A method for C-terminal selenolysis modification of proteins mediated by selenocystamine and its application

Abstract

This invention provides a method for C-terminal selenolytic modification of proteins mediated by selenocysteine ​​and its application, belonging to the field of protein chemical selective modification technology. The method of this invention is based on C-terminal peptide / protein acylhydrazide. First, the acylhydrazide is activated with acetylacetone to form an acylpyrazole. Then, selenocysteine ​​is used to selectively selenolytically modify the C-terminus of the peptide / protein. The resulting more active selenolytic reaction site can be used for selective protein modification, effectively simplifying the reaction steps and operation process, and providing a feasible route for efficient and highly selective modification of protein C-termini. Furthermore, the reaction conditions of this invention are mild, requiring only 1 equivalent of selenocysteine ​​for efficient selenolytic modification, exhibiting good biocompatibility and operability. In addition, the method of this invention has a wide range of substrate applicability, compatible with various functional selenolytic modification reagents and different types of protein substrates, providing a universal technical solution for the targeted modification of peptides / proteins.

Description

Technical Field

[0001] This invention relates to the field of protein chemical selective modification technology, specifically to a method for C-terminal selenolysis modification of proteins mediated by selenocysteine ​​and its application. Background Technology

[0002] With the rapid development of biomedicine and chemical biology, precise and controllable chemical modification of proteins and peptides has become one of the core technologies for achieving functional regulation, constructing novel bioconjugate drugs, and developing advanced biological tools. Post-translational modification and the artificial introduction of functional groups (such as fluorescent probes and drug molecules) can significantly expand the structural and functional diversity of proteins, showing broad application prospects and significant commercial value in disease diagnosis, targeted therapy, bioimaging, and protein function analysis. However, existing modification methods still face serious challenges in achieving site selectivity, biocompatibility, and mild reaction conditions.

[0003] An ideal peptide / protein modification method should possess the following characteristics: strict site selectivity, acting only on specified residues or ends to avoid non-specific modification caused by the repeated occurrence of the same type of amino acid in the sequence; ease of operation and high conversion rate to simplify subsequent purification procedures; high efficiency under aqueous biocompatible conditions to maintain the native conformation and function of the protein; and well-defined and homogeneous modified products. [1] To date, various chemoselective modification techniques have been developed in this field to achieve the functionalization of peptide chains and the N-terminus, C-terminus, and almost all natural amino acid side chains (containing polar or charged functional groups) of proteins. [2] Currently, commonly used bioconjugation strategies mainly rely on the nucleophilic side chains of natural amino acids such as lysine and cysteine. Their inherent nucleophilicity allows for efficient conjugation via nucleophilic substitution or Michael addition of α-halocarbonyl compounds, maleimides, and reagents containing the corresponding functional groups. [3,4] However, the regioselectivity can be insufficient because the same amino acid residue may appear multiple times in a protein sequence. Furthermore, non-natural amino acids can be introduced to provide click chemistry reaction sites; for example, copper-catalyzed azido-alkyne cycloaddition and strain-promoted azido-alkyne cycloaddition reactions, as well as tetrazine chemistry, have also been applied to protein modification. [5,6] However, such methods are mostly limited to non-natural amino acid side chains and are difficult to apply to the precise modification of natural peptides / proteins.

[0004] Currently available C-end modification strategies mainly include the following categories: enzymatic methods (such as transpeptidase sortase). [7] Formicylglycine synthase FGE [8]While possessing good selectivity, these methods are limited by the enzyme's strict requirements on substrate recognition sequences, resulting in low universality, and their activity is easily affected by protein spatial structure; chemoselective methods (such as those based on thioester intermediates, α-keto acid decarboxylation, etc.) [9] While various approaches (such as photocatalysis, etc.) can achieve modification to some extent, they often require pre-activation steps, involve harsh reaction conditions (such as strong acids, high temperatures, or organic phase environments), and result in numerous side reactions, which are detrimental to maintaining protein integrity. Although transition metal catalysis or photocatalysis strategies have made some progress in recent years, they still generally suffer from poor biocompatibility of the catalytic system, the need to introduce non-natural amino acids, or insufficient regioselectivity control. Despite the existence of various technologies attempting C-terminal modification, methods that can achieve highly selective and highly compatible modification under aqueous phase and mild conditions remain relatively scarce. [2] Therefore, developing a novel, efficient modification strategy for the C-terminus of natural proteins that requires mild reaction conditions, does not require the introduction of exogenous amino acids or complex pre-activation, and is applicable to such modifications is of significant scientific importance and has promising application prospects.

[0005] The rapid development of selenium chemistry has brought new opportunities for protein modification. Selenocysteine ​​(Sec), a naturally occurring yet extremely rare amino acid in the proteome, possesses a low pK value in its selenool side chain. a Its high oxidation potential and stronger nucleophilicity compared to cysteine ​​(Cys) endow it with excellent chemoselectivity and regioselectivity in protein chemical modification.

[10] As a result, a variety of innovative applications have emerged in recent years. For example, Zhao et al.

[11] Using Cu(II)-assisted hydrazine compounds to generate aryl / alkyl radicals, functional molecules were selectively introduced into selenocysteine-containing proteins in an aqueous buffer solution close to physiological pH; Angus et al.

[12] An electrochemical selenyl etherification (e-SE) platform was constructed using the reactivity of selenium. Under metal-catalyst-free conditions, site-specific functionalization of the C-terminus of peptides was successfully achieved through electrochemically driven formation of selenyl ether bonds. (Zhao et al.)

[13] By modulating the electronic properties of the substrate, late-stage functionalization of linear, cyclic peptides, and even intact proteins can be achieved in systems containing both cysteine ​​and selenocysteine. These studies clearly demonstrate that sec, with its unique redox and nucleophilic properties, provides a powerful tool for site-selective protein modification and lays the foundation for developing mild, efficient, and biocompatible modification strategies.

[0006] Selenocysteine ​​shares structural similarities with selenocysteine, possessing both reversible diselenylene bond reactivity and strong nucleophilicity of selenools. Under mild reducing conditions, it can release highly reactive selenool species, which then selectively react with C-terminal acylhydrazides in proteins. This approach promises to balance chemoselectivity, regiospecificity, and mild conditions, enabling precise C-terminal functionalization. Compared to selenocysteine, which requires pre-insertion into the protein sequence via codon expansion technology, selenocysteine ​​can be used directly as a small molecule reagent, offering advantages such as simplicity, availability, and low cost. Nevertheless, C-terminal modification techniques that achieve high selectivity, high biocompatibility, and high conversion rates under aqueous conditions remain relatively scarce. Therefore, developing a novel strategy that eliminates the need for exogenous amino acids, does not rely on precious metal catalysis, and can directly, efficiently, and selectively achieve C-terminal selenolysis modification of proteins in aqueous conditions could provide new insights for optimizing peptide / protein targeted modification technologies and significantly expand the applicability of this technology in bioconjugation, therapeutic protein drug research, and applications.

[0007] References:

[0008] [1] Dowman LJ, Kulkarni SS, Alegre-Requena JV, et al. Site-selective photocatalytic functionalization of peptides and proteins atselenocysteine[J]. Nature Communications, 2022, 13(1): 6885.

[0009] [2] Walsh SJ, Bargh JD, Dannheim FM, et al. Site-selective modification strategies in antibody–drug conjugates[J]. Chemical Society Reviews, 2021, 50(2): 1305-1353.

[0010] [3] Boutureira O, Bernardes GJ L. Advances in Chemical ProteinModification[J]. Chemical Reviews, 2015, 115(5): 2174-2195.

[0011] [4] Hoyt E A, Cal P M S D, Oliveira B L, et al. Contemporaryapproaches to site-selective protein modification[J]. Nature ReviewsChemistry, 2019, 3(3): 147-171.

[0012] [5] Sletten E M, Bertozzi C R. Bioorthogonal Chemistry: Fishing forSelectivity in a Sea of Functionality[J]. Angewandte Chemie InternationalEdition, 2009, 48(38): 6974-6998.

[0013] [6] Plass T, Milles S, Koehler C, et al. Genetically Encoded Copper-Free Click Chemistry[J]. Angewandte Chemie International Edition, 2011, 50(17): 3878-3881.

[0014] [7] Harmand T J, Bousbaine D, Chan A, et al. One-Pot Dual Labeling ofIgG 1 and Preparation of C-to-C Fusion Proteins Through a Combination ofSortase A and Butelase 1[J]. Bioconjugate Chemistry, 2018, 29(10): 3245-3249.

[0015] [8] Agarwal P, Kudirka R, Albers A E, et al. Hydrazino-Pictet-Spengler Ligation as a Biocompatible Method for the Generation of StableProtein Conjugates[J]. Bioconjugate Chemistry, 2013, 24(6): 846-851.

[0016] [9] Bloom S, Liu C, Kölmel D K, et al. Decarboxylative alkylation forsite-selective bioconjugation of native proteins via oxidation potentials[J].Nature Chemistry, 2018, 10(2): 205-211.

[0017]

[10] Zhao Z, Laps S, Gichtin J S, et al. Selenium chemistry forspatio-selective peptide and protein functionalization[J]. Nature ReviewsChemistry, 2024, 8(3): 211-229.

[0018]

[11] Zhao Z, Metanis N. Utilizing Copper-Mediated Deprotection ofSelenazolidine for Cyclic Peptide Synthesis[J]. The Journal of OrganicChemistry, 2020, 85(3): 1731-1739.

[0019]

[12] Mackay AS, Maxwell JWC, Bedding MJ, et al. ElectrochemicalModification of Polypeptides at Selenocysteine[J]. Angewandte ChemieInternational Edition, 2023, 62(50): e202313037.

[0020]

[13] Zhao Z, Huang J, Cai Y, et al. Late-Stage Aromatic C–H BondFunctionalization for Cysteine / Selenocysteine ​​Bioconjugation[J]. Journal of the American Chemical Society, 2025, 147(35): 31811-31820. Summary of the Invention

[0021] To overcome the shortcomings and deficiencies of existing peptide / protein C-terminal modification methods in terms of site specificity and modification selectivity, this invention aims to provide a method for selenocysteine-mediated C-terminal selenolysis modification of proteins and its applications. This method achieves efficient and highly selective modification of the protein C-terminus by precisely constructing highly active selenium-specific reaction sites at the C-terminus of peptides / proteins, providing a new approach for targeted protein modification.

[0022] The objective of this invention is achieved through the following technical solution:

[0023] A method for selenocysteine-mediated C-terminal selenolysis modification of proteins, comprising the following steps:

[0024] S1. Dissolve the peptide / protein with a C-terminus acylhydrazine in a buffer solution, add acetylacetone, and react to obtain a first reaction mixture containing a peptide / protein with a C-terminus acylpyrazole group.

[0025] S2. Dissolve selenocysteine ​​in buffer solution, add tris-(2-carboxyethyl)phosphine hydrochloride and ascorbic acid, react to obtain a second reaction mixture containing selenocysteine;

[0026] S3. Add the second reaction mixture obtained in step S2 to the first reaction mixture obtained in step S1, react, and obtain a third reaction mixture containing a selenium group reaction site at the C-terminus.

[0027] S4. Adjust the pH of the third reaction mixture obtained in step S3, add the selenium-based modification reagent, mix well, and react; purify the reaction solution to obtain the target product.

[0028] Further, the amount of buffer solution used in step S1 is calculated based on the concentration of the peptide / protein with a C-terminus hydrazide as 0.1 to 2 mmol / L; preferably 1 mmol / L.

[0029] Further, the amount of acetylacetone used in step S1 is 1.5 to 4 times the equivalent of the peptide / protein with a C-terminus hydrazide; preferably 2.5 times the equivalent.

[0030] Further, the reaction conditions described in step S1 are: temperature 30-45 ℃, rotation speed 800-1500 rpm, and time 15-90 minutes; preferably, temperature 37±3 ℃, rotation speed 1000-1200 rpm, and time 30-60 minutes.

[0031] Further, the amount of selenocysteine ​​used in step S2 is 0.5 to 20 times the equivalent of the peptide / protein with a C-terminus hydrazide; preferably 0.5 to 5 times the equivalent; more preferably 1 time the equivalent.

[0032] Further, the amount of tri-(2-carboxyethyl)phosphonic acid hydrochloride used in step S2 is calculated based on its concentration in the final system as 1 to 20 mmol / L; preferably 1 to 10 mmol / L; more preferably 2 mmol / L.

[0033] Further, the amount of ascorbic acid used in step S2 is calculated based on its concentration in the final system as 1 to 40 mmol / L; preferably 2 to 20 mmol / L; more preferably 4 mmol / L.

[0034] Further, the reaction conditions described in step S2 are: temperature 30-45 ℃, rotation speed 800-1500 rpm, and time 15-90 minutes; preferably, temperature 37±3 ℃, rotation speed 1000-1200 rpm, and time 30-60 minutes.

[0035] Further, the buffer solution formulation in steps S1 and S2 is: 0–7 mol / L guanidine hydrochloride, 0.1–0.3 mol / L phosphate, pH 2.5–3.5; preferably: 0–1 mol / L guanidine hydrochloride, 0.2 mol / L phosphate, pH 3.0.

[0036] Further, the reaction conditions described in step S3 are: temperature 30-45 ℃, rotation speed 800-1500 rpm, and time 1-4 hours; preferably: temperature 37±3 ℃, rotation speed 1000-1200 rpm, and time 2-3 hours.

[0037] Further, the pH adjustment in step S4 is to adjust the pH to a slightly acidic state; preferably, the pH is adjusted to 4.5 to 5.5; more preferably, the pH is adjusted to 5.0 ± 0.2.

[0038] Furthermore, the pH adjustment described in step S4 can be achieved using 1 mol / L sodium hydroxide.

[0039] Further, the reaction conditions described in step S4 are: temperature 30-45 ℃, rotation speed 800-1500 rpm, and time 0.25-2 hours; preferably: temperature 37±3 ℃, rotation speed 1000-1200 rpm, and time 0.25-1 hour.

[0040] Further, the selenium-based modifying agent described in step S4 includes halogenated derivatives and / or maleimide derivatives, etc.; preferably, it includes at least one of biotin-polyethylene glycol-maleimide, azide-C3-iodoacetamide, N-methylmaleimide, dibenzocyclooctylmaleimide, dibenzocyclooctyl-polyethylene glycol-maleimide, iodoacetamide, and iodoacetyl-LC-biotin; more preferably, it is biotin-polyethylene glycol-maleimide.

[0041] Furthermore, the purification described in step S4 is achieved by semi-preparative reversed-phase liquid chromatography, wherein the mobile phase of the semi-preparative reversed-phase liquid chromatography is an acetonitrile / water mixture containing 0.08% trifluoroacetic acid.

[0042] The above-mentioned selenocysteine-mediated C-terminal selenolysis modification method is applied to the selective modification of proteins.

[0043] The present invention has the following advantages and effects compared with the prior art:

[0044] This invention eliminates the need for peptide thioesters or selenoester intermediates in the reaction. The acylpyrazole group can be directly nucleophilically attacked by the selenocysteine ​​group to achieve selenolysis, effectively simplifying the reaction steps and operational procedures. Compared with traditional methods that require thiols or selenools as additives, selenocysteine ​​has significant advantages: First, the raw materials are inexpensive and readily available, and have no unpleasant odor. Compared to the cumbersome operation of removing selenools through multiple extractions, it enables a one-pot reaction for coupling and modification, greatly improving operational convenience. Second, this method has excellent site selectivity, enabling specific modification of the selenogroup in a thiol-coexisting system, achieving precise and efficient modification. Furthermore, the reaction conditions of this invention are mild, requiring only 1 equivalent of selenocysteine ​​for efficient selenolysis, exhibiting good biocompatibility and operability. In addition, the method for C-terminal selenolysis modification of peptides / proteins using selenocysteine ​​described in this invention has a wide range of substrate applicability, compatible with various functional selenoyl modification reagents and different types of protein substrates, providing a universal technical solution for the targeted modification of peptides / proteins. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the reaction process for achieving C-terminal selenolysis modification of peptides / proteins using selenocysteine ​​in the method of the present invention.

[0046] Figure 2 HPLC (A) and ESI-MS (B) schematic diagrams of selenocysteine-mediated coupling of ubiquitin protein with biotin-polyethylene glycol-maleimide; where 1 is ubiquitin hydrazide raw material, 2 is acylpyrazole intermediate, 2' is hydrolysis byproduct, 3 is ubiquitin protein coupled with selenocysteine ​​product, Dimer of 3 is the dimer of ubiquitin protein coupled with selenocysteine ​​product, and 4 is the target product of successful C-terminal modification of ubiquitin protein with biotin-polyethylene glycol-maleimide.

[0047] Figure 3 HPLC (A) and ESI-MS (B) schematic diagrams of the coupling of ZpA963(A42C) protein with azido-C3-iodoacetamide mediated by selenocysteine; where 5 is the ZpA963(A42C) acylhydrazide starting material, 6 is the acylpyrazole intermediate, 7 is the ZpA963(A42C) product coupled with selenocysteine, and 8 is the target product of ZpA963(A42C) protein successfully modified with azido-C3-iodoacetamide at the C-terminus.

[0048] Figure 4 HPLC (A) and ESI-MS (B) schematic diagrams of the C-terminal selenization process of ubiquitin protein mediated by different selenocystamine equivalents; where 1 is ubiquitin hydrazide raw material, 2 is acylpyrazole intermediate, 2' is hydrolysis byproduct, 3 is ubiquitin protein coupled with selenocystamine product, 3' is the dimer of ubiquitin protein coupled with selenocystamine product, and 3* is the dimer of ubiquitin protein coupled with selenocystamine product and another molecule of selenocystamine.

[0049] Figure 5 The reaction process of ubiquitin hydrazide coupling with various selenium-modifying reagents and the ESI-MS characterization of the coupling products are shown. Detailed Implementation

[0050] 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 or polypeptide / protein sequences described herein.

[0051] The terms “polypeptide” and “protein” used in this article are interchangeable and should be interpreted broadly as any compound formed by linking amino acid monomers together with peptide chains. It may also include compounds containing certain amino acid analogs. The number of amino acid monomers in a polypeptide or protein is not particularly limited, and sometimes it is also called “oligopeptide”.

[0052] In this article, the terms "C-terminal acylhydrazine" and "protein with C-terminal acylhydrazine" are used interchangeably, both referring to proteins with a C-terminal acylhydrazine structure.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] The selenocysteine ​​used in the following examples has the following structural formula:

[0056] ;

[0057] In the following examples, the reversed-phase high-performance liquid chromatography (RP-HPLC) instrument used was an Agilent 1260, the chromatographic column was a Welch Ultimate XB-C4 column (300 Å, 5 μm, 4.6 × 250 mm), and the mobile phase was water and acetonitrile (containing 0.1% trifluoroacetic acid).

[0058] In the following examples, the semi-preparative reversed-phase high-performance liquid chromatography (HPLC) used an instrument of Shimadzu LC-16P, a Welch Ultimate XB-C4 column (300 Å, 5 μm, 10 × 250 mm), and a mobile phase of water and acetonitrile (containing 0.08% trifluoroacetic acid).

[0059] The reagent names and abbreviations used in the following examples are as follows:

[0060] ACN: Acetonitrile;

[0061] GdmCl: Guanidine hydrochloride;

[0062] Acac: Acetylacetone;

[0063] HCl: hydrochloric acid;

[0064] NaOH: Sodium hydroxide;

[0065] AscA: Ascorbic acid;

[0066] TCEP: Tris-(2-Carboxyethyl)phosphonic acid hydrochloride;

[0067] A schematic diagram of the reaction process for achieving C-terminal selenolysis modification of peptides / proteins using selenocysteine ​​in the method of this invention is shown below. Figure 1 As shown.

[0068] Example 1 below illustrates the application of the selenocystamine-mediated C-terminal selenolysis modification method proposed in this invention in selective protein modification. Using a protein with a C-terminal hydrazide as the substrate, ubiquitin was selected. Pure ubiquitin hydrazide was obtained through recombinant expression technology and GC hydrazide reaction. The selenocystamine molecule was coupled to the C-terminus of ubiquitin to construct a selenium reaction site. The reagent used to modify the C-terminus of ubiquitin was biotin-polyethylene glycol-maleimide. The amino acid sequence of ubiquitin hydrazide is as follows: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGC-NHNH2.

[0069] Example 2 below demonstrates the application of the selenocystamine-mediated C-terminal selenolysis modification method proposed in this invention in selective protein modification. Using a protein with a C-terminal acylhydrazine as the substrate, the selected protein was the small affinity protein ZpA963(A42C). Pure ZpA963(A42C) acylhydrazine was obtained through recombinant expression technology and GC hydrazolysis. The selenocystamine molecule was coupled to the C-terminus of ZpA963(A42C) to construct a selenium reaction site. The reagent used to modify the selenium group at the C-terminus of ZpA963(A42C) was azide-C3-iodoacetamide. The amino acid sequence of ZpA963(A42C) acylhydrazine is as follows: MGSSHHHHHHLQVDNKFNKETQEASWEIFTLPNLNGRQVAAFISSLLDDPSQSCNLLAEAKKLNDAQAPKGG-NHNH2.

[0070] Example 1: Selenocysteine-mediated C-terminal selenolysis and biotin-polyethylene glycol-maleimide modification of ubiquitin protein

[0071] Dissolve ubiquitin hydrazide in an acidic buffer solution (1 mol / L guanidine hydrochloride, 0.2 mol / L phosphate, pH 3.0) to bring the final concentration of ubiquitin hydrazide to 1 mM; add acetylacetone (2.5 molar equivalents of ubiquitin hydrazide) and react at 37 °C with constant shaking at 1200 rpm for 0.5 hours.

[0072] Separately, selenocysteine ​​(in a molar equivalent of ubiquitin hydrazide) was dissolved in the same acidic buffer solution as above. After complete dissolution, TCEP (reducing agent) and ascorbic acid (deselenoinhibitor) were added for pretreatment, so that the final concentrations of the two in the total system after subsequent mixing with the ubiquitin hydrazide system were 2 mmol / L and 4 mmol / L, respectively. The mixture was reacted at 37 °C and 1200 rpm for 0.5 hours to obtain the pretreated selenocysteine ​​solution.

[0073] The pretreated selenocysteine ​​solution was added to the ubiquitin hydrazide reaction system and the reaction was continued for 3 hours to obtain the coupling product containing the C-terminal selenium group reaction site.

[0074] The pH of the reaction solution was carefully adjusted to 5.0 using 1M NaOH. Biotin-polyethylene glycol-maleimide (twice the molar equivalent of ubiquitin hydrazide) was added, mixed, and reacted at 37 °C with constant shaking at 1200 rpm for 30 min to obtain the target product. The reaction was monitored using reversed-phase HPLC at a range of 20%–45% in 25 min (ACN concentration increased from 20% to 45% within 25 minutes), and the reaction process and molecular weight of the target product were confirmed using ESI-MS. After the reaction, the product was purified using semi-preparative reversed-phase HPLC, following the same method.

[0075] The results are as follows Figure 2 As shown, 1 is ubiquitin hydrazide raw material, 2 is acylpyrazole intermediate, 2' is hydrolysis byproduct, 3 is ubiquitin protein coupled with selenocysteine ​​product, Dimer of 3 is ubiquitin protein coupled with selenocysteine ​​product dimer, and 4 is the target product (conversion rate 91.3%) successfully modified with biotin-polyethylene glycol-maleimide at the C-terminus of ubiquitin protein.

[0076] Example 2: Selenocysteine-mediated C-terminal selenization and azide-C3-iodoacetamide modification of ZpA963 (A42C) protein

[0077] ZpA963(A42C) hydrazide was dissolved in an acidic buffer solution (0.2 mol / L phosphate, pH 3.0) to bring the final concentration of ZpA963(A42C) hydrazide to 1 mM; acetylacetone (2.5 molar equivalents of ubiquitin hydrazide) was added, and the mixture was reacted at 37 °C with constant shaking at 1200 rpm for 1 hour.

[0078] Separately, selenocysteine ​​(in a molar equivalent of ZpA963(A42C) acylhydrazine) was dissolved in the same acidic buffer solution as described above. After complete dissolution, TCEP (reducing agent) and ascorbic acid (deselenoinhibitor) were added for pretreatment, so that the final concentrations of the two in the total system after subsequent mixing with the ZpA963(A42C) acylhydrazine system were 2 mmol / L and 4 mmol / L, respectively. The mixture was reacted at 37 °C and 1200 rpm for 0.5 hours to obtain the pretreated selenocysteine ​​solution.

[0079] The pretreated selenocysteine ​​solution was added to the ZpA963(A42C) hydrazide reaction system and the reaction was continued for 2 hours to obtain the coupling product containing the C-terminal selenium group reaction site.

[0080] The pH of the reaction solution was carefully adjusted to 5.0 using 1M NaOH. Azide-C3-iodoacetamide (at twice the molar equivalent of ZpA963(A42C) hydrazide) was added, mixed thoroughly, and reacted at 37 °C with constant shaking at 1200 rpm for 30 min to obtain the target product. The reaction was monitored using reversed-phase HPLC at a range of 25%–55% in 25 min (ACN concentration increased from 25% to 55% within 25 minutes), and the reaction process and molecular weight of the target product were confirmed using ESI-MS. After the reaction, the product was purified using semi-preparative reversed-phase HPLC, following the same method.

[0081] The results are as follows Figure 3 As shown, 5 is the ZpA963(A42C) acylhydrazine raw material, 6 is the acylpyrazole intermediate, 7 is the ZpA963(A42C) conjugated selenocysteine ​​product, and 8 is the target product (conversion rate 90.3%) successfully modified with azide-C3-iodoacetamide at the C-terminus of ZpA963(A42C) protein.

[0082] Example 3: Selenolysis effect under different selenocystamine equivalent conditions

[0083] Dissolve ubiquitin hydrazide in an acidic buffer solution (1 mol / L guanidine hydrochloride, 0.2 mol / L phosphate, pH 3.0) to bring the final concentration of ubiquitin hydrazide to 1 mM; add acetylacetone (2.5 molar equivalents of ubiquitin hydrazide) and react at 37 °C with constant shaking at 1200 rpm for 0.5 hours.

[0084] Separately, selenocysteine ​​(in amounts equivalent to 5 / 2.5 / 1.5 / 1 / 0.5 molar equivalents of ubiquitin hydrazide) was dissolved in the same acidic buffer solution as described above. After complete dissolution, TCEP (reducing agent) and ascorbic acid (deselenoinhibitor) were added for pretreatment (so that the final concentrations of TCEP in the total system after subsequent mixing with the ubiquitin hydrazide system were 10 / 5 / 3 / 2 / 1 mmol / L, and the final concentrations of ascorbic acid in the total system after subsequent mixing with the ubiquitin hydrazide system were 20 / 10 / 6 / 4 / 2 mmol / L, respectively). The mixture was then reacted at 37 °C with constant temperature shaking at 1200 rpm for 0.5 hours to obtain the pretreated selenocysteine ​​solution.

[0085] The pretreated selenocysteine ​​solution was added to the ubiquitin hydrazide reaction system and the reaction was continued for 3 hours to obtain the coupling product containing the C-terminal selenium group reaction site. The reaction progress was monitored by reversed-phase HPLC and ESI-MS.

[0086] The results are as follows Figure 4 As shown, within the range of 0.5 to 5 molar equivalents of selenocysteine, the conversion rate of the selenolysis reaction remains stable (88.9% to 90.9%), and the coupling effect does not weaken with the decrease of selenocysteine ​​dosage. This feature indicates that the present invention can achieve efficient coupling with low selenocysteine ​​dosage, significantly improving the economy and practicality of the technical solution.

[0087] Example 4: Coupling effects of different selenium-modified reagents

[0088] Dissolve ubiquitin hydrazide in an acidic buffer solution (1 mol / L guanidine hydrochloride, 0.2 mol / L phosphate, pH 3.0) to bring the final concentration of ubiquitin hydrazide to 1 mM; add acetylacetone (2.5 molar equivalents of ubiquitin hydrazide) and react at 37 °C with constant shaking at 1200 rpm for 0.5 hours.

[0089] Separately, selenocysteine ​​(in 1 molar equivalent of ubiquitin hydrazide) was dissolved in the same acidic buffer solution as above. After complete dissolution, TCEP (reducing agent) and ascorbic acid (deselenoinhibitor) were added for pretreatment, so that the final concentrations of the two in the total system after subsequent mixing with the ubiquitin hydrazide system were 2 mmol / L and 4 mmol / L, respectively. The mixture was reacted at 37 °C and 1200 rpm for 0.5 hours to obtain the pretreated selenocysteine ​​solution.

[0090] The pretreated selenocysteine ​​solution was added to the ubiquitin hydrazide reaction system described above, and the reaction was continued for 2-3 hours to obtain the coupling product containing the C-terminal selenium group reaction site.

[0091] The pH of the reaction solution was carefully adjusted to 5.0 using 1M NaOH. Different selenium-modifying reagents (twice the molar equivalent of ubiquitin hydrazide) were added, mixed thoroughly, and reacted at 37 °C with constant shaking at 1200 rpm for 0.25–1 h to obtain the target product. The reaction was monitored using reversed-phase HPLC at a range of 20%–45% in 25 min (ACN concentration increased from 20% to 45% within 25 minutes), and the reaction process and molecular weight of the target product were confirmed using ESI-MS. After the reaction, the product was purified using semi-preparative reversed-phase HPLC, following the same method.

[0092] The experiment tested six halogenated or maleimide derivatives: iodoacetamide, azide-C3-iodoacetamide, iodoacetyl-LC-biotin, N-methylmaleimide, dibenzocyclooctylmaleimide, and biotin-polyethylene glycol-maleimide.

[0093] The results are as follows Figure 5 As shown, all six molecules can be separated by HPLC and the HPLC conversion rate can be calculated. The conversion rate calculation results are 90.1%, 90.5%, 89.9%, 88.6%, 64.1%, and 91.3%, respectively. This result proves that the C-terminal selenium reaction site of the present invention has good versatility and can be compatible with the selective modification of a variety of functional molecules.

[0094] Comparative Example 1: Two molar equivalents of selenocystamine-mediated ZpA963(A42C) hydrazide

[0095] ZpA963(A42C) acylhydrazide was dissolved in an acidic buffer solution (0.2 mol / L phosphate, pH 5.0) to bring the final concentration of ZpA963(A42C) acylhydrazide to 1 mM; azide-C3-iodoacetamide (twice the molar equivalent of ZpA963(A42C) acylhydrazide) was added, mixed well, and reacted at 37 °C with constant shaking at 1200 rpm for 30 min. The reaction progress was monitored by reversed-phase HPLC and ESI-MS.

[0096] Experimental results further confirm that the target modification reaction cannot occur without selenocysteine ​​as a mediating agent. This characteristic fully demonstrates that the modification reaction of the present invention is a C-terminal site-specific reaction mediated by selenocysteine, which can effectively avoid non-specific modification of other sites of peptides / proteins and significantly improve the selectivity and precision of the modification reaction.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. 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 of selenocystamine-mediated C-terminal selenolysis modification of a protein, characterized in that, Includes the following steps: S1. Dissolve the peptide / protein with a C-terminus acylhydrazine in a buffer solution, add acetylacetone, and react to obtain a first reaction mixture containing a peptide / protein with a C-terminus acylpyrazole group. S2. Dissolve selenocysteine ​​in buffer solution, add tris-(2-carboxyethyl)phosphine hydrochloride and ascorbic acid, react to obtain a second reaction mixture containing selenocysteine; S3. Add the second reaction mixture obtained in step S2 to the first reaction mixture obtained in step S1, react, and obtain a third reaction mixture containing a selenium group reaction site at the C-terminus. S4. Adjust the pH of the third reaction mixture obtained in step S3, add the selenium-based modification reagent, mix well, and react; purify the reaction solution to obtain the target product.

2. The method for C-terminal selenolysis modification of proteins mediated by selenocysteine ​​according to claim 1, characterized in that: The amount of buffer solution used in step S1 is calculated based on the concentration of the peptide / protein with a C-terminus hydrazide as 0.1–2 mmol / L; And / or, the amount of acetylacetone used in step S1 is 1.5 to 4 equivalents of the peptide / protein with a C-terminus hydrazide; And / or, the amount of selenocysteine ​​used in step S2 is 0.5 to 20 equivalents of the peptide / protein with a C-terminus hydrazide.

3. The method for C-terminal selenolysis modification of proteins mediated by selenocysteine ​​according to claim 2, characterized in that: The amount of buffer solution used in step S1 is based on a concentration of 1 mmol / L for peptides / proteins with a C-terminus hydrazide. And / or, the amount of acetylacetone used in step S1 is 2.5 times the equivalent of the peptide / protein with a C-terminus hydrazide; And / or, the amount of selenocysteine ​​used in step S2 is 0.5 to 5 equivalents of the peptide / protein with a C-terminus hydrazide.

4. The method for C-terminal selenolysis modification of proteins mediated by selenocysteine ​​according to claim 1, characterized in that: The amount of tri-(2-carboxyethyl)phosphonic acid hydrochloride used in step S2 is calculated based on its concentration in the final system as 1 to 20 mmol / L; And / or, the amount of ascorbic acid used in step S2 is calculated based on its concentration in the final system of 1 to 40 mmol / L.

5. The method for selenocysteine-mediated C-terminal selenolysis modification of proteins according to claim 4, characterized in that: The amount of tri-(2-carboxyethyl)phosphonic acid hydrochloride used in step S2 is calculated based on its concentration in the final system as 1 to 10 mmol / L; And / or, the amount of ascorbic acid used in step S2 is calculated based on its concentration in the final system being 2 to 20 mmol / L.

6. The method for selenocysteine-mediated C-terminal selenolysis modification of proteins according to claim 5, characterized in that: The amount of tri-(2-carboxyethyl)phosphonic acid hydrochloride used in step S2 is based on a concentration of 2 mmol / L in the final system; And / or, the amount of ascorbic acid used in step S2 is calculated based on its concentration of 4 mmol / L in the final system.

7. The method for selenocysteine-mediated C-terminal selenolysis modification of proteins according to claim 1, characterized in that: The reaction conditions described in step S1 are: temperature 30–45 °C, rotation speed 800–1500 rpm, and time 15–90 minutes; And / or, the reaction conditions described in step S2 are: temperature 30-45 °C, rotation speed 800-1500 rpm, and time 15-90 minutes; And / or, the reaction conditions described in step S3 are: temperature 30-45 °C, rotation speed 800-1500 rpm, and time 1-4 hours; And / or, the reaction conditions described in step S4 are: temperature 30–45 °C, rotation speed 800–1500 rpm, and time 0.25–2 hours.

8. The method for selenocysteine-mediated C-terminal selenolysis modification of proteins according to claim 1, characterized in that: The buffer solution described in steps S1 and S2 has the following formulation: 0–7 mol / L guanidine hydrochloride, 0.1–0.3 mol / L phosphate, pH 2.5–3.5; And / or, the pH adjustment described in step S4 is to adjust the pH to a slightly acidic state.

9. The method for C-terminal selenolysis modification of proteins mediated by selenocysteine ​​according to claim 1, characterized in that: The selenium-based modifying agent described in step S4 includes halogenated derivatives and / or maleimide derivatives; further including at least one of biotin-polyethylene glycol-maleimide, azide-C3-iodoacetamide, N-methylmaleimide, dibenzocyclooctylmaleimide, dibenzocyclooctyl-polyethylene glycol-maleimide, iodoacetamide, and iodoacetyl-LC-biotin.

10. The application of the selenocysteine-mediated C-terminal selenolysis modification method of proteins according to any one of claims 1 to 9 in the selective modification of proteins.

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