Deprotection reagent, kit and application thereof

Abstract

The invention belongs to the field of chemical synthesis, and particularly relates to a deprotection reagent, a kit and application of the deprotection reagent and the kit. And an organic base. According to the deprotection reagent, organic unification of mild and efficient deprotection and key side reaction inhibition is achieved through the synergistic effect of the 1-hydroxybenzotriazole and the organic base, specifically, the 1-hydroxybenzotriazole can effectively inhibit amino acid racemization, and more importantly, by capturing a beta-elimination reaction intermediate, the deprotection efficiency of the deprotection reagent is improved. And a protection mechanism aiming at phosphorylated amino acid is constructed together with organic alkali, so that a solid foundation is laid for preparing complex phosphorylated protein / polypeptide with high purity and accurate structure.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis, and particularly relates to deprotection reagents, kits and their applications. Background Technology

[0002] Phosphorylated proteins / peptides are proteins or peptides whose hydroxyl groups (such as serine, threonine, or tyrosine) are modified into acid phosphate esters. This modification is an important post-translational modification that can precisely regulate the structure, activity, stability, and intracellular localization of proteins, and is widely involved in core biological processes such as cell signaling and enzyme activity switching. Due to numerous challenges in extracting phosphorylated proteins / peptides from vivo, including low abundance, high heterogeneity, and extreme difficulty in isolation and purification, chemical synthesis has become a key method for obtaining phosphorylated peptides with specific sequences and small- to medium-scale phosphorylated proteins.

[0003] Currently, the main chemical synthesis methods for phosphorylated proteins / peptides include post-phosphorylation and solid-phase peptide synthesis (SPPS). When using the conventional SPPS strategy to synthesize phosphorylated proteins / peptides, especially when the sequence contains multiple, particularly high-density, phosphorylated serine / threonines, two major challenges are usually faced: (1) Phosphorylated amino acids are prone to β-elimination under alkaline deprotection and acidic cleavage conditions, resulting in irreversible loss of phosphate groups and product degradation; (2) Multi-site modification and long sequence characteristics easily induce peptide chain aggregation and the formation of stubborn secondary structures, which seriously hinders coupling efficiency and significantly reduces product purity and yield.

[0004] Therefore, there is an urgent need to develop a new strategy that can simultaneously inhibit β-elimination and effectively disrupt peptide chain aggregation, which is of great significance for the controllable synthesis of complex phosphorylated peptides with high purity and accurate structure. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides deprotection reagents, kits, and their applications. The deprotection reagent of this invention, through the synergistic effect of 1-hydroxybenzotriazole and an organic base, achieves a harmonious balance between mild and efficient deprotection and inhibition of key side reactions. Specifically, 1-hydroxybenzotriazole not only effectively inhibits amino acid racemization but also, by capturing β-elimination reaction intermediates, constructs a protective mechanism against phosphorylated amino acids together with an organic base, thus laying a solid foundation for preparing high-purity, structurally accurate complex phosphorylated proteins / peptides.

[0006] In a first aspect, the present invention provides a deprotection reagent. According to embodiments of the present invention, the deprotection reagent comprises the following components: 1-hydroxybenzotriazole; and an organic base. The deprotection reagent according to embodiments of the present invention, through the synergistic effect of 1-hydroxybenzotriazole and the organic base, simultaneously inhibits β-elimination side reactions while achieving a combined improvement in deprotection efficiency and stability of phosphorylated protein / peptide synthesis products.

[0007] According to embodiments of the present invention, the above-mentioned deprotecting agent may further include at least one of the following additional technical features: According to an embodiment of the present invention, the deprotecting agent further comprises a solvent.

[0008] According to embodiments of the present invention, the organic base includes one or more of hexamethyleneimine, 1-methylpyrrolidine, piperidine, and morpholine.

[0009] According to an embodiment of the present invention, the solvent is a strongly polar aprotic solvent.

[0010] According to an embodiment of the present invention, the strongly polar aprotic solvent is N-methylpyrrolidone and / or dimethyl sulfoxide.

[0011] According to an embodiment of the present invention, the organic base includes a primary base and one or more auxiliary bases.

[0012] According to an embodiment of the present invention, the primary base is 1-methylpyrrolidine, the auxiliary base is hexamethyleneimine and piperidine, and the solvent is N-methylpyrrolidone and dimethyl sulfoxide.

[0013] According to an embodiment of the present invention, the content of the 1-hydroxybenzotriazole is 10~50 g / L, and the content of the organic base is 220~400 mL / L.

[0014] According to an embodiment of the present invention, the content of 1-hydroxybenzotriazole is 10~50 g / L, the content of the main base is 200~300 mL / L, and the content of the auxiliary base is 20~100 mL / L.

[0015] According to an embodiment of the present invention, the content of 1-hydroxybenzotriazole is 10~50 g / L, the content of 1-methylpyrrolidone is 200~300 mL / L, the content of hexamethyleneimine is 10~50 mL / L, the content of piperidine is 10~50 mL / L, and the balance is the solvent, wherein the solvent is N-methylpyrrolidone and dimethyl sulfoxide, and the volume ratio of N-methylpyrrolidone to dimethyl sulfoxide is (0.5~2):1.

[0016] In a second aspect, the present invention provides a kit. According to an embodiment of the invention, the kit comprises: the deprotecting agent described in the first aspect. The kit according to an embodiment of the invention, by providing the aforementioned deprotecting agent, can gently and efficiently remove protecting groups while effectively inhibiting the β-elimination of phosphorylated amino acids, thereby providing a standardized tool for the synthesis of phosphorylated proteins / peptides.

[0017] According to embodiments of the present invention, the above-described kit may further include at least one of the following additional technical features: According to an embodiment of the present invention, the kit further comprises: pseudoproline dipeptide.

[0018] According to an embodiment of the present invention, the pseudoproline dipeptide has the general formula Fmoc-Xaa-Yaa[Ψ(Me,Me)pro]-OH, wherein Xaa is any natural amino acid and Yaa is serine or threonine.

[0019] According to an embodiment of the present invention, the kit further comprises: phosphorylated amino acids with a protecting group.

[0020] According to embodiments of the present invention, the phosphorylated amino acid with a protecting group includes one or more of Fmoc-Ser(PO(OBzl)OH)-OH, Fmoc-Thr(PO(OBzl)OH)-OH and Fmoc-Tyr(PO(OBzl)OH)-OH.

[0021] In a third aspect, the present invention provides a method for preparing phosphorylated peptides. According to embodiments of the present invention, the preparation method includes the following steps: assembling a target peptide chain; wherein, during the assembly of the target peptide chain, the Fmoc protecting group is removed using the deprotection reagent described in the first aspect or the kit described in the second aspect, and / or, the pseudoproline dipeptide is introduced at a site on the target peptide chain where secondary structure formation is readily achieved using the kit described in the second aspect. The preparation method according to embodiments of the present invention has advantages such as high synthesis efficiency and high product purity. Firstly, it effectively improves the stability of phosphate groups through a mild and efficient deprotection system. Furthermore, it can creatively combine this deprotection system with a pseudoproline dipeptide introduction strategy, thereby further improving the assembly efficiency and sequence accuracy of the target phosphorylated protein / peptide, and effectively solving the problems of numerous side reactions, low coupling efficiency, and poor product purity existing in the synthesis of traditional complex phosphorylated peptides.

[0022] According to embodiments of the present invention, the above preparation method may further include at least one of the following additional technical features: According to an embodiment of the present invention, the assembly of the target peptide chain is performed by solid-phase synthesis.

[0023] According to an embodiment of the present invention, the introduction of the pseudoproline dipeptide is carried out on a solid-phase synthesis support, a coupling agent is used to activate the carboxyl group of the pseudoproline dipeptide, and an organic amine base is used to drive the coupling reaction.

[0024] According to an embodiment of the present invention, the solid-phase synthesis support is a resin.

[0025] According to an embodiment of the present invention, when the pseudoproline dipeptide is introduced, the molar ratio of the solid-phase synthesis support, the pseudoproline dipeptide, the coupling reagent, and the organic amine base added is 1:(2~5):(2~5):(4~10).

[0026] According to an embodiment of the present invention, when the pseudoproline dipeptide is introduced, the temperature is 20~35°C and the reaction time is 45~75 min.

[0027] In a fourth aspect of the invention, the invention proposes the application of the deprotection reagent described in the first aspect, the kit described in the second aspect, or the preparation method described in the third aspect in protein / peptide chemical synthesis, preparation of phosphorylated peptides, and improvement of phosphorylated peptide synthesis efficiency.

[0028] Those skilled in the art will understand that the features and advantages described above for the deprotection reagent, kit, or preparation method also apply to this application and will not be repeated here.

[0029] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0030] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a process flow diagram of the preparation method of phosphorylated peptides in this invention. The diagram shows two phosphorylated peptide synthesis strategies: pseudoproline coupling and "cocktail" deprotection reagents. The key reaction conditions for each step are marked. Figure 2 This is a flowchart comparing the synthesis of phosphorylated polypeptide AT1R-PROX using different deprotection reagents in Example 1 of the present invention. Figure 3 This is a flowchart comparing the synthesis of phosphorylated polypeptide AT1R-PROX using different deprotection reagents in combination with pseudoproline in Example 1 of the present invention. Figure 4The images show the high performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the crude AT1R-PROX peptide sample in Example 1 of the present invention, where, from left to right, they are its high performance liquid chromatogram and electrospray ionization mass spectrum, respectively. Figure 5 The images show the chromatographic purity analysis and electrospray ionization mass spectrometry analysis results of the AT1R-PROX pure product in Example 1 of the present invention. From left to right, they are the chromatographic purity analysis image and the electrospray ionization mass spectrometry image, respectively. Figure 6 The images show the chromatographic purity and electrospray ionization mass spectrometry analysis results of the AT1R-MID pure product in Example 2 of the present invention, where, from left to right, they are the chromatographic purity analysis image and the electrospray ionization mass spectrometry image, respectively. Figure 7 The images show the chromatographic purity and electrospray ionization mass spectrometry analysis results of the AT1R-DIST pure product in Example 3 of the present invention, where, from left to right, they are the chromatographic purity analysis image and the electrospray ionization mass spectrometry image, respectively. Figure 8 The images show the high performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the AT1R-PROX crude peptide sample (microwave synthesized) in Comparative Example 1 of this invention. From left to right, the images show the high performance liquid chromatogram and the electrospray ionization mass spectrum, respectively. Figure 9 The images show the high performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the AT1R-PROX crude peptide sample (SPPS method) in Comparative Example 2 of this invention. From left to right, they are the high performance liquid chromatogram and the electrospray ionization mass spectrum, respectively. Figure 10 The figures show the high performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the AT1R-PROX crude peptide sample (only optimized for deprotection) in Comparative Example 3 of this invention. From left to right, they are its high performance liquid chromatogram and electrospray ionization mass spectrum, respectively. Figure 11 The images show the high performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the AT1R-PROX crude peptide sample (with only pseudoproline introduced) in Comparative Example 4 of this invention. From left to right, they are the high performance liquid chromatogram and the electrospray ionization mass spectrum, respectively. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0033] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0034] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0035] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0036] Terms and Definitions In this article, the term "cocktail deprotecting agent" or "deprotecting agent" refers to a reagent prepared in a specific ratio using 1-hydroxybenzotriazole, one or more organic bases, and a solvent, used to gently and efficiently remove the Fmoc protecting group and inhibit side reactions in the synthesis of phosphorylated proteins / peptides.

[0037] In this paper, the term "peptide aggregation" refers to the phenomenon in the solid-phase synthesis of phosphorylated proteins / peptides where the grown peptide chains form ordered secondary structures such as β-sheets due to intermolecular or intramolecular interactions (such as hydrogen bonds and hydrophobic interactions), resulting in a decrease in resin swelling and encapsulation of subsequent reaction sites, thereby significantly reducing coupling efficiency.

[0038] In this paper, the term "β-elimination" refers to the chemical process by which phosphorylated serine or phosphorylated threonine undergoes a side reaction under alkaline conditions, resulting in the loss of a phosphate group and the formation of a dehydrogenated amino acid byproduct.

[0039] Deprotection reagent This invention proposes a deprotection reagent. According to embodiments of the invention, the deprotection reagent comprises the following components: 1-hydroxybenzotriazole; and an organic base. The deprotection reagent of this invention achieves a harmonious balance between mild and efficient deprotection and inhibition of key side reactions through the synergistic effect of 1-hydroxybenzotriazole and the organic base. Specifically, 1-hydroxybenzotriazole not only effectively inhibits amino acid racemization but also, by capturing β-elimination reaction intermediates, constructs a protective mechanism against phosphorylated amino acids together with the organic base, thus laying a solid foundation for preparing high-purity, structurally accurate complex phosphorylated proteins / peptides.

[0040] It should be noted that 1-hydroxybenzotriazole (HOBt) plays an indispensable role in the deprotection reagent described in this invention, mainly including the following aspects: (1) inhibiting racemization: In the strongly alkaline environment generated by the removal of the protecting group, the activated amino acid is very easy to racemize, resulting in the generation of diastereomeric impurities. The addition of 1-hydroxybenzotriazole can effectively inhibit this side reaction, thereby improving the purity of the final product; (2) capturing β-elimination intermediates and stabilizing phosphate groups: Under alkaline deprotection conditions, phosphorylated serine / threonine will generate highly active dehydroalanine (Dha) intermediates through β-elimination reaction. HOBt, as a highly efficient nucleophile, can rapidly undergo Michael addition reaction with the intermediate, thereby effectively blocking its irreversible conversion into a stable byproduct, and temporarily "capturing" and stabilizing the phosphate group in the form of an O-acylated isomer; in the subsequent coupling step, this captured group can be replaced by the newly added amino acid nucleophile, thereby largely restoring the original phosphorylated serine / threonine structure and significantly reducing the loss rate of the phosphate group; (3) Synergistic enhancement of deprotection efficiency: 1-hydroxybenzotriazole can work together with the organic base in the deprotection reagent of the present invention to form a highly efficient catalytic cycle; among them, HOBt interacts with the carbonate part of the Fmoc protecting group through hydrogen bonds, activating the group and making it more susceptible to nucleophilic attack by the organic base; at the same time, the organic base (such as piperidine) is responsible for synergistic action with HOBt to jointly promote the cleavage and removal of the Fmoc group; this synergistic effect allows the deprotection reaction to proceed rapidly and thoroughly under relatively mild alkaline conditions, reducing the destructive action time on acid-sensitive phosphate groups.

[0041] According to an embodiment of the present invention, the deprotecting agent further comprises a solvent. Thus, using the solvent as a medium ensures that the components in the deprotecting agent are uniformly and sufficiently dispersed, thereby improving the uniformity of its application in the deprotection reaction.

[0042] According to embodiments of the present invention, the organic base includes one or more of hexamethyleneimine, 1-methylpyrrolidine, piperidine, and morpholine. Thus, by preferentially selecting the specific type of organic base, it can achieve better deprotection effects (e.g., mildness) when used in combination with 1-hydroxybenzotriazole.

[0043] It should be noted that the organic base is not limited to the types of organic bases specifically listed above. Any organic base that can synergize with 1-hydroxybenzotriazole to achieve both efficient removal of the protecting group and mild alkaline conditions with low β-elimination risk, such as other cyclic secondary amines (e.g., morpholine, pyrrolidine) or other sterically hindered tertiary amines (e.g., N-methylmorpholine, N,N'-dimethylpiperazine), is within the scope of protection of this invention.

[0044] According to an embodiment of the present invention, the solvent is a strongly polar aprotic solvent. Therefore, by preferentially selecting a strongly polar aprotic solvent as the solvent for the deprotection reagent of the present invention, it can both fully swell the solid-phase synthesis support (such as resin) and not interfere with the base-catalyzed deprotection reaction, thus providing a more stable reaction environment for subsequent application in the deprotection reaction.

[0045] In this paper, the term "strongly polar aprotic solvent" refers to a solvent with a high dielectric constant (>30) and no acidic protons (dissociable protons). It is characterized by high polarity but low nucleophilicity, and can effectively dissolve polar molecules and salts, while not undergoing proton transfer reactions with strong bases.

[0046] According to an embodiment of the present invention, the strongly polar aprotic solvent is N-methylpyrrolidone and / or dimethyl sulfoxide.

[0047] It should be noted that the strongly polar aprotic solvent is not limited to the strongly polar aprotic solvent types specifically listed above. Any strongly polar aprotic solvent type that is compatible with other components of the deprotection reagent and does not interfere with the base-catalyzed deprotection reaction, such as other amide solvents (e.g., N,N-dimethylacetamide), lactone solvents (e.g., γ-valerolactone), or any mixture thereof, is within the scope of protection of this invention.

[0048] According to an embodiment of the present invention, the organic base includes a primary base and one or more auxiliary bases.

[0049] In this document, the term "main base" refers to the organic base component with the highest working concentration that performs the main deprotection function in the deprotection reagent described in this invention. Its core role is to provide the necessary basic alkaline environment for the removal of the Fmoc protecting group and is the main driving force for the deprotection reaction.

[0050] In this document, the term "auxiliary base" refers to one or more organic base components present at a lower concentration in the deprotection reagent of the present invention, which work synergistically with the main base to optimize the deprotection reaction kinetics and selectivity. Its function is to assist in regulating the basicity of the reaction system or the reaction process, thereby ensuring efficient deprotection while further enhancing the inhibitory effect on side reactions such as β-elimination of phosphorylated amino acids and racemization of amino acids.

[0051] According to an embodiment of the present invention, the main base is 1-methylpyrrolidone, the auxiliary base is hexamethyleneimine and piperidine, and the solvent is N-methylpyrrolidone and dimethyl sulfoxide. Thus, by combining a specifically selected organic base, solvent, and 1-hydroxybenzotriazole, an optimal deprotection system is constructed. Specifically, this system uses mildly alkaline 1-methylpyrrolidone as the main base to provide the basic deprotection motive force, highly active piperidine as a reaction promoter to ensure deprotection efficiency, and hexamethyleneimine as an alkaline regulator. These three components form a functionally complementary composite base system, maintaining a high reaction rate while creating a mildly alkaline environment unfavorable to β-elimination. Simultaneously, the mixed solvent of N-methylpyrrolidone and dimethyl sulfoxide, through its synergistic swelling effect, effectively disrupts the secondary structure of the peptide chain and improves resin accessibility, providing ideal mass transfer conditions for the deprotection reaction. 1-hydroxybenzotriazole fully utilizes its dual function of inhibiting racemization and neutralizing β-elimination active intermediates in the above optimized system, ultimately achieving a simultaneous improvement in deprotection efficiency and product purity.

[0052] According to an embodiment of the present invention, the content of the 1-hydroxybenzotriazole is 10~50 g / L, and the content of the organic base is 220~400 mL / L. Therefore, by optimizing the concentration range of 1-hydroxybenzotriazole and organic base in the deprotection reagent, an optimal balance between suppressing side reactions and ensuring the reaction rate is achieved. For example, the content of 1-hydroxybenzotriazole is 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L, preferably 20-40 g / L, and more preferably 20-30 g / L; the content of organic base is 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 mL / L, preferably 250-320 mL / L, and more preferably 280-300 mL / L.

[0053] According to an embodiment of the present invention, the content of 1-hydroxybenzotriazole is 10~50 g / L, the content of the main base is 200~300 mL / L, and the content of the auxiliary base is 20~100 mL / L. Therefore, by optimizing the concentration range of 1-hydroxybenzotriazole, the main base, and the auxiliary base in the deprotection reagent, the application effect of the deprotection reagent can be further optimized. For example, the content of 1-hydroxybenzotriazole is 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L, preferably 20-40 g / L, more preferably 20-30 g / L; the content of the main base is 200 mL / L, 210 mL / L, 220 mL / L, 230 mL / L, 240 mL / L, 250 mL / L, 260 mL / L, 270 mL / L, 280 mL / L, 290 mL / L, or 300 mL / L, preferably 230-280 mL / L, more preferably 250-280 mL / L; the content of the auxiliary base is 20 g / L. mL / L, 30 mL / L, 40 mL / L, 50 mL / L, 60 mL / L, 70 mL / L, 80 mL / L, 90 mL / L, 100 mL / L, preferably 30~60 mL / L, more preferably 40~50 mL / L.

[0054] According to an embodiment of the present invention, the content of 1-hydroxybenzotriazole is 10~50 g / L, the content of 1-methylpyrrolidone is 200~300 mL / L, the content of hexamethyleneimine is 10~50 mL / L, the content of piperidine is 10~50 mL / L, and the balance is the solvent, wherein the solvent is N-methylpyrrolidone and dimethyl sulfoxide, and the volume ratio of N-methylpyrrolidone to dimethyl sulfoxide is (0.5~2):1. Therefore, by optimizing and controlling the concentration of each component, the deprotection reagent can effectively suppress side reactions while achieving the deprotection reaction more gently and efficiently. For example, the content of 1-hydroxybenzotriazole is 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, preferably 20-40 g / L, more preferably 25-35 g / L; the content of 1-methylpyrrolidine is 200 mL / L, 210 mL / L, 220 mL / L, 230 mL / L, 240 mL / L, 250 mL / L, 260 mL / L, 270 mL / L, 280 mL / L, 290 mL / L, 300 mL / L, preferably 230-280 mL / L, more preferably 250-280 mL / L; the content of hexamethyleneimine is 10 mL / L, 15 g / L, 20 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, preferably 20-40 g / L, more preferably 25-35 g / L. The concentrations of piperidine are 10 mL / L, 20 mL / L, 25 mL / L, 30 mL / L, 35 mL / L, 40 mL / L, 45 mL / L, and 50 mL / L, preferably 15-30 mL / L, and more preferably 20-25 mL / L; the piperidine content is 10 mL / L, 15 mL / L, 20 mL / L, 25 mL / L, 30 mL / L, 35 mL / L, 40 mL / L, 45 mL / L, and 50 mL / L, preferably 15-30 mL / L, and more preferably 20-25 mL / L. mL / L; in the solvent, the volume ratio of the N-methylpyrrolidone to the dimethyl sulfoxide is 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, preferably (0.8~1.5):1, more preferably (1~1.2):1.

[0055] Those skilled in the art should understand that the concentration units “g / L” and “X% (w / v)”, and “mL / L” and “Y% (v / v)” can be equivalently converted. Specifically, “X% (w / v)” is equivalent to X×10 g / L, and Y% (v / v) is equivalent to Y×10 mL / L. For example, 1% (w / v) is equivalent to 10 g / L, and 20% (v / v) is equivalent to 200 mL / L.

[0056] Reagent test kit This invention provides a kit. According to embodiments of the invention, the kit includes the aforementioned deprotection reagent. The kit according to embodiments of the invention, by providing the aforementioned deprotection reagent, can gently and efficiently remove protecting groups while effectively inhibiting the β-elimination of phosphorylated amino acids, thereby providing a standardized tool for the synthesis of phosphorylated proteins / peptides.

[0057] According to an embodiment of the present invention, the kit further comprises: a pseudoproline dipeptide. Thus, the rigid cyclic structure of the pseudoproline dipeptide can effectively disrupt the formation of secondary structures such as β-sheets, enabling efficient linkage of subsequent amino acids, especially sterically hindered phosphorylated amino acids. When used in conjunction with the aforementioned deprotection reagent, it not only inhibits side reactions and peptide chain aggregation but also significantly improves synthesis efficiency and product purity by enhancing the overall synthetic microenvironment.

[0058] In this paper, the term "pseudoproline dipeptide" refers to a special dipeptide derivative used as a structural unit in solid-phase polypeptide synthesis. Its rigid ring can effectively disrupt the regular folding and hydrogen bond network of the growing peptide chain during synthesis, thereby significantly inhibiting the formation of secondary structures such as β-sheets and fundamentally solving the problem of low coupling efficiency caused by peptide chain aggregation.

[0059] According to an embodiment of the present invention, the pseudoproline dipeptide has the general formula Fmoc-Xaa-Yaa[Ψ(Me,Me)pro]-OH, wherein Xaa is any natural amino acid and Yaa is serine or threonine.

[0060] In the term “Fmoc-Xaa-Yaa[Ψ(Me,Me)pro]-OH”, “-Yaa[Ψ(Me,Me)pro]” refers to the connection between the side chain hydroxyl group of serine or threonine and the nitrogen atom of the modified proline through an oxazolidine ring, forming a rigid five-membered ring structure. This rigid ring is the key to effectively disrupting the β-sheet of the peptide chain and preventing peptide chain aggregation.

[0061] For example, the pseudoproline dipeptide may be Fmoc-Leu-Ser[Ψ(Me,Me)pro]-OH or Fmoc-Val-Ser[Ψ (Me,Me)pro]-OH, Fmoc-Leu-Thr[Ψ(Me,Me)pro]-OH, Fmoc-Phe-Ser[Ψ(Me,Me)pro]-OH, etc.

[0062] It should be noted that pseudoproline dipeptide plays an indispensable role in the kit described in this invention, mainly including the following aspects: (1) Structural regulation: Through its unique rigid structure of the oxazolidine ring, pseudoproline dipeptide can effectively break the regular arrangement of the hydrogen bond network in the growing peptide chain, fundamentally destroying the formation of secondary structures such as β-sheet. This characteristic solves the most critical peptide chain aggregation problem in the synthesis of long-sequence and high-density modified peptides, allowing subsequent amino acids to be fully close to the reaction site; (2) Sequence universality: Through the design of variable sites in the general formula Fmoc-Xaa-Yaa[Ψ(Me,Me)pro]-OH of pseudoproline dipeptide, it can flexibly adapt to different sequence rings. The environment can play a role whether it is located before hydrophobic fragments, between multiple phosphorylation sites, or other key regions that are prone to secondary structure formation; (3) In-situ protection: pseudoproline dipeptide plays a role as a stable structural element in the synthesis process, and can be completely converted into the natural dipeptide sequence in the final cleavage step, ensuring that the structure of the final product is completely consistent with the natural protein sequence and there is no non-natural residue; (4) Synergistic effect: when pseudoproline dipeptide is used in combination with the aforementioned deprotection reagent, pseudoproline dipeptide can further improve the reaction efficiency when the deprotection reagent is applied by maintaining the peptide chain in an extended state; at the same time, the mild reaction environment provided by the deprotection reagent also ensures the stability of the pseudoproline dipeptide structure.

[0063] According to an embodiment of the present invention, the kit further comprises phosphorylated amino acids with a protecting group. Thus, the kit provides a standard protocol for preparing complex phosphorylated peptides.

[0064] In this paper, the term "phosphorylated amino acid with a protecting group" refers to serine, threonine, or tyrosine derivatives used in solid-phase polypeptide synthesis whose side-chain phosphate groups are stabilized by an appropriate protecting group.

[0065] According to embodiments of the present invention, the phosphorylated amino acid with a protecting group includes one or more of Fmoc-Ser(PO(OBzl)OH)-OH, Fmoc-Thr(PO(OBzl)OH)-OH and Fmoc-Tyr(PO(OBzl)OH)-OH.

[0066] It should be noted that the phosphorylated amino acid with the protecting group is not limited to the benzyl ester (Bzl) protected form specifically listed above; under the concept of this invention, other protecting group forms with similar functions (such as alkyl esters, aryl esters, etc.) known to those skilled in the art, such as but not limited to tert-butyl ester (tBu), p-methoxybenzyl ester (PMB), etc., can also be considered for achieving the purpose of this invention, and all fall within the protection scope of this invention.

[0067] Preparation method This invention proposes a method for preparing phosphorylated peptides. According to embodiments of the invention, the preparation method includes the following steps: assembling a target peptide chain; wherein, during the assembly of the target peptide chain, the Fmoc protecting group is removed using the aforementioned deprotection reagent or the aforementioned kit, and / or, the pseudoproline dipeptide is introduced at sites on the target peptide chain where secondary structures are easily formed using the aforementioned kit. The preparation method according to embodiments of the invention has advantages such as high synthesis efficiency and high product purity. Firstly, it effectively improves the stability of phosphate groups through a mild and efficient deprotection system. Furthermore, it can creatively combine this deprotection system with a pseudoproline dipeptide introduction strategy to further improve the assembly efficiency and sequence accuracy of the target phosphorylated protein / peptide, and effectively solve the problems of numerous side reactions, low coupling efficiency, and poor product purity existing in the synthesis of traditional complex phosphorylated peptides.

[0068] The process flow for preparing the phosphorylated polypeptide of this invention is as follows: Figure 1 .

[0069] According to an embodiment of the present invention, the assembly of the target peptide chain is performed by solid-phase synthesis.

[0070] If the assembly of the target peptide chain is carried out by solid-phase synthesis, it mainly includes steps such as resin pretreatment, peptide chain fragment synthesis, introduction of pseudoproline dipeptide, and coupling of phosphorylated amino acids.

[0071] According to embodiments of the present invention, the introduction of the pseudoproline dipeptide is carried out on a solid-phase synthesis support, using a coupling reagent to activate the carboxyl group of the pseudoproline dipeptide, and using an organic amine base to drive the coupling reaction. Thus, by explicitly using a coupling reagent to activate the carboxyl group and coordinating with an organic amine base to drive the reaction, a standardized synthetic process for the target peptide chain is established.

[0072] According to an embodiment of the present invention, the solid-phase synthesis support is a resin.

[0073] It should be noted that the solid-phase synthesis support is not limited to the resin materials specifically listed above. Those skilled in the art will understand that any insoluble matrix material with specific functional groups (such as amino, carboxyl, hydroxyl, etc.) suitable for the Fmoc solid-phase peptide synthesis strategy, such as cellulose and its derivatives, polyamide polymers, surface-modified polyethylene or polystyrene resins, and magnetic microspheres, can be used in the method described in this invention and are all within the scope of protection of this invention.

[0074] In this paper, the term "coupling reagent" refers to a chemical reagent used in solid-phase peptide synthesis to activate the carboxyl group of an amino acid (or peptide fragment), making it more readily react with the amino group to form an amide bond (peptide bond). By forming a highly reactive intermediate (such as an ester or anhydride) with the carboxyl group, it significantly reduces the activation energy required to form a peptide bond, thereby driving the condensation reaction to proceed efficiently and rapidly.

[0075] For example, the coupling reagents include, but are not limited to, phosphonium salts (such as HATU, HBTU, PyBOP), ureonium salts (such as TBTU, HCTU), and carbodiimides (such as DIC, EDC).

[0076] In this paper, the term "organic amine base" refers to an organic nitrogen-containing basic compound used in the coupling reaction step of peptide synthesis to neutralize acidic protons (such as HX released during the formation of urea salt byproducts) generated during carboxyl activation and to maintain the reaction system in a suitable alkaline environment to promote peptide bond formation.

[0077] For example, organic amine bases suitable for the coupling reaction include (but are not limited to) sterically hindered amine bases such as N,N-diisopropylethylamine (DIEA) and N-methylmorpholine (NMM).

[0078] According to an embodiment of the present invention, when the pseudoproline dipeptide is introduced, the molar ratio of the solid-phase synthesis support, the pseudoproline dipeptide, the coupling reagent, and the organic amine base added is 1:(2~5):(2~5):(4~10). Therefore, by optimizing and controlling the ratio of each component, the full activation and efficient coupling of the pseudoproline dipeptide can be ensured while avoiding the unnecessary addition of reagents. For example, when introducing the pseudoproline dipeptide, the molar ratio of the solid-phase synthesis support, the pseudoproline dipeptide, the coupling reagent, and the organic amine base is 1:2:2:4, 1:2:2:5, 1:2:2:6, 1:2:2:7, 1:2:2:8, 1:2:2:9, 1:2:2:10, 1:3:2:4, 1:3:2:5, 1:3:2:6, 1:3:2:7, 1:3:2:8, 1:3:2:9, 1:3:2:10, 1:3:2:4, 1:3:2:5, 1:3:2:6, 1:3:2:7, 1:3:2:8, 1:3:2:9, 1:3:2:1 0, 1:5:2:4, 1:5:2:5, 1:5:2:6, 1:5:2:7, 1:5:2:8, 1:5:2:9, 1:5:2:10, 1:2:4:4, 1:2:4:5, 1:2:4:6, 1:2:4:7, 1:2:4:8, 1:2:4:9, 1:2:4:10, 1:2:5:4, 1:2:5:5, 1:2:5:6, 1:2:5:7, 1:2:5:8, 1:2:5:9, 1:2:5:10, preferably 1:(2~3):(2~4):(4~8), more preferably 1:(2~2.5):(3~4):(4~6).

[0079] For example, the coupling agent may be 2-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and / or 1-hydroxy-7-azabenzotriazole (HOAt); when the coupling agent is a mixture of HATU and HOAt, the two can exert a synergistic activation effect, which is particularly suitable for the carboxyl activation of amino acids (such as pseudoproline dipeptides) with large steric hindrance or difficult coupling. In a specific embodiment, HATU and HOAt can be mixed in an equimolar ratio and used as a coupling agent; for example, the organic amine base is N,N-diisopropylethylamine (DIEA).

[0080] According to embodiments of the present invention, when introducing the pseudoproline dipeptide, the temperature is 20-35°C and the reaction time is 45-75 min. Thus, by optimizing and controlling the reaction temperature and time, a sufficient reaction rate is ensured while effectively avoiding side reactions that may be caused by high temperatures, thereby achieving an optimal balance between reaction efficiency and product integrity. For example, when introducing the pseudoproline dipeptide, the temperature is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C, preferably 25°C-32°C, more preferably 30°C; the reaction time is 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, or 75 min, preferably 55-70 min, more preferably 60 min.

[0081] application This invention proposes the application of the aforementioned deprotection reagents, kits, or preparation methods in protein / peptide chemical synthesis, preparation of phosphorylated peptides, and improvement of phosphorylated peptide synthesis efficiency.

[0082] Those skilled in the art will understand that the features and advantages described above for the deprotection reagent, kit, or preparation method also apply to this application and will not be repeated here.

[0083] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. All reagents used are of analytical grade, and all reagents or instruments whose manufacturers are not specified are commercially available conventional products.

[0084] Example 1: Synthesis and Analysis of Phosphorylated Peptide AT1R-PROX 1. Target peptide The phosphorylated polypeptide AT1R-PROX, used for chemical semi-synthesis, was synthesized, and its amino acid sequence is shown in SEQ ID NO: 1: GKpSHpSNLpSpTKMSTLSYRPSDNVSSSTKK (SEQ ID NO: 1) A comparison of the synthesis procedures for the phosphorylated peptide AT1R-PROX using different deprotection reagents is shown in [link to documentation]. Figure 2 A comparison of the synthesis procedures for phosphorylated peptide AT1R-PROX using different deprotection reagents combined with pseudoproline is shown in [link to documentation]. Figure 3 .

[0085] 2. Synthesis of the target polypeptide (1) Resin pretreatment Weigh 0.1 mmol (degree of substitution 0.33 mmol / g) of Rink Amide-AM resin (purchased from Tianjin Nankai Hecheng Technology Co., Ltd.), place it in a solid-phase synthesis reactor, add 15 mL of N,N-dimethylformamide (DMF, purchased from Chengdu Kelong Chemical Co., Ltd.), and allow it to swell at room temperature for 10 min to obtain the resin.

[0086] (2) Microwave-assisted synthesis of C-terminal fragments Using a Liberty Blue™ fully automated continuous peptide synthesizer, with microwave synthesis parameters set (program setting: 0.2 mmol scale), peptide-based resin A was synthesized using the resin treated in step (1) as a carrier. The amino acid sequence of peptide-based resin A is shown in SEQ ID NO: 2. YRPSDNVSSSTKK (SEQ ID NO: 2); The specific parameter settings for microwave synthesis are as follows: Coupling reaction: microwave power 28 W, temperature 90℃, reaction time 110 s; the coupling reagent used was a DMF solution of 1.0 M ethyl 2-oxime cyanoacetate (Oxyma) / 1.0 MN,N'-diisopropylcarbodiimide (DIC); Deprotection reaction: microwave power 30 W, temperature 90℃, reaction time 50 s; the deprotection reagent used was 20% piperidine / DMF solution.

[0087] After each amino acid coupling and deprotection cycle was completed, the resin bed was automatically rinsed with DMF. After the microwave synthesis was completed, the resin was removed from the container, thoroughly washed with DMF (3×5 mL) and dichloromethane (DCM) (6×5 mL), and dried for later use to obtain peptide resin loaded with C-terminal sequences. Subsequent steps were then switched to manual solid-phase synthesis.

[0088] The protective amino acids used in steps (1) to (2) are calculated from the resin. The amino acid coupling parameters of microwave-synthesized peptide resin A are shown in Table 1.

[0089] Table 1. Amino acid coupling parameters of microwave-synthesized peptide resin A

[0090] (3) Introduction of pseudoproline dipeptide units After synthesizing peptide resin A, amino acids are sequentially coupled from the C-terminus to the N-terminus to obtain peptide resin B. The amino acid sequence of peptide resin B is shown in SEQ ID NO: 3. LSYRPSDNVSSSTKK (SEQ ID NO: 3); The specific steps for this coupling process are as follows: Resin swelling: Take the peptide resin A loaded with C-terminal sequence obtained in step (2), place it in a solid-phase synthesis reaction tube, add 5 mL DMF, bubble with nitrogen at room temperature for 5 min to swell, and after swelling is complete, filter to remove DMF to obtain swollen C-terminal sequence peptide resin.

[0091] Coupled pseudoproline: To efficiently activate the carboxyl group of pseudoproline dipeptide, a dual activation system combining 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and N-hydroxy-7-azabenzotriazole (HOAt) is adopted. Based on the molar amount of resin, the following reagents were added according to the molar ratio of resin: pseudoproline (purchased from Jiangsu Jitai Peptide Technology Co., Ltd.): HATU: HOAt: N,N-diisopropylethylamine (DIEA) = 1:2:2:2:4. The corresponding reagents were accurately weighed into dry sample vials, 3 mL of DMF was added to dissolve and vortexed, and the mixture was allowed to stand for 1 min to activate. Finally, DIEA solution (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was added. After mixing evenly, the mixture was quickly transferred to a synthesis tube containing swollen C-terminal peptide resin. The total time from adding DIEA solution to adding the mixture containing DIEA solution into the synthesis tube was controlled within 1~2 min to obtain the best coupling efficiency. The synthesis tube was then placed in a 30℃ constant temperature shaker and the reaction was carried out at a speed of 135 r / min for 1 h.

[0092] Washing: After coupling is complete, wash the resin three times each with DMF, DCM and DMF (5 mL each time).

[0093] Deprotection: After the resin temperature drops to room temperature, deprotection is performed twice (5 min and 10 min respectively) using 20% ​​(v / v) piperidine / DMF solution in a constant temperature shaker at 30℃ (135 r / min). The specific operation procedure is as follows: add 20% piperidine / DMF solution (5 mL), react for 5 min, filter to remove the reaction solution, wash the resin twice with DMF (5 mL each time) to completely remove residual piperidine and byproducts, add fresh 20% piperidine / DMF solution (5 mL), and continue to react for 10 min.

[0094] Secondary washing: After deprotection is completed, wash the resin three times each with DMF, DCM and DMF (5 mL each time), and dry it to obtain peptide resin B, ready for the next coupling step.

[0095] (4) Coupling and deprotection of common amino acids before the first phosphorylated amino acid (pT) After synthesizing peptide resin B, amino acids are sequentially coupled from the C-terminus to the N-terminus until the first phosphorylated amino acid (pT) is reached, to obtain peptide resin C. The amino acid sequence of peptide resin C is shown in SEQ ID NO: 4. KMSTLSYRPSDNVSSSTKK (SEQ ID NO: 4); The specific steps for this coupling process are as follows: Resin swelling: Take the peptide resin B obtained in step (3), place it in a solid-phase synthesis reaction tube, add 5 mL DMF, bubble with nitrogen at room temperature for 5 min to swell, and after swelling is complete, filter to remove DMF to obtain swollen peptide resin B.

[0096] Coupling reaction: Based on the molar amount of resin, add the corresponding reagents in a molar ratio of resin:corresponding amino acid:HATU:HOAt:DIEA = 1:5:5:5:10. Accurately weigh the corresponding reagents into a dry sample bottle, add 3 mL of DMF to dissolve and vortex, let stand for 1 min to activate, and finally add DIEA solution. After mixing evenly, quickly transfer it to a synthesis tube containing swollen peptide resin B. Ensure that the total time from adding DIEA solution to adding the mixture containing DIEA solution into the synthesis tube is controlled within 1~2 min to obtain the best coupling efficiency. Then place the synthesis tube at 55℃ under nitrogen protection and react for 20 min. After the reaction is complete, filter to remove the reaction solution, and then wash the resin twice with DMF (5 mL each time). To ensure complete coupling, the above coupling-washing operation needs to be repeated once (a total of 2 rounds of coupling reaction).

[0097] Washing: The washing procedure is the same as in step (3).

[0098] Deprotection: The operation is the same as the deprotection step in step (3).

[0099] Secondary washing: The operation is the same as the secondary washing step in step (3). After drying, peptide resin C is obtained.

[0100] (5) Coupling of phosphorylated amino acids and key deprotection After synthesizing peptide resin C, the first phosphorylated amino acid (pT) is coupled to obtain peptide resin D, wherein the amino acid sequence of peptide resin D is shown in SEQ ID NO: 5: pTKMSTLSYRPSDNVSSSTKK (SEQ ID NO: 5); Resin swelling: Take the peptide resin C obtained in step (4), place it in a solid-phase synthesis reaction tube, add 5 mL DMF, bubble with nitrogen at room temperature for 5 min to swell, and after swelling is complete, filter to remove DMF to obtain swollen peptide resin C.

[0101] Coupling reaction: Based on the molar amount of resin, add the corresponding reagents in a molar ratio of resin:corresponding amino acid:HATU:HOAt:DIEA = 1:5:5:5:10. Accurately weigh the corresponding reagents into a dry sample bottle, add 3 mL of DMF to dissolve and vortex, let stand for 1 min to activate, and finally add DIEA solution. After mixing evenly, quickly transfer it to a synthesis tube containing swollen peptide resin C. Ensure that the total time from adding DIEA solution to adding the mixture containing DIEA solution into the synthesis tube is controlled within 1~2 min to obtain the best coupling efficiency. Then place the synthesis tube at 55℃ under nitrogen protection and react for 20 min. After the reaction is complete, filter to remove the reaction solution, and then wash the resin twice with DMF (5 mL each time). To ensure complete coupling, the above coupling-washing operation needs to be repeated twice (a total of 3 rounds of coupling reaction).

[0102] Washing: The washing procedure is the same as in step (3).

[0103] Deprotection: After the resin temperature drops to room temperature, deprotection is carried out twice (5 min and 10 min respectively) in a constant temperature shaker (135 r / min) at 30℃ using a "cocktail" deprotection reagent. The specific operation procedure is as follows: add "cocktail" deprotection reagent (5 mL), react for 5 min, filter to remove the reaction solution after completion, wash the resin twice with DMF (5 mL each time) to completely remove residual piperidine and by-products, add fresh "cocktail" deprotection reagent (5 mL), and continue to react for 10 min. The formulation of the "cocktail" deprotection reagent is: 2% 1-hydroxybenzotriazole (HOBt, w / v), 2% hexamethyleneimine (v / v), 25% 1-methylpyrrolidine (v / v), 2% piperidine (v / v), and the remaining solvent is a mixed solution of NMP / DMSO (volume ratio 1:1).

[0104] Secondary washing: The operation is the same as the secondary washing step in step (3). After drying, peptide resin D is obtained.

[0105] All subsequent coupling and deprotection of phosphorylated amino acids follow this standard procedure.

[0106] (6) Coupling and deprotection of ordinary amino acids / phosphorylated amino acids following the first phosphorylated amino acid (pT) After synthesizing peptide resin D, the ordinary amino acid following the first phosphorylated amino acid (pT) is coupled (for ordinary amino acids, refer to step (4), and the deprotection reagent "20% piperidine / DMF" is adjusted to "cocktail deprotection reagent"). When encountering phosphorylated amino acids, refer to step (5). Finally, the phosphorylated polypeptide AT1R-PROX is prepared, and its amino acid sequence is shown in SEQ ID NO: 1.

[0107] Coupling reaction: The operation steps are the same as those in step (4) of the coupling reaction.

[0108] Washing: The washing procedure is the same as in step (3).

[0109] Deprotection: The operation is the same as the deprotection step in step (5).

[0110] Secondary washing: The operation is the same as the secondary washing step in step (3). After the last amino acid is deprotected, the resin is thoroughly washed with DMF (3×5 mL) and DCM (6×5 mL) in sequence to completely replace the solvent system. After drying, the finished peptide resin is obtained for later use.

[0111] The protective amino acids used in steps (3) to (6) are counted from the amino acid at the end of microwave synthesis. The corresponding amino acid coupling parameters of the manually synthesized partial sequence are shown in Table 2.

[0112]

[0113] (7) Peptide cleavage and resin removal Prepare the cleavage reagent (volume ratio): trifluoroacetic acid (TFA): phenol: water: anisole: ethylene dithiol (EDT) = 82.5:5:5:5:2.5; Take a small amount of the finished peptide resin obtained in step (6) into a 10 mL centrifuge tube, add an appropriate amount of cleavage reagent to ensure that the resin is completely submerged, tighten the cap, and stir the reaction at room temperature for 2 h to achieve complete cleavage of the peptide from the resin and simultaneous removal of the side chain protecting groups, and obtain the reaction mixture.

[0114] (8) Termination of reaction and polypeptide precipitation After the cutting reaction in step (7) is completed, dilute the reaction mixture with 10 times the volume of ice-cold ether, centrifuge at 4000 r / min for 3 min, carefully discard the supernatant, repeat 3 times to completely remove TFA, phenol, EDT and other residues, place the obtained white or off-white solid precipitate in a fume hood and dry at room temperature to obtain the AT1R-PROX crude peptide sample.

[0115] 3. Analysis of target peptide samples (1) Analysis of crude peptide samples of AT1R-PROX High-performance liquid chromatography (HPLC) analysis: 1 mg of the crude AT1R-PROX peptide sample obtained in step 2 was dissolved in 250 μL of pure water, filtered through a 0.22 μm filter membrane, and 50 μL was injected for reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. A ReproSil Gold 300 C18 column (250 × 4.6 mm, 5 μm) was used. The mobile phase consisted of A: an aqueous solution containing 0.1% TFA and B: an acetonitrile solution containing 0.1% TFA. The flow rate was set to 0.9 mL / min, and the detection wavelength was 214 nm.

[0116] The elution gradient settings for high performance liquid chromatography analysis are shown in Table 3.

[0117] Table 3 Elution gradient settings for high performance liquid chromatography analysis

[0118] Electrospray ionization mass spectrometry (ESI-MS) analysis: The main peak of the crude AT1R-PROX peptide sample obtained by high performance liquid chromatography analysis in step 3-(1) was collected, and then 5~8 μL of the collected solution was analyzed by ESI-MS.

[0119] The high-performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the AT1R-PROX crude peptide sample are shown in the figure. Figure 4 .

[0120] The results showed that the high-performance liquid chromatography (HPLC) retention time of the crude AT1R-PROX peptide sample was 19.26 min, with a sharp main peak. Mass spectrometry analysis revealed that the molecular ion peak detected at the elution time corresponding to the main peak was related to the target peptide AT1R-PROX ([M+H)). + The result is consistent with the theoretical value (3348.34), confirming the successful synthesis of the target product, whose peak area accounts for approximately 23.39%.

[0121] (2) Quality verification of AT1R-PROX pure product The crude AT1R-PROX peptide sample obtained in step 2 was subjected to semi-preparative HPLC treatment to obtain pure AT1R-PROX. Quality verification was then performed, and the specific steps are as follows: The crude AT1R-PROX peptide sample was dissolved in an appropriate amount of water, filtered through a 0.22 μm filter membrane, and purified using a semi-preparative C18 column with an acetonitrile elution gradient of 10-69%. The eluent corresponding to the target main peak was collected and freeze-dried to obtain a white flocculent solid, which is the pure AT1R-PROX product.

[0122] The results of chromatographic purity analysis and electrospray ionization mass spectrometry analysis of AT1R-PROX are shown in the figure. Figure 5 .

[0123] The results showed that the analytical chromatographic purity of AT1R-PROX was not less than 99%, and the mass spectrometry analysis showed that its molecular weight was completely consistent with the theoretical value, indicating that the high-purity target phosphorylated peptide was successfully obtained.

[0124] Example 2: Synthesis and Analysis of Phosphorylated Peptide AT1R-MID 1. Target peptide The phosphorylated polypeptide AT1R-MID, used for chemical semi-synthesis, was synthesized, and its amino acid sequence is shown in SEQ ID NO: 6: GKSHSNLSTKMpSpTLpSpYRPpSDNVSSSTKK (SEQ ID NO: 6) 2. Synthesis of the target polypeptide Obtain the crude AT1R-MID peptide sample. This step is the same as step 2 in Example 1, except that: The amino acid sequence of the target phosphorylated peptide was changed from SEQ ID NO: 1 to SEQ ID NO: 6, and the order and type of amino acid addition were adjusted accordingly. Specifically, a pseudoproline dipeptide unit (Fmoc-Ser(tBu)-Ser[Psi(Me,Me)Pro]-OH) was introduced at sites prone to aggregation, and the corresponding phosphorylated amino acids (pS, pT, pY) were accurately introduced. Apart from the necessary adjustments made due to the change in the amino acid sequence of the target phosphorylated peptide, all other conditions such as synthesis strategy, reagents, temperature, and time were the same as in Example 1.

[0125] 3. Purification and quality analysis of target peptide samples Take the crude AT1R-MID peptide sample obtained in step 2 and purify and analyze it according to the method described in step 3 of Example 1. The specific operation is the same as step 3 in Example 1 to obtain a white flocculent pure AT1R-MID.

[0126] The chromatographic purity and electrospray ionization mass spectrometry analysis results of AT1R-MID are shown in the figure. Figure 6 .

[0127] The results showed that the chromatographic purity of AT1R-MID, as determined by analytical HPLC, was no less than 99%; mass spectrometry analysis confirmed that its molecular weight was consistent with the theoretical value ([M+H)). + The result is consistent with the theoretical value of 3428.34, confirming the successful acquisition of the high-purity target product.

[0128] Example 3: Synthesis and Analysis of Phosphorylated Peptide AT1R-DIST 1. Target peptide The phosphorylated polypeptide AT1R-DIST, used for chemical semi-synthesis, was synthesized, and its amino acid sequence is shown in SEQ ID NO: 7: GKSHSNLSTKMSTLSYRPSDNVpSpSpSpTKK (SEQ ID NO: 7) 2. Synthesis of the target polypeptide Obtain the crude AT1R-DIST peptide sample. This step is the same as step 2 in Example 1, except that: The amino acid sequence of the target phosphorylated polypeptide was adjusted from SEQ ID NO: 1 to SEQ ID NO: 7, and the order and type of amino acid addition were adjusted accordingly. Specifically, based on the structural characteristics of the high-density phosphorylation sequence at the C-terminus, pseudoproline dipeptide units (Fmoc-Leu-Ser[Psi(Me,Me)Pro]-OH) were introduced at the corresponding sites where aggregation is likely to occur, and in particular, four consecutive phosphorylated serine (pS) units were introduced into the C-terminus of the peptide chain. Meanwhile, the synthesis strategy, reagents, temperature, time and all other conditions were the same as in Example 1.

[0129] 3. Purification and quality analysis of target peptide samples The crude AT1R-DIST peptide sample obtained in step 2 was purified and subjected to quality analysis. This step is the same as step 3 in Example 1, with the only difference being: During the HPLC purification process, the acetonitrile elution gradient was specifically optimized from 10-69% to 10-99% to address the significant changes in the polarity of AT1R-DIST, thus achieving effective separation of the target product from impurities. The theoretical molecular weight detected by mass spectrometry was adjusted to the theoretical value corresponding to this sequence.

[0130] The chromatographic purity and electrospray ionization mass spectrometry analysis results of AT1R-DIST are shown in the figure. Figure 7 .

[0131] The results showed that the final chromatographic purity of AT1R-DIST, as determined by analytical HPLC, was approximately 73.8%; mass spectrometry analysis confirmed that its molecular weight was consistent with the theoretical value ([M+H)). + (Theoretical value: 3348.34) is consistent. Although the four consecutive phosphorylation sites at its C-terminus greatly increased the difficulty of synthesis and purification, resulting in a lower final yield and purity than AT1R-PROX and AT1R-MID, the method of this invention still successfully obtained this highly challenging target product, demonstrating the effectiveness of this strategy in the face of extremely challenging sequences.

[0132] The following Comparative Examples 1 to 4 are based on the synthesis method of the phosphorylated polypeptide AT1R-PROX described in Example 1. Except for the experimental conditions explicitly stated below, the other reagents, raw materials, feed amounts, reaction equipment, operating procedures and other conditions are exactly the same as those in Example 1.

[0133] Comparative Example 1: Microwave synthesis of phosphorylated peptide AT1R-PROX 1. Target peptide A phosphorylated polypeptide AT1R-PROX for chemical semi-synthesis was synthesized, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0134] 2. Synthesis of the target polypeptide A microwave solid-phase synthesis strategy was used to obtain crude AT1R-PROX peptide samples (microwave synthesis). This step is the same as step 2 in Example 1, with the only difference being: The entire synthesis was performed using microwave-assisted synthesis, without introducing pseudoproline dipeptide units. Furthermore, in the step of removing the Fmoc protecting group, a 20% (v / v) piperidine / DMF solution was used as the deprotection agent throughout the process, and the "cocktail" deprotection agent of this invention was not used. 3. Purification and quality analysis of target peptide samples Since the synthesis failed, no preparation and purification were performed. The crude AT1R-PROX peptide sample was directly analyzed by HPLC and ESI-MS to evaluate the synthesis results.

[0135] The high-performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the crude AT1R-PROX peptide sample (microwave synthesized) are shown in the figure below. Figure 8 .

[0136] The results showed that no obvious main product peak appeared in the high-performance liquid chromatography (HPLC) chromatogram of the AT1R-PROX crude peptide sample (microwave synthesized), and its mass spectrum was at the target molecular weight ([M+H)). + No corresponding molecular ion peak was detected near the theoretical value (3348.34).

[0137] The above results demonstrate that for such complex multi-site phosphorylated peptides, the traditional microwave-assisted synthesis combined with conventional piperidine / DMF deprotection strategy is completely ineffective and cannot yield the target product.

[0138] Comparative Example 2: Synthesis of phosphorylated peptide AT1R-PROX using conventional SPPS method 1. Target peptide A phosphorylated polypeptide AT1R-PROX for chemical semi-synthesis was synthesized, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0139] 2. Synthesis of the target polypeptide A microwave-assisted method combined with the traditional SPPS method was used to obtain crude AT1R-PROX peptide samples (SPPS method). This step is the same as step 2 in Example 1, with the only difference being: No pseudoproline dipeptide units were introduced to disrupt the secondary structure of the peptide chain throughout the process, and in the step of removing the Fmoc protecting group, a 20% (v / v) piperidine / DMF solution was used as the deprotecting agent throughout the process. The "cocktail" deprotecting agent of the present invention was not used to inhibit side reactions such as β-elimination.

[0140] 3. Purification and quality analysis of target peptide samples The crude AT1R-PROX peptide sample obtained in step 2 (SPPS method) was purified and analyzed for quality. This step is the same as step 3 in Example 1. The same purification and analysis methods were used in this step to directly compare the quality differences of the synthesized crude peptides.

[0141] The results of high performance liquid chromatography and electrospray ionization mass spectrometry analysis of AT1R-PROX crude peptide samples (SPPS method) are shown in the figure below. Figure 9 .

[0142] The results showed that the main peak of the AT1R-PROX crude peptide sample (SPPS method) was not significant in the high-performance liquid chromatography (HPLC) chromatogram, while the impurity peaks were numerous and strong. The calculated analytical chromatographic purity was only 13.58%. Although mass spectrometry analysis could detect the target molecular weight ([M+H)... + The theoretical value is 3348.34), but the signal-to-noise ratio is low, and there are a large number of impurity ion peaks in the mass spectrum.

[0143] The above results show that although the traditional SPPS method can barely obtain the target product, it suffers from serious side reactions, extremely poor product purity, and low synthesis efficiency.

[0144] Comparative Example 3: Synthesis of phosphorylated peptide AT1R-PROX by optimizing only the deprotection strategy 1. Target peptide A phosphorylated polypeptide AT1R-PROX for chemical semi-synthesis was synthesized, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0145] 2. Synthesis of the target polypeptide A microwave-assisted deprotection strategy combined with a "cocktail" deprotection reagent was used to obtain crude AT1R-PROX peptide samples (deprotection optimized only). This step is the same as step 2 in Example 1, with the only difference being: No pseudoproline dipeptide units were introduced throughout the process. The microwave-assisted synthesis of the first 19 amino acids used 20% piperidine / DMF for deprotection; the synthesis of the last 9 amino acids replaced the deprotection reagent with the "cocktail" deprotection reagent of this invention.

[0146] 3. Purification and quality analysis of target peptide samples The crude AT1R-PROX peptide sample obtained in step 2 (only optimized for deprotection) was purified and analyzed for quality. This step is the same as step 3 in Example 1. The same purification and analysis methods were used in this step to fairly compare the quality of crude peptide samples under different synthesis strategies.

[0147] The high-performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the crude peptide sample of AT1R-PROX (optimized deprotection only) are shown in the figure below. Figure 10 .

[0148] The results showed that the main peak of the HPLC chromatogram of the crude peptide sample of AT1R-PROX (only optimized for deprotection) was improved, and its analytical chromatographic purity was increased to 16.75%, which was 3.17 percentage points higher than that of Comparative Example 2 (conventional SPPS) at 13.58%. Mass spectrometry analysis showed that the target product signal was enhanced.

[0149] The above results indicate that using a "cocktail" deprotection reagent alone can effectively inhibit side reactions such as β-elimination and improve product purity to a certain extent. However, due to the unresolved peptide chain aggregation problem, the overall improvement in synthesis efficiency remains very limited.

[0150] Comparative Example 4: Synthesis of phosphorylated peptide AT1R-PROX using only pseudoproline strategy 1. Target peptide A phosphorylated polypeptide AT1R-PROX for chemical semi-synthesis was synthesized, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0151] 2. Synthesis of the target polypeptide A microwave-assisted pseudoproline introduction strategy was used to obtain crude AT1R-PROX peptide samples (with only pseudoproline introduced). This step is the same as step 2 in Example 1, with the only difference being: A pseudoproline dipeptide structural unit (Fmoc-Leu-Ser[Ψ(Me,Me)pro]-OH) was introduced at a key site where the peptide chain is prone to forming secondary structures. However, in the step of removing the Fmoc protecting group, a 20% (v / v) piperidine / DMF solution was still used as the deprotecting agent throughout the process, and the "cocktail" deprotecting agent of the present invention was not used.

[0152] 3. Purification and quality analysis of target peptide samples The crude AT1R-PROX peptide sample obtained in step 2 (with only pseudoproline introduced) was purified and analyzed for quality. This step is the same as step 3 in Example 1. The same purification and analysis methods were used in this step to fairly compare the quality of crude peptide samples under different synthesis strategies.

[0153] The high performance liquid chromatography and electrospray ionization mass spectrometry analysis results of the AT1R-PROX crude peptide sample (with pseudoproline only) are shown in the figure. Figure 11 .

[0154] The results showed that the main peak of the HPLC chromatogram of the crude peptide sample of AT1R-PROX (with only pseudoproline introduced) was significantly improved, and its analytical chromatographic purity was increased to 20.91%, which was 7.33 percentage points higher than that of Comparative Example 2 (traditional SPPS). Mass spectrometry analysis showed that the target product signal was significantly enhanced and the impurity peaks were reduced.

[0155] The above results indicate that the strategy of introducing pseudoproline alone can effectively disrupt peptide chain aggregation and improve coupling efficiency, which has a significant effect on improving the purity of the synthesis. However, since the traditional reagent with strong basicity is still used in the deprotection step, many impurity peaks can still be seen in its high performance liquid chromatogram, which is speculated to be related to side reactions such as β-elimination that were not completely suppressed.

[0156] Example 4: Comparison of purity of phosphorylated peptide AT1R-PROX synthesized by different methods The crude peptide samples of AT1R-PROX obtained in Example 1, the crude peptide samples of AT1R-PROX obtained in Comparative Examples 2-4 (SPPS method), the crude peptide samples of AT1R-PROX (only optimized deprotection), and the crude peptide samples of AT1R-PROX (only pseudoproline introduced) were respectively analyzed for the target peptide samples. The analysis steps are the same as steps 3-(1) in Example 1.

[0157] Table 4 shows the comparison of chromatographic purity of crude AT1R-PROX phosphorylated peptides synthesized by different methods.

[0158] Table 4. Comparison of chromatographic purity of crude AT1R-PROX phosphorylated peptides synthesized by different methods.

[0159] The results show: (1) Combination strategy to achieve the highest purity: The AT1R-PROX synthesized by the combination strategy of "cocktail" deprotection reagent + pseudoproline has the highest purity, reaching 23.39%; (2) The effect of a single strategy is limited: optimizing the deprotection reagent alone can increase the purity from 13.58% to 16.75%; introducing pseudoproline alone can increase it to 20.91%. This shows that although the two strategies are effective in themselves, they both have certain bottlenecks.

[0160] The above results demonstrate that the "cocktail" deprotection reagent + pseudoproline combination strategy of this invention simultaneously solves two major bottlenecks in the synthesis of phosphorylated peptides from the source. First, the unique deprotection reagent effectively inhibits key side reactions such as β-elimination, ensuring the stability of the phosphate group and the correctness of the product structure. Second, the introduction of pseudoproline units successfully solves the problem of low coupling efficiency caused by peptide chain aggregation, ensuring the complete extension of the sequence.

[0161] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0162] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A deprotection reagent characterized in that, The deprotection reagent comprises the following components: 1-hydroxybenzotriazole; an organic base.

2. The deprotection reagent according to claim 1, characterized in that, The deprotection reagent further comprises a solvent; Optionally, the organic base comprises one or more of hexamethylendiamine, 1-methylpyrrolidine, piperidine and morpholine; Optionally, the solvent is a strong polar aprotic solvent; Optionally, the strong polar aprotic solvent is N-methylpyrrolidone and / or dimethyl sulfoxide.

3. The deprotection reagent according to claim 1 or 2, characterized in that, The organic base comprises a primary base and one or more auxiliary bases; Optionally, the primary base is 1-methylpyrrolidine, the auxiliary base is hexamethylendiamine and piperidine, and the solvent is N-methylpyrrolidone and dimethyl sulfoxide; Optionally, the content of 1-hydroxybenzotriazole is 10-50 g / L, and the content of the organic base is 220-400 mL / L; Optionally, the content of 1-hydroxybenzotriazole is 10-50 g / L, the content of the primary base is 200-300 mL / L, and the content of the auxiliary base is 20-100 mL / L; Optionally, the content of 1-hydroxybenzotriazole is 10-50 g / L, the content of 1-methylpyrrolidine is 200-300 mL / L, the content of hexamethylendiamine is 10-50 mL / L, the content of piperidine is 10-50 mL / L, and the rest is the solvent, which is N-methylpyrrolidone and dimethyl sulfoxide, wherein the volume ratio of N-methylpyrrolidone to dimethyl sulfoxide is (0.5-2):

1.

4. A kit characterized in that, The kit comprises: The deprotection reagent of any one of claims 1-3.

5. The kit of claim 4, wherein The kit further comprises: a pseudo-proline dipeptide; Optionally, the general formula of the pseudo-proline dipeptide is Fmoc-Xaa-Yaa[Ψ(Me,Me)pro]-OH, wherein Xaa is any natural amino acid, and Yaa is serine or threonine.

6. The kit of claim 4, wherein The kit further comprises: a phosphorylated amino acid with a protecting group; Optionally, the phosphorylated amino acid with a protecting group comprises one or more of Fmoc-Ser(PO(OBzl)OH)-OH, Fmoc-Thr(PO(OBzl)OH)-OH and Fmoc-Tyr(PO(OBzl)OH)-OH.

7. A method for preparing a phosphorylated polypeptide, characterized by, The preparation method comprises the following steps: performing assembly of a target peptide chain; wherein, in the process of assembly of the target peptide chain, the deprotection reagent of any one of claims 1-3 or the kit of any one of claims 4-6 is used to remove the Fmoc protecting group, and / or the kit of any one of claims 4-6 is used to introduce the pseudo-proline dipeptide at a site prone to forming a secondary structure of the target peptide chain.

8. The production method according to claim 7, characterized by, The assembly of the target peptide chain is performed by solid-phase synthesis; Optionally, when the pseudo-proline dipeptide is introduced, the carboxyl group of the pseudo-proline dipeptide is activated by using a coupling reagent on a solid-phase synthesis carrier, and an organic amine base is used to drive the coupling reaction; Optionally, the solid-phase synthesis carrier is a resin; Optionally, the molar ratio of the solid-phase synthesis carrier, the pseudo-proline dipeptide, the coupling reagent, and the organic amine base added amount is 1: (2-5): (2-5): (4-10) when the pseudo-proline dipeptide is introduced. Optionally, the temperature is 20-35℃ and the reaction time is 45-75 min when the pseudo-proline dipeptide is introduced.

9. Use of the deprotection reagent according to any one of claims 1-3, the kit according to any one of claims 4-6, or the preparation method according to claim 7 or 8 in protein / polypeptide chemical synthesis, preparation of phosphorylated polypeptides, or improving the synthesis efficiency of phosphorylated polypeptides.

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