Method for imidazole-mediated mild and precise modification of n-terminal cys of polypeptides / proteins and applications thereof

Abstract

The application discloses an imidazole-mediated method for mild and precise modification of N-terminal Cys of polypeptides / proteins and application thereof, and has the advantages of simple operation, compatibility with hydrazide, N a variety of commercial and easily synthesized acyl donor forms such as hydroxysuccinimide ester, p-nitrophenyl ester, perfluorophenyl ester, acid anhydride, compatibility with the use of reducing agents, low feeding equivalent of acyl donor molecules, realization of selective modification in the range of polypeptide / protein concentration from μmol / L to mmol / L, one-pot bifunctional modification of N-terminal cysteine residues of polypeptides / proteins, and no influence of the thiol of cysteine residues in the sequence on the modification reaction. The application is friendly to living cells and organisms, and can realize the modification of membrane proteins of biological entities such as living phages, cells and the like.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, specifically to a method for mild and precise N-terminal Cys modification of peptides / proteins mediated by imidazole, and its application. Background Technology

[0002] Post-translational modifications of proteins are protein regulatory mechanisms that have developed in organisms over a long evolutionary process. They play a crucial role in many aspects of protein structure, stability, diversity, and biological function, and are an important area of ​​research in protein chemical biology. Post-translational modifications of proteins in organisms are precise, and the sites of modification are specific. By mimicking and learning from the post-translational modifications of proteins that have evolved over a long period of time in living organisms, proteins can be artificially modified to obtain precisely modified proteins (for antibody-drug conjugates, this means having an accurate and unique drug-antibody ratio, which is one of the most important quality indicators of antibody-drug conjugates and has become a transformative strategy for elucidating biological mechanisms and engineering functional biomolecules for diagnostic and therapeutic applications (Nat. Chem. 2016, 8, 103–113; Nat. Rev. Chem. 2019, 3, 147–171; Nat. Rev. Drug Discov. 2017, 16, 315–337; Chin. Chem. Lett. 2018, 29, 1017–1021).

[0003] Currently, various protein modification technologies have been developed, including -NH2 at the N-terminus of proteins, -SH for Cys, and -SeH for selenocysteine ​​(Sec) and -SeH for Lys. ε -NH2 is a common nucleophilic group in protein sequences, exhibiting good reactivity and becoming a preferred site for protein modification technology development (Nat. Rev. Chem. 2019, 3, 147–171; Chem. Soc. Rev. 2015, 44, 5495–5551); among these sites, the N-terminal -NH2 is unique in proteins. α -NH2, with a lower p K a(6-8, can still maintain nucleophilicity at physiological pH), and is usually free, does not participate in the formation of protein functional domains, and is a preferred site for protein modification. It is easy to modify and the modification does not affect the function of the protein (RSCChem. Biol. 2023, 4, 56–64); among them, the 1,2-aminothiol group of N-terminal Cys (NCys) has the dual nucleophilicity, combining the advantages of N-terminal -NH2 and -SH, and can achieve bifunctional modification of proteins. Currently, formulations using benzaldehyde (Chem. Sci. 2016, 7, 4589–4593; Chem. Sci. 2016, 7, 5052–5058; Angew. Chem. Int. Ed. 2020, 59, 14246–14250), nitriles (Angew. Chem. Int. Ed. 2009, 48, 9658–9662; Chem. Sci. 2022, 13, 8349–8354) and some other functional groups have been developed (J. Am. Chem. Soc. 2020, 142, 5097–5103; J. Am. Chem. Soc. 2025, 147, ) Methods for the specific modification of NCys (1612–1623) are available, but the limited commercial availability and / or synthetic complexity of these reagents limit their widespread use.

[0004] N 2-hydroxysuccinimide (NHS) esters are a class of widely available and highly reactive acyl donors, and are among the most commonly used acyl donors in protein modification techniques. A large number of commercially available NHS esters are available for researchers to choose from, and NHS esters can be easily obtained through the coupling reaction between the carboxyl group and NHS (Chem. Soc. Rev. 2015, 44, 5495–5551). However, when NHS esters are directly applied to protein modification, due to their strong reactivity, they can react with N-terminal -NH2, Cys' -SH, Sec' -SeH, and Lys' -SeH. εThe non-selective modification occurs at all sites, including -NH2, resulting in severe off-target effects. Researchers have developed other acyl donors to address the uneven modification of NHS esters, such as squaric acid derivatives with weaker activity than NHS esters (J. Am. Chem. Soc. 2023, 145, 25056–25060). However, this sacrifices the advantages of NHS esters, such as their wide availability, ease of preparation, and rapid reaction rate. In recent years, researchers have successfully achieved selective modification of NHS esters using NCys via natural chemical linkage (NCL) technology (J. Am. Chem. Soc. 2018, 140, 9374–9378; Eur. J. Med. Chem. 2023, 260). (115747), but these methods require high concentrations of thiol reagents, such as sodium 2-mercaptoethanesulfonate or 4-mercaptophenylacetic acid, and also require high concentrations of NHS esters or reducing agents (tris(2-chloroethyl) phosphate (TCEP)). This makes it impossible to achieve one-pot bifunctional modification of NCys (i.e., after modifying -NH2 via the NCL method, directly adding a maleimide-containing reagent to achieve one-pot bifunctional modification of NCys without any further treatment) and live-cell modification (thiol reagents and high concentrations of reducing agents are not cell-friendly). In fact, one-pot bifunctional modification would greatly improve the efficiency and economy of modification technology. Meanwhile, live-cell modification plays an extremely important role in many aspects such as disease diagnosis, drug development, and biological mechanism research, and is a goal that people urgently hope to achieve, a problem that urgently needs to be solved in current research, and a direction of effort.

[0005] Therefore, there is an urgent need to develop a mild and efficient NCys modification method that can achieve modification reactions that do not depend on reducing agents and denaturing conditions, do not require the participation of thiol reagents, and are compatible with live cell modification, thereby laying the foundation for the further widespread application of NCys modification methods. Summary of the Invention

[0006] To address the shortcomings and deficiencies of existing NCys (N-terminal cysteine) modification technologies, and to achieve selective NCys modification under mild and economical conditions, while ensuring that the method is cell-friendly and capable of modifying cell surface proteins, thereby further expanding the application scope of NCys modification technology, the primary objective of this invention is to provide an imidazole-mediated method for mild and precise NCys modification of peptides / proteins.

[0007] Another object of the present invention is to provide an application of the above method for modifying membrane proteins on the surface of biological entities such as bacteriophages and cells that maintain their activity.

[0008] This invention is the first to discover that under the conditions of 50 mmol / L NaH2PO4, 20 mmol / L TCEP, and pH 6.5, the NCys-containing affinity zHER2 (i.e., NCys-zHER2, denoted as P1, with the amino acid sequence: CVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKINDAQAPK (Sequence 1)) reacts with 8 equivalents of biotin-NHS ester to produce a complex mixture containing one or more biotin groups, such as... Figure 1 As shown; however, under the same conditions of 2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 20 mmol / L TCEP, and pH 6.5, the reaction yielded a high-yield product modified with only one biotin- (denoted as 1). After 4 hours of reaction, the high-performance liquid chromatography (HPLC) yield of product 1 (i.e., the yield of the corresponding product calculated based on the peak integral of HPLC) was 81%. Figure 2 As shown, and through liquid chromatography-secondary mass spectrometry (LC-MS / MS) analysis, it was confirmed that biotin- in product 1 is located at the N-terminus, that is, the reaction is selective modification, which demonstrates the important role of imidazole in promoting site-selective modification.

[0009] Based on the above findings, this invention explores the reaction conditions for variables such as the equivalent amount of acyl donor molecules, TCEP concentration, imidazole concentration, pH, and substrate protein concentration involved in the reaction, and determines that: (1) the optimal reaction conditions for the modification reaction are: 2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 2 mmol / L TCEP, and pH 6.5, wherein the imidazole concentration can maintain selectivity in the modification reaction within the range of 1 to 2.5 mol / L, and the imidazole concentration is preferably 2.5 mol / L; (2) the reaction is compatible with a range of NCys peptide / protein substrate concentrations from μmol / L to mmol / L, preferably 0.05 to 5 mmol / L; (3) the reaction is compatible with a wide range of acyl donor amounts, preferably 1.2 to 20 times the NCys peptide / protein concentration.

[0010] The general flowchart of this invention is shown below. Figure 3 The technical solution adopted is:

[0011] A method for mild and precise NCys modification of peptides / proteins mediated by imidazole, comprising the following steps:

[0012] A polypeptide / protein containing NCys and / or a non-natural amino acid with a 1,2-aminothiol group on its side chain is dissolved in a standard modification reaction buffer, and an acyl donor is added to the buffer to react and obtain a single-modification product in which the -NH2 of the 1,2-aminothiol group in the polypeptide / protein is linked to the acyl donor molecule via an amide bond; the standard modification reaction buffer contains imidazole.

[0013] Furthermore, the method also includes the following steps:

[0014] After obtaining the single-modified product, the maleimide--containing molecule was added to the reaction mixture for a one-pot reaction to obtain a double-modified product in which the -NH2 of the 1,2-aminothiol group in the polypeptide / protein is linked to the acyl donor molecule via an amide bond and the -SH is linked to the maleimide-containing molecule.

[0015] Further, the standard modified reaction buffer solution has the following composition: 1–2.5 mol / L imidazole, 40–60 mmol / L NaH2PO4, 1–20 mmol / L TCEP, pH 6.5–7.2; preferably: 2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 2 mmol / L TCEP, pH 6.5.

[0016] Furthermore, for polypeptides / proteins containing NCys, there are no special requirements for their sequence other than NCys; for polypeptides / proteins containing non-natural amino acids with 1,2-aminothiol groups on their side chains, there are no special requirements for their sequence other than containing non-natural amino acids with 1,2-aminothiol groups on their side chains; Cys in the sequence (regardless of whether the thiol group of Cys exists in the form of -SH or disulfide bond) or Lys does not affect the selectivity of the modification reaction; before the reaction, it must be ensured that the -SH of the 1,2-aminothiol group of NCys or non-natural amino acids is in a free state. The reaction is compatible with NCys polypeptide / protein substrate concentrations ranging from μmol / L to mmol / L, preferably 0.05–5 mmol / L.

[0017] Furthermore, the acyl donor is any one of a variety of commercially available or easily synthesized forms, such as acyl hydrazide, active ester, or acid anhydride.

[0018] Furthermore, the active ester is an NHS ester ( N Any one of the following: 1-hydroxysuccinimide ester, PNP ester (p-nitrophenyl ester), and PFP ester (pentafluorophenyl ester).

[0019] When the acyl donor is in the form of an acylhydrazine, it first needs to be activated by adding acetylacetone (ACAC) to a buffer solution at pH 3 and reacting at 37°C. N-Acyl-3,5-dimethylpyrazole form, or activated by reaction with NaNO2 at -15°C to N Only after being in the form of an acyl azide can it undergo modification reactions with the 1,2-aminothiol groups on the side chains of non-natural amino acids in peptides / proteins.

[0020] Furthermore, the amount of the acyl donor is 1.2 to 20 times the equivalent of the peptide / protein concentration, preferably 1.2 times the equivalent.

[0021] Furthermore, the reaction conditions are: shaking reaction at 37°C for 2–5 hours. The completion time of the reaction varies when different acyl donors are used for modification.

[0022] Furthermore, the amount of the maleimide-containing molecule fed is 1.2 to 20 times the equivalent of the peptide / protein concentration, preferably 1.2 times the equivalent.

[0023] Furthermore, the conditions for the one-pot reaction are: shaking reaction at 37°C for 0.5–2 hours. The completion time of the reaction varies when different molecules containing maleimide- are used for modification.

[0024] Furthermore, both the acyl donor and the maleimide-containing molecule are first treated with NMP (N-methyl- ... N Prepare a stock solution of methylpyrrolidone for later use.

[0025] A method for labeling / modifying proteins on the surface of biological entities involves incubating a biological entity containing a polypeptide / protein with non-natural amino acids having NCys and / or side chains with 1,2-aminothiol groups with an acyl donor in a standard modification reaction buffer solution to label / modify the surface of the biological entity; wherein the standard modification reaction buffer solution contains imidazole.

[0026] Furthermore, the biological entities mentioned include cells, bacteriophages, etc.

[0027] Furthermore, the cells mentioned include prokaryotic cells and eukaryotic cells.

[0028] Furthermore, the modification / labeling includes any one or more of small molecule-peptide / protein conjugates, peptide / protein-peptide / protein conjugates, etc., such as biotin labeling, fluorescent labeling, sulfation modification, phosphorylation modification, and succinylation modification; in this case, the acyl donor is any one or more of acylhydrazide, active ester, acid anhydride, etc., containing biotin groups, fluorescent groups, sulfate groups, phosphate groups, or succinic anhydride groups.

[0029] When the biological entity is a bacteriophage, the bacteriophage displays a peptide sequence of CX. nC is a phage display peptide library, where X is any amino acid and n is the number of any amino acid X.

[0030] When the biological entity is a bacteriophage, the standard modification reaction buffer solution consists of 2.5 mol / L imidazole and 2 mmol / L TCEP; the concentration of the acyl donor in the system is 1 ± 0.5 mmol / L, and the incubation time is 30 ± 5 minutes.

[0031] When the biological entity is a cell, the cell is first transiently transfected with a plasmid containing an N-terminal signal peptide, a Cys residue, and other functional sequences. As the plasmid is expressed and the signal peptide is cleaved in the cell, a cell is obtained in which the Cys residue becomes the first amino acid residue of the mature protein on the cell surface. Then, the cell is incubated with an acyl donor in a standard modification reaction buffer solution.

[0032] When the biological entity is a cell, the standard modification reaction buffer solution consists of 1 mol / L imidazole and 1 mmol / L TCEP; the concentration of the acyl donor in the system is 100±50 μmol / L, and the incubation time is 30±5 minutes.

[0033] The present invention has the following advantages and effects compared with the prior art:

[0034] The method provided by this invention is mild and cell-friendly, and has the potential to be applied to live cell modification. The inventors have successfully used this method to achieve live cell modification through experiments. Using the method of this invention, selective in-situ modification of NCys (or 1,2-aminothiol groups on non-natural amino acids within the membrane protein sequence) on the surface of various active biological entities, such as bacteriophages (viruses), bacteria (prokaryotic cells), and mammalian cells (eukaryotic cells), can be performed without causing inactivation or death of the aforementioned biological entities.

[0035] The method provided by this invention not only solves the problem that existing methods for modifying NCys with NHS esters cannot achieve one-pot thiol modification, but is also compatible with a variety of common commercially available acyl donors such as hydrazides, NHS esters, PNP esters, PFP esters, and acid anhydrides. The method is simple, efficient, and highly convenient to use.

[0036] The method provided by this invention can be used to construct small molecule-peptide / protein conjugates, peptide / protein-peptide / protein conjugates, etc., and shows good application prospects in the mild and precise modification of peptide / protein NCys and the preparation of related conjugates.

[0037] In summary, the method provided by this invention has the following advantages:

[0038] Specificity: Site-specific (1,2-aminothiol group) modification;

[0039] Mildness: Non-denaturing conditions, neutral pH (6.5–7.2), wide substrate concentration range;

[0040] Flexibility: Wide concentration range (μmol / L to mmol / L) of non-natural amino acids containing NCys and / or side chains with 1,2-aminothiol groups, compatible with a variety of acyl donor molecules;

[0041] Simplicity: Enables one-pot bifunctional modification of peptides / proteins containing NCys and / or non-natural amino acids with 1,2-aminothiol groups on their side chains.

[0042] Speed: The two-step modification reaction can be completed in 2–5 hours and 0.5–1 hour, respectively;

[0043] Economic efficiency: Low feed amount of modified molecules (≥1.2 equivalents) and high yield of modified products. Attached Figure Description

[0044] Figure 1 The reaction progress monitoring graph (monitored by HPLC) shows the modification reaction of P1 (1 mmol / L) with biotin-NHS ester (D5, 8 equivalents) under the conditions of 50 mmol / L NaH2PO4, 20 mmol / L TCEP, and pH 6.5.

[0045] Figure 2 The reaction progress monitoring graph (monitored by HPLC) shows the modification reaction of P1 (1 mmol / L) with biotin-NHS ester (D5, 8 equivalents) under the conditions of 2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 20 mmol / L TCEP, and pH 6.5.

[0046] Figure 3 This is a schematic diagram of the modification reaction process of the present invention;

[0047] Figure 4 A schematic diagram and modification yield diagram of a phage display peptide library with the display sequence CX7C selectively modified using biotin-GSGΓWGETGEGΓG acylhydrazide peptide (D4, where Γ is the non-natural amino acid penicillamine).

[0048] Figure 5 A schematic diagram and characterization results of selective modification of HEK293T cell surface proteins using Cy5-NHS ester (D8);

[0049] Figure 6The following is a structural diagram of the imidazole, biotin-, acylhydrazine group, ACAC, acyl-3,5-dimethylpyrazole group, acyl azide group, NHS ester, PNP ester, PFP ester, acid anhydride group, and maleimide- involved in the examples (the "" in the structural formula is missing from the original text). (Used to describe the position of group substitution);

[0050] Figure 7 The structures and designations of the acyl hydrazides, NHS esters, PNP esters, PFP esters, and acyl donor molecules in the form of acid anhydrides involved in the examples are shown in the diagram.

[0051] Figure 8 The diagram shows the structure (sequence) and symbol of the NCys polypeptide / protein involved in the examples;

[0052] Figure 9 The diagram shows the structure and designation of the maleimide- molecule involved in the examples;

[0053] Figure 10 The diagram shows the structure and designation of coupling elements 1 to 11.

[0054] Figure 11 The diagram shows the structure and designation of coupling elements 12-18.

[0055] Figure 12 The diagram shows the structure and designation of the double-modified products 19–21. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings. However, in addition to the specific implementation options described and given in the embodiments below, various alternative methods may be used, and it should be further understood that the present invention is not limited to the specific methods described herein and the polypeptide / protein sequences mentioned.

[0057] Examples 1-28 below illustrate the application of the method of the present invention in single-pot modification and one-pot thiol modification of different NCys peptides / proteins using different acyl donors, as well as its application in live cell modification. In the examples, the reversed-phase high-performance liquid chromatography (RP-HPLC) instrument used was an Agilent 1260, with either a Welch Ultimate XB-C4 column (5 μm, 300 Å, 4.6 × 250 mm) or a Welch Ultimate XB-C18 column (5 μm, 120 Å, 4.6 × 250 mm), and the mobile phase was water and acetonitrile (containing 0.1% trifluoroacetic acid).

[0058] The terms “peptide” and “protein” as used below refer to compounds formed by any amino acid monomers (including non-natural amino acids) linked together by peptide bonds; “NCys peptide / protein” refers to a peptide / protein with a Cys terminus at the N-terminus, where the -SH of the Cys terminus is in an unprotected free state.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0060] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0061] The structural formulas of imidazole, biotin-, acylhydrazine group, ACAC, acyl-3,5-dimethylpyrazole group, acyl azide group, NHS ester, PNP ester, PFP ester, acid anhydride group, and maleimide- used and involved in the following examples are as follows: Figure 6 As shown.

[0062] The acyl hydrazide, NHS ester, PNP ester, PFP ester, and anhydride acyl donor molecules involved in the following examples have the following structures and designations: Figure 7 As shown.

[0063] The NCys polypeptides / proteins involved in the following examples have the following structures (sequences) and codes: Figure 8 As shown.

[0064] The structures and designations of maleimide- molecules involved in the following examples are as follows: Figure 9 As shown.

[0065] The structures and codes of couplings 1 to 11 involved in the following embodiments are as follows: Figure 10 As shown; the structures and codes of couplings 12-18 are as follows. Figure 11 As shown; the structures and codes of the double-modified products 19–21 are as follows. Figure 12 As shown.

[0066] I. Specific Examples of Modification Using Different Acyl Donors

[0067] (a) Acyl donors in the form of hydrazides

[0068] Example 1: D1 is activated by ACAC and then modified with P1

[0069] First, D1 (2.4 mmol / L) was activated with ACAC (1.5 equivalents) for 2 hours under conditions of 50 mmol / L NaH2PO4 and pH 3.0. The reaction mixture was then lyophilized to obtain the activated product. ND1 in the form of acyl-3,5-dimethylpyrazole (denoted as D1-a) was prepared by dissolving P1 in a standard modification reaction buffer (2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 2 mmol / L TCEP, pH 6.5) to prepare a 1 mmol / L P1 solution. The solution was then transferred to the D1-a tube, making the concentration of D1-a 1.2 times that of P1. After reacting at 37 °C for 3 hours, the small molecule-protein conjugate 1 was obtained with a yield of 79% by HPLC. The relative molecular mass was determined by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS), with a theoretical value of 7035.0 and a measured value of 7034.9.

[0070] Example 2: D1 was activated with NaNO2 and then modified with P1

[0071] First, D1 (2.4 mmol / L) was activated with NaNO2 (10 equivalents) for 15 minutes at 50 mmol / L NaH2PO4 and pH 3.0. The reaction mixture was then directly freeze-dried to obtain the activated product. N D1 in the form of acyl azide (denoted as D1-b) was prepared by dissolving P1 in a standard modification reaction buffer (2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 2 mmol / L TCEP, pH 6.5) to prepare a P1 solution with a concentration of 1 mmol / L. The solution was then transferred to the D1-b tube, making the concentration of D1-b 1.2 times that of P1. After reacting at 37 °C for 3 hours, the small molecule-protein conjugate 1 was obtained with a yield of 95% by HPLC.

[0072] Example 3: D2 is activated by NaNO2 and then modified into P1

[0073] The preparation method is the same as in Example 2, except that the D2 concentration is 4.8 mmol / L, and the activation is... N The concentration of D2 (denoted as D2-b) in the α-acyl azide form was 2.4 times that of P1. After reacting for 3 hours, the peptide-protein conjugate 2 was obtained with an 85% HPLC yield. The relative molecular mass was determined by LC-ESI-MS, with a theoretical value of 8724.0 and a measured value of 8723.0.

[0074] Example 4: D3 activated by ACAC then modified P1

[0075] The preparation method is the same as in Example 1, except that ACAC is added at 2.5 times the equivalent amount and activated for 1 hour to obtain... ND3 in the form of acyl-3,5-dimethylpyrazole was reacted for 3 hours to obtain polypeptide-protein conjugate 3 with a yield of 74% by HPLC. The relative molecular mass was determined by LC-ESI-MS, with a theoretical value of 8272.3 and a measured value of 8271.6.

[0076] Example 5: D4 activated by ACAC then modified with P1

[0077] D4 is a ubiquitin protein with a C-terminal acylhydrazine, and its sequence is as follows:

[0078] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRG (sequence 2)

[0079] The preparation method was the same as in Example 1, except that the concentration of D4 was 0.5 mmol / L. After reacting for 5 hours, protein-protein conjugate 4 was obtained with a yield of 61% by HPLC. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 15298.5 and a measured value of 15297.2.

[0080] (ii) Acyl donors in NHS ester form

[0081] Example 6: D5 modification of P1

[0082] First, P1 was dissolved in a standard modification reaction buffer (2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 2 mmol / L TCEP, pH 6.5) to prepare a P1 solution with a concentration of 1 mmol / L. Then, 1.2 equivalents of D2 were added (D5 was prepared as a stock solution with NMP beforehand). After reacting at 37°C for 2.5 hours, the small molecule-protein conjugate 1 was obtained with an HPLC yield of 91%.

[0083] Example 7: D6 modification of P1

[0084] The preparation method was the same as in Example 6. After reacting for 5 hours, the small molecule-protein conjugate 5 was obtained with a yield of 66% by HPLC. The relative molecular mass was measured by LC-ESI-MS. The theoretical value was 7082.0 and the measured value was 7081.8.

[0085] Example 8: D7 modification of P1

[0086] The preparation method was the same as in Example 6. After reacting for 3 hours, the small molecule-protein conjugate 6 was obtained with a yield of 70% by HPLC. The relative molecular mass was measured by LC-ESI-MS. The theoretical value was 6862.7 and the measured value was 6862.0.

[0087] Example 9: D8 modification of P1

[0088] The preparation method was the same as in Example 6, except that the concentration of TCEP was 5 mmol / L. After reacting for 8 hours, the small molecule-protein conjugate 7 was obtained with an HPLC yield of 88%. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 7432.5 and a measured value of 7433.0.

[0089] (iii) Acyl donors in PNP ester form

[0090] Example 10: D9 modification of P1

[0091] The preparation method was the same as in Example 6. After reacting for 2.5 hours, small molecule-protein conjugate 1 was obtained with a HPLC yield of 72%.

[0092] Example 11: D10 modification of P1

[0093] The preparation method was the same as in Example 6. After reacting for 4 hours, small molecule-protein conjugate 8 was obtained with a yield of 71% by HPLC. The relative molecular mass was measured by LC-ESI-MS. The theoretical value was 6906.9 and the measured value was 6905.9.

[0094] (iv) Acyl donors in PFP ester form

[0095] Example 12: D11 modification of P1

[0096] The preparation method was the same as in Example 6. After reacting for 2 hours, small molecule-protein conjugate 1 was obtained with an HPLC yield of 66%.

[0097] Example 13: D12 modification of P1

[0098] The preparation method was the same as in Example 6. After reacting for 5 hours, the small molecule-protein conjugate 9 was obtained with an HPLC yield of 89%. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 7102.1 and a measured value of 7101.7.

[0099] (v) Acyl donors in the form of acid anhydrides

[0100] Example 14: D13 modification of P1

[0101] The preparation method was the same as in Example 6. After reacting for 10 hours, small molecule-protein conjugate 10 was obtained with a yield of 72% by HPLC. The relative molecular mass was measured by LC-ESI-MS. The theoretical value was 6912.8 and the measured value was 6911.5.

[0102] Example 15: D14 modification of P1

[0103] The preparation method was the same as in Example 6. After reacting for 1 hour, the small molecule-protein conjugate 11 was obtained with an HPLC yield of 83%. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 6908.8 and a measured value of 6907.9.

[0104] II. Specific Examples of NCys Peptide / Protein Expansion

[0105] In this embodiment, for NCys peptide substrates (P2-P5), the reaction progress was monitored by HPLC, the HPLC yield was calculated, and the substances corresponding to the HPLC peaks were identified by LC-ESI-MS. For protein substrates (P1, P6-P8), the reaction solution was sampled at different times and directly injected into an LC-ESI-MS instrument equipped with a Xevo G3 QTOF mass spectrometer (ESI source; the instrument is manufactured by Waters Corporation, USA) to monitor the reaction progress. The conversion rate of the protein substrate to the product was calculated based on the relative area of ​​the deconvoluted mass spectrum peaks of the corresponding products.

[0106] (I) NCys peptide substrate expansion

[0107] Example 16: D5 modification of P2

[0108] The preparation method was the same as in Example 6. After reacting for 7 hours, the small molecule-peptide conjugate 12 was obtained with a yield of 84% by HPLC. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 1661.6 and a measured value of 1662.0. It is worth noting that, in addition to NCys, the P2 sequence also contains two Cys (their -SH groups are in a free state and have not formed disulfide bonds). After the modification reaction, the single modified product 12 was the only modified product, indicating that the modification reaction was not affected by the Cys in the sequence.

[0109] Example 17: D5 modification of P3

[0110] The preparation method was the same as in Example 6, except that the concentration of P3 was 0.5 mmol / L. After reacting for 3 hours, the small molecule-peptide conjugate 13 was obtained with an HPLC yield of 87%. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 1963.0 and a measured value of 1964.0.

[0111] Example 18: D5 modification of P4

[0112] The preparation method was the same as in Example 6. After reacting for 3 hours, small molecule-peptide conjugate 14 was obtained with a yield of 72% by HPLC. The relative molecular mass was measured by LC-ESI-MS. The theoretical value was 3415.0 and the measured value was 3414.3.

[0113] Example 19: D5 modification of P5

[0114] The preparation method was the same as in Example 6, except that the concentration of TCEP was 5 mmol / L. After reacting for 10 hours, the small molecule-peptide conjugate 15 was obtained with a yield of 68% by HPLC. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 11179.9 and a measured value of 11178.8.

[0115] (II) NCys protein substrate expansion

[0116] Example 20: D5 modification of P1 (reaction progress monitored by LC-MS)

[0117] The preparation method is the same as in Example 6, except that the reaction process is monitored by LC-MS. After 2 hours of reaction, P1 generates small molecule-protein conjugate 1 with a conversion rate of >95%.

[0118] Example 21: D5 modification of P6 (reaction progress monitored by LC-MS)

[0119] P6 is an azurin containing NCys, and its sequence is as follows:

[0120] CAEC 4 SVDIQGNDQMQFNTNAITVDKSC 27 KQFTVNLSHPGNLPKNVMGHNWVLSTAADMQGVVTDGMASGLDKDYLKPDDSRVIAHTKLIGSGEKDSVTFDVSKLKEGEQYMFFC 113 TFPGHSALMKGTLTLK (sequence 3)

[0121] In addition to NCys, the P6 sequence also contains 3 Cys, namely C 4 C 27 and C 113 And C 4 With C 27 Disulfide bonds were formed between them, C 113 The sidechain is free-SH.

[0122] The preparation method was the same as in Example 6, except that the imidazole concentration was 1 mol / L, and the reaction progress was monitored by LC-MS. After 2 hours of reaction, P6 was converted into small molecule-protein conjugate 16 with a conversion rate of >95%. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 14273.3 and a measured value of 14273.8. This example demonstrates that this method is not affected by intra-sequence Cys (regardless of whether the thiol group of Cys exists in the form of -SH or disulfide bond).

[0123] Example 22: D5 modification of P7 (reaction progress monitored by LC-MS)

[0124] P7 is a small ubiquitin-like modified protein (SUMO) with NCys, and its sequence is as follows:

[0125] CASDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGSHHHHHH (Sequence 4)

[0126] The preparation method was the same as in Example 6, except that the concentration of P7 was 0.5 mmol / L and the concentration of TCEP was 5 mmol / L. The reaction process was monitored by LC-MS. After 3 hours of reaction, P7 was converted into small molecule-protein conjugate 17 with a conversion rate of >95%. The relative molecular mass was measured by LC-ESI-MS. The theoretical value was 12441.0 and the measured value was 12441.4.

[0127] Example 23: D2 modification of P8 (reaction progress monitored by LC-MS)

[0128] P8 is a multidrug resistance regulatory protein (LmrR) of *Streptococcus lactis* with NCys, and its sequence is as follows:

[0129] CASAEIPKEMLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQDGIISSYWGDESQGGRRKYYRLTEIGHENMRLAFESWSRVDKIIENLEANKKSEAIKGSHHHHHH (Sequence 5)

[0130] The preparation method was the same as in Example 6, except that the concentration of P8 was 0.3 mmol / L and the concentration of TCEP was 5 mmol / L. The reaction progress was monitored by LC-MS. After 3 hours of reaction, P8 was converted into small molecule-protein conjugate 18 with a conversion rate of >95%. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 14770.0 and a measured value of 14769.3. It is particularly noteworthy that, due to the incomplete cleavage of the pelB signal peptide of the expressed pelB-C-LmrR fusion protein, the purified P8 expression yielded a mixture. However, only P8 in the mixture was modified to obtain small molecule-protein conjugate 18, while other impurities without NCys did not react. This demonstrates the good selectivity of this method, which can selectively modify NCys-containing proteins in the protein mixture.

[0131] III. Specific Examples of One-Pot Thiol Modification

[0132] Example 24: One-pot thiol modification of P1 using D5 and M1

[0133] First, P1 was dissolved in a standard modification reaction buffer (2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 2 mmol / L TCEP, pH 6.5) to prepare a 1 mmol / L P1 solution. Then, 1.2 equivalents of D5 (prepared as a stock solution with NMP beforehand) were added. The reaction was carried out at 37°C for 2 hours to obtain a single-modified product. Then, 1.2 equivalents of M1 (prepared as a stock solution with NMP beforehand) were added directly to the reaction mixture. After reacting at 37°C for 30 minutes, the target double-modified product 19 was obtained with a 95% HPLC yield. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 7146.1 and a measured value of 7146.6. The reaction solution was then passed through a 0.22 μm syringe filter and injected into a semi-preparative HPLC system, where the target double-modified product 19 was obtained with a separation yield of 64%.

[0134] Example 25: One-pot thiol modification of P1 using D3 and M2

[0135] First, D3 (4.8 mmol / L) was activated with NaNO2 (10 equivalents) for 15 minutes at 50 mmol / L NaH2PO4 and pH 3.0. The reaction mixture was then lyophilized to obtain D3-b. Then, P1 was dissolved in a standard modification reaction buffer (2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 5 mmol / L TCEP, pH 6.5) to prepare a 1 mmol / L solution. The P1 solution was prepared and then transferred to a D3-b tube, making the D3-b concentration 2.4 times that of P1. After reacting at 37°C for 3 hours, a single-modified product was obtained. Then, 1.2 equivalents of M2 (prepared as a stock solution with NMP beforehand) were added directly to the reaction mixture. After reacting at 37°C for 1 hour, the target double-modified product 20 was obtained with a 58% HPLC yield. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 10040.7 and a measured value of 10039.7. The reaction solution was filtered through a 0.22 μm syringe and injected into a semi-preparative HPLC system, where the target double-modified product 20 was obtained with a 30% separation yield.

[0136] Example 26: One-pot thiol modification of P1 using D8 and M2

[0137] First, P1 was dissolved in a standard modification reaction buffer (2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 2 mmol / L TCEP, pH 6.5) to prepare a 1 mmol / L P1 solution. Then, 1.2 equivalents of D8 (using a prepared NMP solution of D8) were added. After reacting at 37°C for 2 hours, a single-modified product was obtained. Then, 1.2 equivalents of M2 (prepared as a stock solution with NMP) were added directly to the reaction mixture. After reacting at 37°C for 1 hour, the target double-modified product 21 was obtained with a 76% HPLC yield. The relative molecular mass was measured by LC-ESI-MS, with a theoretical value of 8749.1 and a measured value of 8749.1. The reaction solution was filtered through a 0.22 μm syringe and injected into a semi-preparative HPLC system, where the target double-modified product 21 was obtained with a 55% separation yield.

[0138] IV. Specific Examples of Modifying Bacteriophage Surface Proteins

[0139] Example 27: Modification of phage surface proteins

[0140] This method is used to modify proteins on the surface of bacteriophages, such as... Figure 4 As shown. First, the N-terminal biotin-conjugated acylhydrazide peptide D4 was synthesized, and then activated by ACAC to... N The acyl-3,5-dimethylpyrazole form (denoted as D4-a) was purified, and then D4-a was incubated with a phage display peptide library with the display peptide sequence CX7C (X being any amino acid) for 2 hours in the presence of 2.5 mol / L imidazole and 2 mmol / L LTCEP. The modification yield was calculated after screening with streptavidin immobilized on the surface of magnetic beads, and the modification yield was 58%, indicating successful modification. This shows that the method is phage-friendly and will not cause phage inactivation or death, and can be applied to the modification of phage surface proteins. Further optimization of the suitable conditions for labeling bacteriophages revealed that when bacteriophages were incubated with 1 mmol / L D4-a in the presence of 2.5 mol / L imidazole and 2 mmol / L TCEP for 30 minutes, a modification yield of 75% was obtained. This is comparable to the modification yield of previously reported enzyme-mediated ligation reactions (the modification yield of OaAEP1 enzyme was 68% (Org. Lett., 2024, 26, 2601-2605), and the modification yield of SrtA enzyme was 73% (Chem. Sci., 2024, 15, 9649-9656)).

[0141] V. Specific Examples of Modifying Live Cell Surface Proteins

[0142] Example 28: Modification of mammalian living cell surface proteins

[0143] This method was used to selectively fluorescently label NCys on the surface membrane proteins of mammalian living cells, such as... Figure 5 As shown. HEK293T cells were transiently transfected with a plasmid containing the model protein C-HA-Nlgn3-mCherry (amino acid sequence shown in Sequence 6, nucleotide sequence shown in Sequence 7) containing an N-terminal signal peptide, Cys residues, an HA tag, and Nlgn3 (a member of the neural connecton family) and the red fluorescent protein mCherry. The plasmid was pCMV, and the insertion site was [insert insertion site here]. Pac I and Nhe I); With the expression of this plasmid and the cleavage of the signal peptide in HEK293T cells, the Cys residue becomes the first amino acid residue of the mature protein on the cell surface; the cells were labeled with the fluorescently labeled molecule Cy5-NHS ester (D8) (100 μmol / L) for 30 minutes under 1 mol / L imidazole and 1 mmol / L TCEP conditions, then washed three times with live-cell imaging solution, and cultured in culture medium for 2 hours before confocal laser scanning microscopy imaging. Confocal microscopy analysis was performed only on transfected cells (i). Figure 5 Cy5 modification was observed in cells indicated by the solid arrow (i.e., untransfected cells) and not in untransfected cells (i.e., cells indicated by the solid arrow). Figure 5 No Cy5 modification was observed on the cells indicated by the hollow arrow, confirming the selectivity of Cy5 labeling; the fluorescence of Cy5 highly overlapped with the fluorescence of mCherry in the membrane protein, indicating selective modification of the C-HA-Nlgn3-mCherry membrane protein on the surface of HEK293T cells.

[0144] Examples 27 and 28 illustrate that this method is friendly to active bacteriophages and mammalian cells, and will not cause the above-mentioned biological entities to become inactive or die. It can be applied to the modification of NCys on the membrane proteins on the surface of these biological entities and has broad application prospects.

[0145] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for mild, precise modification of NCys of a polypeptide / protein mediated by imidazole, characterized in that: It comprises the following steps: dissolving the polypeptide / protein containing NCys and / or unnatural amino acids with 1,2-aminothiol groups in the side chain in a standard modification reaction buffer solution, and adding an acyl donor to the solution to obtain a single modification product of the -NH2 of the 1,2-aminothiol group in the polypeptide / protein connected to the acyl donor molecule through an amide bond by reaction; the standard modification reaction buffer solution comprises 1-2.5 mol / L imidazole, 40-60 mmol / L NaH2PO4, 1-2 mmol / L TCEP, and has a pH of 6.5-7.2; The acyl donor is any of the hydrazide, active ester and anhydride forms; when the acyl donor is in the hydrazide form, it is first required to be activated by the addition of acetylacetone in a buffer solution at pH 3 at 37 °C to form N -acyl-3,5-dimethylpyrazole form, or by the addition of NaNO2at -15 °C to form N -acyl azide form before it can undergo a modification reaction with the NCys of the polypeptide / protein and / or the 1,2-aminothiol groups of the side chains of the non-natural amino acids in the sequence; the active ester is any one of NHS ester, PNP ester, and PFP ester; the reaction conditions are: oscillation reaction at 37℃, and the reaction time is 2-5 hours.

2. The method for mild and precise modification of NCys of polypeptide / protein mediated by imidazole according to claim 1, characterized in that: after obtaining the single modification product, a maleimide-containing molecule is added to the reaction mixture to perform a one-pot reaction, so as to obtain a double modification product of the -NH2 of the 1,2-aminothiol group in the polypeptide / protein connected to the acyl donor molecule through an amide bond and the -SH connected to the maleimide-containing molecule; the one-pot reaction conditions are: oscillation reaction at 37℃, and the reaction time is 0.5-2 hours.

3. The method for mild and precise modification of NCys of polypeptide / protein mediated by imidazole according to claim 2, characterized in that: the concentration of the polypeptide / protein ranges from 0.05 mmol / L to 5 mmol / L; the feeding amount of the acyl donor is 1.2-20 times the equivalent of the polypeptide / protein concentration; the feeding amount of the maleimide-containing molecule is 1.2-20 times the equivalent of the polypeptide / protein concentration.

4. The method for mild and precise modification of NCys of polypeptide / protein mediated by imidazole according to claim 2, characterized in that: the standard modification reaction buffer solution comprises 2.5 mol / L imidazole, 50 mmol / L NaH2PO4, 2 mmol / L TCEP, and has a pH of 6.5; the feeding amount of the acyl donor is 1.2 times the equivalent of the polypeptide / protein concentration; the feeding amount of the maleimide-containing molecule is 1.2 times the equivalent of the polypeptide / protein concentration.

5. The method for mild and precise modification of NCys of polypeptide / protein mediated by imidazole according to any one of claims 1-4, characterized in that: The acyl donor and the maleimide-containing molecule are both first used N - methylpyrrolidone is formulated into a mother liquor for later use.

6. A method of biological entity surface protein labelling / modification, characterised by: the biological entity of the polypeptide / protein containing NCys and / or unnatural amino acids with 1,2-aminothiol groups in the side chain is incubated with an acyl donor in a standard modification reaction buffer solution to perform surface protein labeling / modification of the biological entity; and the standard modification reaction buffer solution contains imidazole. The label / modification includes any one or more of biotin label, fluorescent label, sulfation modification, phosphorylation modification or succinylation modification; at this time, the acyl donor is any one or more of hydrazide, active ester, acid anhydride containing biotin group, fluorescent group, sulfate group, phosphate group or succinic anhydride group; The biological entity includes at least one of cell and bacteriophage; When the biological entity is bacteriophage, the standard modification reaction buffer solution is composed of 2.5 mol / L imidazole and 2 mmol / L TCEP; the concentration of the acyl donor in the system is 1±0.5 mmol / L, and the incubation time is 30±5 minutes; When the biological entity is cell, the standard modification reaction buffer solution is composed of 1 mol / L imidazole and 1 mmol / L TCEP; the concentration of the acyl donor in the system is 100±50 μmol / L, and the incubation time is 30±5 minutes.

7. The method for labeling / modifying surface protein of biological entity according to claim 6, characterized in that: The cell includes prokaryotic cell and eukaryotic cell.

8. The method for labeling / modifying surface protein of biological entity according to claim 6, characterized in that: The phage has a displayed peptide sequence of CX n C, wherein X is any amino acid and n is the number of any amino acid X. The cell is first transiently transfected with plasmid containing N-terminal signal peptide, Cys residue and other functional sequences, and then incubated with acyl donor in standard modification reaction buffer solution after the Cys residue becomes the first amino acid residue of mature protein on the cell surface with the expression of plasmid in the cell and the removal of signal peptide.

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