terminal histidine has a structure as shown in formula (I), the o-benzoquinone has a structure as shown in formula (II), and the polypeptide or protein product containing oxazine has a structure as shown in formula (III); wherein R is a peptide chain; R 1 , R 2 each independently is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy. The method has specificity for polypeptide or protein with N-terminal histidine, and can selectively label polypeptide and protein with N-terminal histidine in a complex system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical synthesis, and particularly relates to a method for specifically modifying N-terminal histidine of polypeptide or protein. BACKGROUND

[0002] Precise chemical modification of polypeptide or protein is one of the research hotspots in the field of chemical biology in recent years. Site-specific modification of polypeptide or protein can regulate or change its properties and functions, and endow polypeptide or protein with new physiological functions, such as prolonging half-life, labeling target receptors, regulating protein-protein interaction, etc., which is of great significance to the study of various biological processes and drug development in the field of pharmaceutical chemistry and biochemistry.

[0003] The prior art (NHS-Esters as Versatile Reactivity-Based Probes for Mapping Proteome-Wide Ligandable Hotspots) uses NHS-ester (N-hydroxysuccinimide ester) to modify lysine, serine, threonine and tyrosine amino acid residues in the protein. Due to the strong reactivity of NHS-ester, the modification reaction has low specificity.

[0004] At present, the selective modification of polypeptide or protein at a single site mostly depends on gene code extension technology or biological coupling enzyme. Due to the relatively complex technical route, the application range of these methods is limited to a certain extent. In contrast, the direct chemical coupling method of protein is simple in operation and low in cost, and is favored in many application scenarios. However, most of the current chemical coupling methods only have chemical selectivity, and often lack selectivity at a single site. For example, the Chinese patent document with publication number CN117343124A discloses a visible light-mediated cysteine-based chemical modification method of polypeptide and protein. Based on the cysteine residue, the thiol group is selectively activated by reacting with an activated reagent, and then the thiol group is removed under photocatalysis to generate free radicals to realize the chemical modification of polypeptide and protein. There are still many unsolved challenges in the accurate labeling of polypeptide and protein at a single site.

[0005] N-terminal modification refers to the process of chemical modification at the amino terminus (N-terminal) of a polypeptide chain or protein. It is an important form of post-translational modification of proteins, which plays a crucial role in regulating the structure, stability, functional localization and final fate of proteins. Common N-terminal modifications include formylation, acetylation, ubiquitination, myristoylation, etc. In-depth study of these N-terminal modifications not only deepens our understanding of the basic laws of life, but also opens up new avenues for developing new diagnostic methods and therapeutic drugs for cancer, neurological diseases and rare diseases.

[0006] Most of the current N-terminal modification methods have no good selectivity for amino acids, and there is no report on selective modification of N-terminal histidine. Therefore, it is of great significance to develop selective modification of polypeptides or proteins with N-terminal histidine. SUMMARY

[0007] The application provides a method for selective modification of polypeptides or proteins with N-terminal histidine.

[0008] The technical scheme of the application is as follows:

[0009] The method for selective modification of polypeptides or proteins with N-terminal histidine comprises: reacting polypeptides or proteins with N-terminal histidine and o-benzoquinone to generate an oxazine-containing polypeptide or protein product.

[0010] The polypeptides or proteins with N-terminal histidine have a structure as shown in formula (I), the o-benzoquinone has a structure as shown in formula (II), and the oxazine-containing polypeptide or protein product has a structure as shown in formula (III).

[0011] ;

[0012] wherein, R is a peptide chain; R 1 , R 2 each independently is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy.

[0013] Preferably, R is a peptide chain.

[0014] R 1 , R 2 each independently is selected from hydrogen, C1-C5 alkyl, C1-C5 alkoxy, ; n is an integer of 0-3.

[0015] Preferably, R is or , m Xs are independently any amino acid, and m is a positive integer.

[0016] Preferably, the o-benzoquinone is selected from:

[0017] .

[0018] Preferably, the molar amount of the o-benzoquinone is 1-300 times, further preferably 1-30 times, the molar amount of the polypeptides or proteins.

[0019] Preferably, the solvent used in the reaction is at least one of water, acetonitrile, dimethyl sulfoxide, a phosphate buffer solution, a Tris-hydrochloric acid buffer solution, a HEPES buffer solution, and a PIPES buffer solution.

[0020] Preferably, the molar amount of the solvent is 100-10000 times the molar amount of the polypeptide or protein; further preferably 5000-10000 times.

[0021] Preferably, the reaction is carried out at room temperature, and the reaction time is 0.5-25h.

[0022] In the method of the present application, the polypeptide or protein with N-terminal histidine is oxidatively coupled with the o-benzoquinone in the presence of the o-benzoquinone and the solvent to obtain the polypeptide or protein with N-terminal oxazine. In the method of the present application, when the amino acid at the N-terminal of the polypeptide or protein is not histidine, no or little polypeptide or protein with N-terminal oxazine is obtained, and the method of the present application is specific to the polypeptide or protein with N-terminal histidine.

[0023] Compared with the prior art, the present application has the following advantages:

[0024] (1) The method of the present application can specifically modify the polypeptide or protein with N-terminal histidine, and the residues of other amino acids in the polypeptide or protein are retained, so that the polypeptide or protein with N-terminal histidine can be selectively labeled in a complex system.

[0025] (2) The method of the present application has the advantages of simple and readily available raw materials, mild reaction conditions, and wide applicability of substrates, which meets the requirements of developing green and environmentally friendly chemistry. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A bar chart of oxazine products of the reaction of polypeptides S1-S18 with N-terminal different amino acids and o-benzoquinone Q3 in Example 1;

[0027] Figure 2 A liquid-liquid phase diagram of the reaction of polypeptide S1 with N-terminal histidine and o-benzoquinone Q3 in Example 1;

[0028] Figure 3 An X-ray structure diagram of the single crystal of oxazine product P19 in Example 2;

[0029] Figure 4 A nuclear magnetic hydrogen spectrum diagram of oxazine product P19 in Example 2;

[0030] Figure 5 A nuclear magnetic carbon spectrum diagram of oxazine product P19 in Example 2;

[0031] Figure 6 A liquid-liquid phase diagram of the reaction of polypeptide S20 and o-benzoquinone Q6 in Example 3;

[0032] Figure 7 A liquid phase diagram of oxazine product P20' in Example 3;

[0033] Figure 8 Liquid chromatogram of the oxazine product P20" of Example 3;

[0034] Figure 9 Secondary mass spectrum MS / MS chromatogram of the oxazine product of Example 3;

[0035] Figure 10 Liquid chromatogram of the reaction of the polypeptide S21 and the quinone Q3 of Example 4;

[0036] Figure 11 Liquid chromatogram of the oxazine product P21 of Example 4;

[0037] Figure 12 Reaction scheme of the polypeptides S1, S8, S10, S22, S23 and the quinone Q4 of Example 5;

[0038] Figure 13 Liquid chromatogram of the reaction of the polypeptides S1, S8, S10, S22, S23 and the quinone Q4 of Example 5;

[0039] Figure 14 Liquid chromatogram of the oxazine product P24' of Example 5;

[0040] Figure 15 Liquid chromatogram of the oxazine product P24" of Example 5;

[0041] Figure 16 Reaction scheme of the proteins S24, S25 and the quinone Q6 of Example 6;

[0042] Figure 17 Western Blot and Coomassie Brilliant Blue (CBB) of the reaction of the proteins S24, S25 and the quinone Q6 of Example 6. DETAILED DESCRIPTION

[0043] The embodiments of the present application provide a method for modifying N-terminal histidine of a polypeptide or protein, comprising the following steps: reacting a polypeptide or protein (I) with N-terminal histidine in the presence of a quinone (II) and a solvent to generate an oxazine-containing polypeptide or protein (III);

[0044] ;

[0045] wherein R is a peptide chain;

[0046] R 1 , R 2 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy.

[0047] According to an embodiment of the present application, the above-mentioned o-benzoquinone (II) is selected from one of the following, but not limited to:

[0048] .

[0049] According to an embodiment of the present application, the above-mentioned solvent is selected from one of the following, but not limited to: water, acetonitrile, dimethyl sulfoxide, phosphate buffer solution, Tris-hydrochloric acid buffer solution, HEPES buffer solution, MOPS buffer solution, PIPES buffer solution.

[0050] According to an embodiment of the present application, the molar amount of the o-benzoquinone is 100% to 30000% of the molar amount of the reaction substrate.

[0051] According to an embodiment of the present application, the molar amount of the solvent in the method step is 500000% to 1000000% of the molar amount of the reaction substrate.

[0052] According to an embodiment of the present application, in the method step, the reaction is stirred or left to stand at room temperature, and the reaction time is 0.5 h to 25 h.

[0053] The present application will be further described in detail below with reference to the accompanying drawings and examples, it should be noted that the following examples are intended to facilitate the understanding of the present application, and do not limit the present application in any way.

[0054] Example 1:

[0055] ;

[0056] Take 18 0.5 milliliter centrifuge tubes, and sequentially add polypeptides S1-S18 (3 mM) with different N-terminal amino acids, o-benzoquinone Q3 (15 mM), and phosphate buffer solution with pH = 5.1, and shake the reaction on a shaking table at room temperature for 9 hours. After the reaction is completed, stop stirring, extract with ether, dilute with water, and measure LCMS. Analytical liquid chromatography method: 2% to 60% acetonitrile running for 11 minutes, flow rate 0.3 mL / min, mobile phase containing 0.1% formic acid, λ = 230 nm, column temperature 40 o C, Hedera C18 3 μ m, 2.1 × 100 mm chromatographic column. The reaction results of different polypeptides are shown in Figure 1 , polypeptide S1 with N-terminal histidine can obtain oxazine products (P1' and P1") with a liquid phase yield of 84% (as shown in Figure 2 , the liquid phase chromatogram of the reaction solution of polypeptide S1), while polypeptides with other N-terminal amino acids only obtain a small amount of corresponding oxazine products or no corresponding oxazine products are generated, indicating that the reaction has good selectivity for polypeptides with N-terminal histidine.

[0057] wherein S1-S18 are represented by one letter abbreviations for amino acids:

[0058] S1: NH2-HYSKEASAL-CONH2;

[0059] S2: NH2-YYSKEASAL-CONH2;

[0060] S3: NH2-FYSKEASAL-CONH2;

[0061] S4: NH2-LYSKEASAL-CONH2;

[0062] S5: NH2-MYSKEASAL-CONH2;

[0063] S6: NH2-GYSKEASAL-CONH2;

[0064] S7: NH2-AYSKEASAL-CONH2;

[0065] S8: NH2-VYSKEASAL-CONH2;

[0066] S9: NH2-IYSKEASAL-CONH2;

[0067] S10: NH2-DYSKEASAL-CONH2;

[0068] S11: NH2-EYSKEASAL-CONH2;

[0069] S12: NH2-NYSKEASAL-CONH2;

[0070] S13: NH2-QYSKEASAL-CONH2;

[0071] S14: NH2-SYSKEASAL-CONH2;

[0072] S15: NH2-TYSKEASAL-CONH2;

[0073] S16: NH2-KYSKEASAL-CONH2;

[0074] S17: NH2-RYSKEASAL-CONH2;

[0075] S18: NH2-WYSKEASAL-CONH2.

[0076] Example 2:

[0077] ;

[0078] In an 8-mL reaction vial equipped with a magnetic stir bar, histidine S19 (0.1 mmol), o-benzoquinone Q2 (0.1 mmol), phosphate buffer solution (pH = 5.1) (1 mL), and acetonitrile (1 mL) were added successively. The reaction was stirred at room temperature for 2 h. After the reaction was completed, the stirring was stopped, and the reaction was extracted with water and ethyl acetate. The organic phase was collected, and concentrated under reduced pressure to give a crude reaction liquid. Column chromatography (dichloromethane:methanol = 20:1) was used to separate the product to give white solid P19 (3.1 mg, 9% isolated yield). Single crystals of P19 were obtained by crystallization. The crystal structure of the oxazine product P19 is shown in Figure 3 .

[0079] 1 H NMR (600 MHz, DMSO- d 6 ) δ 12.11 (s, 1H), 7.54 (s, 1H), 7.23 (s, 1H),7.07 (s, 1H), 6.48 (s, 1H), 6.17 (s, 1H), 3.77 (s, 3H), 3.75 (s, 3H), 1.30(s, 9H). 13 C NMR (150 MHz, DMSO- d 6 ) δ 163.56, 161.15, 147.85, 145.42, 136.51,136.02, 131.61, 130.12, 127.06, 125.42, 116.27, 100.61, 67.28, 56.16, 52.81,34.40, 30.10. HRMS (ESI-TOF) m / z calcd. for C 18 H 22 N3O4 ([M + H] + ): 344.1605, found: 344.1600.

[0080] Example 3:

[0081] ;

[0082] In an 8 mL reaction vial with magnetic stir bar, polypeptide S20 (3 mM), o-benzoquinone Q6 (15 mM), and pH = 5.1 phosphate buffer solution were added sequentially. The reaction was stirred at room temperature for 22 hours. After the reaction was completed, stirring was stopped, and the reaction was extracted with ether. Semi-preparative chromatography was performed to obtain white solid P20 (2.52 mg, 63%). Semi-preparative chromatography method: 20% to 36% acetonitrile over 5 minutes, 36% to 43% acetonitrile over 25 minutes, flow rate 5 mL / min, mobile phase containing 0.1% trifluoroacetic acid, Shimadzu C18 5 μ m, 20 x 250 mm column. Analytical liquid chromatography method: 2% to 60% acetonitrile over 11 minutes, flow rate 0.3 mL / min, mobile phase containing 0.1% formic acid, λ = 230 nm, column temperature 40 o C, Hedera C18 3 μ m, 2.1 x 100 mm column. As Figure 6 shown in the liquid chromatogram of the reaction mixture, the product (P20' and P20") was obtained in 77% liquid yield, Figure 7 and Figure 8 are the liquid chromatograms of the pure product obtained by semi-preparative separation, the retention time of polypeptide product P20' is t R = 6.23 min, and the retention time of polypeptide product P20" is t R = 6.81 min.

[0083] HRMS (ESI-TOF) m / z calcd. for C 65 H 86 N 15 O 14 S ([M+H] + ) 1332.6194, found: ([M+H] + ) 1332.6182.

[0084] The secondary MS / MS spectra of the product (P20' and P20") are shown in Figure 9 , indicating that the reaction is a modification of the N-terminal amino acid of the polypeptide.

[0085] Example 4:

[0086] ;

[0087] In an 8 mL reaction vial with magnetic stir bar, polypeptide S21 (1 mM), o-benzoquinone Q3 (10 mM), and phosphate buffer solution pH = 5.1 were added sequentially. The reaction was stirred at room temperature for 9 hours. After the reaction was completed, stirring was stopped, and the reaction was extracted with ether. Semi-preparative chromatography was performed to obtain white solid P21 (1.24 mg, 62%). Semi-preparative chromatography method: 20% to 70% acetonitrile over 30 minutes at a flow rate of 5 mL / min with 0.1% trifluoroacetic acid in the mobile phase, Shimadzu C18 5 μ m, 20 x 250 mm column. Analytical liquid chromatography method: 20% to 70% acetonitrile over 10 minutes at a flow rate of 0.3 mL / min with 0.1% trifluoroacetic acid in the mobile phase, λ = 230 nm, column temperature 40 o C, Hedera C18 3 μ m, 2.1 x 100 mm column. As shown in the liquid chromatogram of the reaction mixture, the product P21 was obtained in 79% liquid yield, Figure 10 the liquid chromatogram of the pure product obtained from semi-preparative chromatography, the retention time of polypeptide product P21 Figure 11 t R = 7.25 min.

[0088] HRMS (ESI-TOF) m / z calcd. for C 147 H 242 N 43 O 47 ([M+3H] 3+ ) 1121.6027; ([M+4H] 4+ ) 841.4539; ([M+5H] 5+ ) 673.3646, found: ([M+3H] 3+ ) 1121.6044; ([M+4H] 4+ ) 841.4539; ([M+5H] 5+ ) 673.3650.

[0089] Example 5:

[0090] The reaction process is shown in Figure 12 ​As shown, S1 (NH2-HYSKEASAL-CONH2), S8 (NH2-VYSKEASAL-CONH2), S10 (NH2-DYSKEASAL-CONH2), S22 (NH2-TKPRPGP-COOH), S23 (NH2-SIKVAV-COOH), and o-quinone Q4 (10 mM) were added sequentially to an 8 mL reaction flask equipped with a magnetic stirrer. Each peptide had a concentration of 2 mM in a phosphate buffer solution at pH 5.1. The reaction was stirred at room temperature for 14 hours. After the reaction was complete, stirring was stopped, and the mixture was extracted with diethyl ether. Figure 13 LCMS analysis showed that only peptide S1 reacted, generating the oxazine product P22 (including P22' and P22"). Subsequently, compound P22 was extracted from the mixture using an azide-treated resin, and a Click reaction occurred in the presence of copper sulfate to give compound P23. Finally, desulphurization and semi-preparative purification yielded the oxazine product P24 (including P24' and P24"). Figure 14 and 15 The figures show the liquid phase chromatograms of compounds P24' and P24', respectively. This reaction flow demonstrates that the method of this invention can be used to separate and purify the oxazine product of peptides with an N-terminus histidine in a mixed peptide product. This provides an efficient method for subsequent purification of the oxazine product of peptides with an N-terminus histidine. Semi-preparative chromatography method: run in 20% to 70% acetonitrile for 30 minutes at a flow rate of 5 mL / min, with a mobile phase containing 0.1% trifluoroacetic acid, Shimadzu C18 5. μ m, 20 × 250 mm column. Analytical liquid chromatography method: run in 2% to 60% acetonitrile for 11 minutes, flow rate 0.3 mL / min, mobile phase containing 0.1% trifluoroacetic acid. λ = 230 nm, column temperature 40 o C, Hedera C18 3 μ m, 2.1 × 100 mm chromatographic column.

[0091] Example 6:

[0092] The reaction process is as follows Figure 16 As shown, phosphate buffer solution was added sequentially to a 200 μL centrifuge tube. Protein S24, with its N-terminus histidine, or protein S25, with its N-terminus methionine, were added (20 μL). μ Different concentrations of ortho-quinone Q6 (0–2.0 mM) were reacted and allowed to stand at room temperature for 1 hour. After the reaction, a small amount of the reaction solution was subjected to Western blotting and Coomassie Brilliant Blue assays. Figure 17 The results showed that the reaction had good selectivity for proteins with histidine residues at the N-terminus, and the modification became more pronounced with increasing concentration of ortho-quinone.

[0093] The above-described embodiments illustrate the technical solutions and beneficial effects of the present application. It should be understood that the above-described embodiments are only specific embodiments of the present application and are not intended to limit the present application. Any modifications, supplements, and equivalent replacements made within the principle range of the present application should be included in the protection range of the present application.

Claims

1. A method for N-terminal histidine-specific modification of a polypeptide or protein, characterized in that, The application relates to a method for preparing a polypeptide or protein product containing an oxazine. The polypeptide or protein with a histidine at the N-terminal end has a structure as shown in formula (I), the o-benzoquinone has a structure as shown in formula (II), and the polypeptide or protein product containing an oxazine has a structure as shown in formula (III). The o-benzoquinone is selected from the group consisting of: wherein R is a peptide chain; R 1 , R 2 each of the R groups is independently selected from the group consisting of hydrogen, C1-C5 alkyl, C1-C5 alkoxy, ； n is an integer from 0 to 3; R 1 , R 2 groups are not simultaneously hydrogen.

2. The method of N-terminal histidine-specific modification of a polypeptide or protein according to claim 1, wherein, The molar amount of the o-benzoquinone is 1-300 times the molar amount of the polypeptide or protein. 。 3. The method of N-terminal histidine-specific modification of a polypeptide or protein according to claim 1, wherein, The molar amount of the o-benzoquinone is 1-30 times the molar amount of the polypeptide or protein.

4. The method of N-terminal histidine-specific modification of a polypeptide or protein according to claim 1, wherein, The solvent used in the reaction is at least one of water, acetonitrile, dimethyl sulfoxide, a phosphate buffer solution, a Tris-hydrochloric acid buffer solution, a HEPES buffer solution and a PIPES buffer solution.

5. The method of N-terminal histidine-specific modification of a polypeptide or protein according to claim 1, wherein, The molar amount of the solvent is 100-10000 times the molar amount of the polypeptide or protein.

6. The method of N-terminal histidine-specific modification of a polypeptide or protein according to claim 1 or 5, characterized in that, The molar amount of the solvent is 5000-10000 times the molar amount of the polypeptide or protein.

7. The method of N-terminal histidine-specific modification of a polypeptide or protein according to claim 6, wherein, The reaction is carried out at room temperature, and the reaction time is 0.5-25 h.

8. The method of N-terminal histidine-specific modification of a polypeptide or protein according to claim 1, wherein, ​

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