Ultrasonic-assisted polypeptide C-terminal amidation method and application thereof

Abstract

The invention provides an ultrasonic-assisted polypeptide C-terminal amidation method and application thereof. The method comprises the following steps: mixing a substrate polypeptide, a reducing agent and a buffer solution to obtain a reaction system; performing ultrasonic treatment on the reaction system for reaction to obtain a polypeptide C-terminal amidated product; the reducing agent comprises one or more of tris (2-carbonyl ethyl) phosphorus hydrochloride, triphenylphosphine tri-m-sodium sulfonate or triphenylphosphine; the structures of the substrate polypeptide and the amino acid residue at the C end of the substrate polypeptide are as shown in a formula I, and the structures of the product and the amino acid residue at the C end of the product are as shown in a formula II, r2 is a sequence composed of other amino acids except the amino acid at the C terminal in the substrate polypeptide; r is a sequence composed of other amino acids except the amino acid at the C terminal in the product; x1 is SH or SeH; and X2 is OH or NH2. The problem that in the prior art, the large-scale synthesis cost of polypeptide C-terminal amidation is too high can be solved, and the method is suitable for the field of polypeptide synthesis.

Description

Technical Field

[0001] This invention relates to the field of peptide synthesis, and more specifically, to a method for ultrasound-assisted C-terminal amidation of peptides and its application. Background Technology

[0002] Post-translational modifications of peptides are among the key factors determining their biological activity, stability, and drug-likeness. C-terminal amidation, along with phosphorylation and acetylation, is a common and functionally significant modification. Extensive clinical data demonstrate that C-terminal amidation can increase peptide activity by more than an order of magnitude (e.g., the receptor agonist activity of the amide form of human pancreatic amyloid peptides is approximately 15 times higher than that of the carboxylic acid form); reduce overall net negative charge, improve membrane permeability and tissue distribution; significantly enhance resistance to exopeptidases (carboxypeptidase, cathepsin, etc.), and prolong plasma half-life by 2–10 times.

[0003] For this reason, dozens of peptide drugs approved worldwide, including Tirzepatide, Pramlintide, Exenatide, and Cetrorelix Acetate, all use the C-terminal amide form as the active pharmaceutical ingredient (API).

[0004] The main technical routes for C-terminal amidation of existing peptide technologies and their drawbacks are as follows:

[0005] (1) Solid-phase synthesis directly introducing amide resin method

[0006] Using Rink Amide, Sieber, or PAL resin, C-terminal -CON is directly provided after the last amino acid coupling. This method is a mature process, but it consumes a lot of solvent and generates a lot of waste liquid. At the same time, amino acids and coupling reagents are prone to producing racemic impurities, which makes subsequent purification difficult and results in low yield, which does not conform to the concept of green chemistry.

[0007] (2) In vitro enzymatic oxidation method

[0008] Using glycine, which extends from the C-terminus of the target peptide, as a precursor, the product is catalyzed in two steps by peptidyl glycine α-amidyl monooxygenase (PAM) (hydroxylation → product generation). PAM is only expressed in mammalian cell lines, and purification requires four-step chromatography, resulting in high enzyme costs. Furthermore, the enzymatic introduction of exogenous proteins necessitates validation of residual enzymes, cofactors, and viral clearance during the API stage, leading to a significant workload in quality research and limiting its large-scale application.

[0009] (3) Electrochemical oxidative decarboxylation-ammonolysis

[0010] Malins et al. (J. Am. Chem. Soc. 2021, 143, 11811-11819, 2021) achieved the electro-oxidation of C-terminal α-keto acid intermediates of peptides on a graphite felt anode, followed by reaction with ammonia to form -CON This method requires an external power source and has poor compatibility with amino acids containing sensitive side chain functional groups, especially in sequences containing Tyr, His, and Trp, which limits its scale-up production.

[0011] (4) Photocatalytic Cys-Extension-Elimination Method

[0012] This method uses a precursor of cysteine ​​extended from the C-terminus of the target peptide sequence as a substrate, and obtains a C-terminal amidated peptide through a substitution reaction of the C-terminal cysteine ​​with a light-emitting agent, a photocatalytic elimination reaction, and a cleavage reaction. This route requires three consecutive steps, the intermediates have poor stability, the second step requires a custom-designed photoreactor, the route is complex, costly, and time-consuming, making it unsuitable for large-scale production.

[0013] (5) Photo-driven decarboxylation and oxygenation of chalcogenide bonds (Se / Te catalysis)

[0014] Shenpeng et al. (Angew. Chem. Int. Ed. 2025, e202502233) used diphenyldiselendide as a Lewis acid catalyst and oxygen as an oxidant to obtain C-terminal amidated peptides via a thiobond-catalyzed, light-driven decarboxylation-oxygenation reaction. This method requires protecting groups on the amino acid side chains and is difficult to react with sequences containing Cys, His, and Trp, thus limiting its application.

[0015] In summary, existing technologies suffer from drawbacks such as "high cost, high waste liquid, high racemization, scarce enzyme sources, multiple steps, long processing time, demanding equipment, or poor substrate compatibility." There is still a lack of a mild, low-cost, relatively short reaction time, and scale-up suitable amidation method for peptide C-terminus. Summary of the Invention

[0016] The main objective of this invention is to provide a method for ultrasound-assisted C-terminal amidation of peptides and its application, in order to solve the problem of excessively high cost in the large-scale synthesis of C-terminal amidation of peptides in the prior art.

[0017] To achieve the above objectives, according to a first aspect of the present invention, a method for ultrasound-assisted C-terminal amidation of a peptide is provided, the method comprising: mixing a substrate peptide, a reducing agent, and a buffer to obtain a reaction system; and subjecting the reaction system to ultrasound treatment to obtain a product with C-terminal amidation of the peptide.

[0018] The reducing agent includes one or more of tris(2-carbonylethyl)phosphine hydrochloride, sodium triphenylphosphine tri-m-sulfonate, or triphenylphosphine; the structure of the substrate polypeptide and the C-terminal amino acid residue of the substrate polypeptide is shown below:

[0019]

[0020] Formula I;

[0021] The structural formulas of the product and its C-terminal amino acid residues are shown below:

[0022]

[0023] Formula II;

[0024] Among them, R 1 For H or Me;

[0025] R 2 It is the sequence of amino acids in the substrate polypeptide, excluding the C-terminal amino acid;

[0026] R represents the sequence of amino acids in the product other than the C-terminal amino acid;

[0027] X 1 It is SH or SeH;

[0028] X 2 It can be OH or NH2.

[0029] Furthermore, the frequency of the ultrasonic treatment is 20-100 kHz.

[0030] Furthermore, the power density of the ultrasonic treatment was 0.5-2 W / mL.

[0031] Furthermore, the pulse mode of the ultrasonic treatment has a duty cycle of 30-80%.

[0032] Furthermore, the reducing agent includes tris(2-carbonylethyl)phosphohydrochloride.

[0033] Furthermore, the concentration of the substrate peptide is 0.1–2 mM.

[0034] Furthermore, the concentration of the reducing agent is 20~200 mM.

[0035] Furthermore, the reaction temperature is 25~45 ℃.

[0036] Furthermore, the reaction time is 2 to 15 hours.

[0037] Furthermore, the reaction time is 2 to 5 hours.

[0038] Furthermore, the pH of the reaction is 8.0~9.0.

[0039] Furthermore, the pH of the reaction was 9.0.

[0040] Furthermore, the buffer solution includes NaH2PO4 or KH2PO4.

[0041] Furthermore, the concentration of the buffer solution is 0.1~5 M.

[0042] Furthermore, the method also includes: sonicating the reaction system after mixing the substrate peptide, reducing agent, buffer and auxiliary agent to obtain the product with C-terminal amidation of the peptide.

[0043] Furthermore, the additives include TiO2.

[0044] Furthermore, the concentration of the adjuvant is 0.1~2 mg / mL.

[0045] To achieve the above objectives, according to a second aspect of the present invention, the application of the above-described ultrasound-assisted C-terminal amidation method for polypeptides in the preparation of polypeptide drugs is provided.

[0046] By employing the technical solution of this invention, and with the assistance of ultrasonic treatment, and using reducing agents such as tris(2-carbonylethyl)phosphohydrochloride (TCEP), sodium triphenylphosphine tri-m-sulfonate (TPPTS), or triphenylphosphine (PPh3), the C-terminus of the substrate peptide is subjected to desulfurization and decarboxylation reactions of cysteine, cysteine ​​amide, penicillamine, or selenocysteine, achieving a one-step amidation reaction to obtain the C-terminal amidation product. Compared with existing technologies, the peptide C-terminal amidation method of this application is more efficient and simpler, has good substrate compatibility, and a simple reaction setup. It does not require photochemical or electrochemical equipment or enzyme catalysts; it only requires a "one-step" method to achieve the amidation of the peptide C-terminus and further obtain the corresponding product. The synthesis efficiency is high, the cost is low, it conforms to the concept of green development, and it is more suitable for large-scale industrial production. Attached Figure Description

[0047] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0048] Figure 1 The UPLC detection results of the reaction system in Example 1 of this application are shown.

[0049] Figure 2 The UPLC detection results of the reaction system in Example 2 of this application are shown.

[0050] Figure 3The UPLC detection results of the reaction system in Example 3 of this application are shown.

[0051] Figure 4 The UPLC detection results of the reaction system in Example 4 of this application are shown.

[0052] Figure 5 The UPLC detection results of reaction system 1 in Example 5 of this application are shown.

[0053] Figure 6 The UPLC detection results of reaction system 2 in Example 5 of this application are shown.

[0054] Figure 7 The UPLC detection results of reaction system 3 in Example 5 of this application are shown.

[0055] Figure 8 The UPLC detection results of reaction system 4 in Example 5 of this application are shown. Detailed Implementation

[0056] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0057] Terminology Explanation:

[0058] Cysteine ​​amide: A derivative obtained by amidation of the carboxyl group of cysteine, namely Cys-N The structure is: HS-C -CH(N )-CON Compared to the parent cysteine, it retains the free sulfhydryl group (-SH), but the C-terminus is changed to -CON. .

[0059] As mentioned in the background art, the existing methods for amidation of the C-terminus of peptides have drawbacks such as complex preparation methods, high costs, and difficulty in large-scale application. Based on this, the inventors in this application attempt to provide a method for amidation of the C-terminus of peptides, and thus propose a series of protection schemes in this application.

[0060] In a first typical embodiment of this application, a method for ultrasound-assisted C-terminal amidation of a peptide is provided. The method includes: mixing a substrate peptide, a reducing agent, and a buffer to obtain a reaction system; subjecting the reaction system to ultrasound treatment to obtain a product with C-terminal amidation of the peptide; the reducing agent includes one or more of tris(2-carbonylethyl)phosphine hydrochloride, tris(3-hydroxypropyl)phosphine, sodium triphenylphosphine tri-m-sulfonate, or triphenylphosphine.

[0061] The structures of the substrate peptide and its C-terminal amino acid residues are shown below:

[0062]

[0063] Formula I;

[0064] The structural formulas of the product and its C-terminal amino acid residues are shown below:

[0065]

[0066] Formula II;

[0067] Among them, R 1 For H or Me; R 2 R is the sequence of amino acids in the substrate polypeptide excluding the C-terminal amino acid; R is the sequence of amino acids in the product excluding the C-terminal amino acid; X 1 For SH or SeH; X 2 It can be OH or NH2.

[0068] Existing methods for C-terminal amidation of peptides have revealed significant drawbacks in industrial scale-up: solid-phase methods require excessive reagents and large amounts of organic solvents, resulting in high costs, large amounts of waste liquid, and the risk of racemization; enzymatic methods rely on PAM expressed in mammalian cells, with expensive enzyme sources and complex quality control; electrochemical methods have poor compatibility with sensitive residues such as Tyr, His, and Trp; and photocatalytic routes require dedicated photoreactors, multi-step operations, or fully protected side chains, resulting in large equipment investment and unstable intermediates, which limits large-scale applications.

[0069] This application utilizes one or more of tris(2-carbonylethyl)phosphine hydrochloride, sodium triphenylphosphine tri-m-sulfonate, or sodium triphenylphosphine tri-m-sulfonate as a reducing agent to selectively induce a free radical-coordinated desulfurization and decarboxylation reaction of cysteine, cysteine ​​amide, penicillamine, or selenocysteine ​​at the C-terminus of a polypeptide, directly yielding the C-terminal amidation product in one step. The selenium hydroxyl group of selenocysteine ​​is readily oxidized to form a diselenate bond. If the amino acid at the C-terminus of the polypeptide is an oxidically coupled selenocysteine ​​(containing a diselenate bond), the reaction described in this application can also be achieved. The reaction formula for the amidation of the C-terminus of the polypeptide under the assistance of ultrasonic treatment is shown below:

[0070] .

[0071] Compared to existing methods for C-terminal amidation of peptides, this method utilizes the high-frequency vibrations generated by ultrasound propagation in a liquid during ultrasonic treatment to induce cavitation, releasing localized high temperatures and triggering free radical reactions. This further improves the mixing efficiency of the reactants, allowing the reaction to occur within a shorter reaction time, promoting intermolecular exchange and transfer, and increasing the efficiency of the chemical reaction. Therefore, this method avoids enzyme catalysis and photocatalysis steps, and has a wider substrate applicability. It is applicable to amidation reactions where the amino acid closest to the C-terminus of the substrate peptide (the penultimate amino acid in the NC-terminal sequence, i.e., the second amino acid at the C-terminus) is any one of the following amino acids: Ala, Pro, Asp, Asn, Lys, Met, Ser, Trp, Tyr, Arg, and His. Furthermore, this method is particularly suitable for peptide sequences with low activity under enzymatic methods where the C-terminal cysteine ​​is adjacent to a proline. Compared to existing technologies, this method requires only one reaction step to obtain the target product, is simple to operate, improves reaction yield, reduces production costs, and is suitable for large-scale production, providing a concise and efficient new method for synthesizing C-terminal amidated peptides.

[0072] In a preferred embodiment, the frequency of the ultrasonic treatment is 20-100 kHz (including but not limited to 20, 30, 40, 50, 60, 70, 80, 90 or 100 kHz).

[0073] This application sets the ultrasonic treatment frequency in the range of 20-100 kHz. This frequency range was chosen based on the efficiency of promoting the synergistic desulfurization and decarboxylation reaction of C-terminal cysteine, penicillamine, or selenocysteine ​​radicals. Ultrasound in this frequency range can effectively induce the formation of free radicals, accelerating the desulfurization and decarboxylation processes, thereby increasing the yield of C-terminal amidated peptides. Too low a frequency may fail to adequately stimulate intermolecular energy transfer, while too high a frequency may lead to excessive heating of the reaction system, affecting the stability of the amino acid side chains and the selectivity of the reaction. The aforementioned ultrasonic treatment frequency range promotes reaction efficiency and is relatively mild, making it applicable to various C-terminal amidation reactions of peptides.

[0074] In a preferred embodiment, the power density of the ultrasonic treatment is 0.5-2.0 W / mL (including but not limited to 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 W / mL).

[0075] The ultrasonic power density of this application is 0.5-2 W / mL. Within this power density range, ultrasound can provide sufficient energy to activate TCEP to initiate the desulfurization and decarboxylation reaction, while avoiding unnecessary side reactions and thermal damage to peptides, which is more conducive to the formation of C-terminal amidation of peptides.

[0076] In a preferred embodiment, the pulse mode of the ultrasonic treatment has a duty cycle of 30-80%; preferably, the reactor for the ultrasonic treatment includes, but is not limited to, a bath type, a probe type, or a flow tank type reactor, which can be flexibly selected by those skilled in the art according to actual needs, and this application does not impose any restrictions.

[0077] In another preferred embodiment of the invention, the duty cycle of the ultrasonic pulse mode is controlled between 30% and 80%. The duty cycle refers to the ratio of the ultrasonic "on" time to the total cycle time, and it can regulate the duration and intensity distribution of ultrasonic impact on the reaction system. Using a duty cycle within the above range allows the reaction system to be subjected to continuous and effective ultrasonic impact for a sufficiently long time. Simultaneously, appropriate rest intervals prevent excessive heat accumulation in the reaction system, promoting reaction controllability and peptide structure stability.

[0078] In a preferred embodiment, the reducing agent comprises tris(2-carbonylethyl)phosphohydrochloride.

[0079] The preferred reducing agent in this application is tris(2-carbonylethyl)phosphohydrochloride (TCEP). As a highly efficient reducing agent, TCEP can selectively promote the radical-mediated desulfurization and decarboxylation of C-terminal cysteine, penicillamine, cysteine ​​amide, or selenocysteine ​​in peptides under mild conditions (such as the ultrasonic and weakly alkaline environment of the reaction system in this application), thereby directly yielding the amidated product. The advantages of TCEP lie in its compatibility with various amino acid side chains, reducing restrictions on peptide sequences, and its good reactivity under ultrasonic conditions, which can effectively improve the yield of the amidation reaction and reduce the occurrence of side reactions.

[0080] In a preferred embodiment, the concentration of the substrate peptide is 0.1 to 2 mM (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mM), preferably 1 mM.

[0081] This application precisely controls the concentration of the substrate peptide within the range of 0.1 to 2 mM, more preferably 1 mM. Lower concentrations may not be sufficient to trigger a adequate reaction rate, while excessively high concentrations may lead to molecular crowding in the reaction system, affecting the efficient recognition and action of TCEP on the C-terminus of the target peptide. The above-mentioned concentration range of the substrate peptide can avoid the above problems and optimize the yield and purity of the amidation reaction.

[0082] In a preferred embodiment, the concentration of the reducing agent is 20~200 mM (including but not limited to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 55, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74). 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 84, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mM), preferably 50~100 mM.

[0083] In this application, the concentration of the reducing agent is preferably set at 20-200 mM, more specifically, preferably 50-100 mM. Within this concentration range, TCEP can generate sufficient reducing activity to promote the C-terminal amidation reaction. Furthermore, a concentration of 50-100 mM provides a better balance, avoiding unnecessary side reactions caused by excessively high reducing agent concentrations, and promoting the complete progress of the reaction, further improving the yield and purity of the target amidated peptide.

[0084] In a preferred embodiment, the reaction temperature is 25~45℃ (including but not limited to 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃), preferably 35~40℃.

[0085] In a preferred embodiment, the reaction time is 2 to 15 hours (including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 hours), preferably 2 to 5 hours.

[0086] The reaction temperature of this application needs to be controlled within the range of 25~45 ℃, and more preferably 35~40 ℃, and the reaction time is controlled within 2~15 h, preferably 2~5 h.

[0087] Within the reaction temperature range of this application, the reaction system maintains sufficient fluidity, thereby promoting effective collisions between peptide molecules and the reducing agent, further increasing the reaction rate, reducing the potential damage to the peptide structure caused by high temperatures, and enabling the product to maintain its integrity and biological activity. The amidation reaction time in this application is controlled between 2 and 15 hours, preferably 2 to 5 hours, which is beneficial for balancing reaction efficiency and product stability. Within this reaction time, the amidation reaction of this application can achieve better yield and purity.

[0088] In a preferred embodiment, the pH of the reaction is 8.0 to 9.0 (including but not limited to 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0), preferably 9.0.

[0089] The pH value of the reaction in this application is controlled between 8.0 and 9.0, and more preferably, pH 9.0. Under a weakly alkaline environment (pH = 8.0–9.0), TCEP is activated as a reducing agent, promoting the free radical-mediated desulfurization and decarboxylation reaction of C-terminal cysteine, penicillamine, or selenocysteine. Especially at pH = 9.0, the amidation reaction in this application exhibits better product formation efficiency, placing the reaction system in a chemical state more favorable for amide bond formation.

[0090] In a preferred embodiment, the buffer solution comprises NaH₂PO₄-NaOH or KH₂PO₄-NaOH. NaH₂PO₄ is preferred.

[0091] The buffer system of this application includes either NaH₂PO₄-NaOH buffer or KH₂PO₄-NaOH buffer. The choice of buffer plays a crucial role in maintaining the pH stability of the reaction system, especially for pH-sensitive chemical reactions such as C-terminal amidation. Both NaH₂PO₄-NaOH and KH₂PO₄-NaOH exhibit good stability under weakly alkaline conditions, and the inventors of this application have found that these two buffers have good compatibility with peptide molecules and TCEP. Considering reagent cost, NaH₂PO₄ is preferred as the buffer in this application. In the buffer system of this application, the C-terminal amidation reaction of the peptide can proceed with high efficiency without being affected by fluctuations in reaction conditions in product formation.

[0092] In a preferred embodiment, the concentration of the buffer solution is 0.1~5 M (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 or 5 M), preferably 0.1~1 M.

[0093] The buffer concentration in this application is controlled within the range of 0.1–5 M, which is beneficial for maintaining the pH stability of the reaction system and promoting effective contact between peptide molecules and TCEP. Lower buffer concentrations may not effectively maintain the pH conditions required for the reaction, while excessively high concentrations may introduce unnecessary ionic strength into the system, affecting the reaction. When the buffer concentration is within the aforementioned range, the pH of the reaction system remains stable, while simultaneously promoting sufficient contact between peptide molecules and TCEP, thus improving the selectivity and efficiency of the reaction. This reaction environment is most favorable for the efficient execution of C-terminal amidation reactions, further promoting the quality and yield of the product, and providing a technical basis for the widespread application of this invention in the biochemical field.

[0094] In a preferred embodiment, the method further includes: sonicating the reaction system after mixing the substrate peptide, reducing agent, buffer, and auxiliary agent to obtain a product with C-terminal amidation of the peptide.

[0095] In a preferred embodiment, the additive includes TiO2.

[0096] In a preferred embodiment, the concentration of the adjuvant is 0.1 to 2 mg / mL (including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mg / mL).

[0097] In another preferred embodiment of the present invention, the auxiliary agent TiO2 (titanium dioxide) is added to the reaction system, which can further improve the efficiency of the C-terminal amidation reaction. Under ultrasonic and weakly alkaline conditions, the presence of TiO2 can promote the generation of free radicals and accelerate the target reaction. In this application, the concentration range of the auxiliary agent is set to 0.1~1 mg / mL, at which the reaction system exhibits better yield and purity of C-terminal amidation product.

[0098] In a second typical embodiment of this application, the above-described ultrasound-assisted C-terminal amidation method for polypeptides is provided for the preparation of polypeptide drugs.

[0099] Unless otherwise specified, the reagents used in the embodiments of this application are all commercially available products.

[0100] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0101] Example 1

[0102] At room temperature (15–25 °C), prepare 3 mL of a mixed aqueous solution of 0.2 M NaH₂PO₄ and 50 mM TCEP. Adjust the pH to 8.0 with 4 M NaOH solution. Then, add IWTKDHEEVYEAC-OH (SEQ ID NO: 1) (5 mg, 3 μmol) to the above mixed solution. The final concentration of the substrate peptide shown in SEQ ID NO: 1 is 1 mM. The system is a colorless and transparent liquid. Place it in an ultrasonic bath (frequency 20 kHz, power density 1 W / mL, pulse mode 50% duty cycle) at 40 °C in the dark for 3 h. Then, add 10 mM sodium ascorbate to terminate the reaction. Perform UPLC analysis on the reaction system. The product percentage (Area%) is 45%. The UPLC spectrum is shown below. Figure 1 As shown.

[0103] Example 2

[0104] At room temperature (15–25 °C), prepare 3 mL of a mixed aqueous solution of 0.2 M NaH₂PO₄ and 50 mM TCEP. Adjust the pH to 8.0 with 4 M NaOH solution, add 1.5 mg TiO₂ (particle size 20 nm), and then add IWTKDHEEVYEAC-OH (SEQ ID NO: 1) (5 mg, 3 μmol) to the above mixed solution. The final concentration of the substrate peptide shown in SEQ ID NO: 1 is 1 mM. The system is a colorless and transparent liquid. Place it in an ultrasonic bath (frequency 20 kHz, power density 1 W / mL, pulse mode 50% duty cycle) at 40 °C in the dark for 3 h. Then, add 10 mM sodium ascorbate to terminate the reaction. Perform UPLC analysis on the reaction system. The product percentage (Area%) is 54%. The UPLC spectra are shown below. Figure 2 As shown.

[0105] Example 3

[0106] At room temperature (15-25℃), prepare 3 mL of a mixed aqueous solution of 0.2 M KH₂PO₄ and 50 mM TCEP. Adjust the pH to 8.0 with 4 M NaOH solution. Then, add IWTKDHEEVYEAC-OH (SEQ ID NO: 1) (5 mg, 3 μmol) to the above mixed solution. The final concentration of the substrate peptide shown in SEQ ID NO: 1 is 1 mM. The system is a colorless and transparent liquid. Place it in an ultrasonic bath (frequency 20 kHz, power density 1 W / mL, pulse mode 50% duty cycle) at 40℃ in the dark for 3 h. Then, add 10 mM sodium ascorbate to terminate the reaction. Perform UPLC analysis on the reaction system. The product percentage (Area%) is 56%. The UPLC chromatogram is shown below. Figure 3 As shown.

[0107] Example 4

[0108] At room temperature (15–25 °C), prepare 3 mL of a mixed aqueous solution of 0.2 M KH₂PO₄ and 50 mM TCEP. Adjust the pH to 8.0 with 4 M NaOH solution, add 1.5 mg TiO₂ (particle size 20 nm), and then add IWTKDHEEVYEAC-OH (SEQ ID NO: 1) (5 mg, 3 μmol) to the above mixed solution. The final concentration of the substrate peptide shown in SEQ ID NO: 1 is 1 mM. The system is a colorless and transparent liquid. Place it in an ultrasonic bath (frequency 20 kHz, power density 1 W / mL, pulse mode 50% duty cycle) at 40 °C in the dark for 3 h. Then, add 10 mM sodium ascorbate to terminate the reaction. Perform UPLC analysis on the reaction system. The product percentage (Area%) is 54%. The UPLC chromatogram is shown below. Figure 4 As shown.

[0109] Example 5

[0110] pH screening for one-step amidation reaction of TCEP under ultrasound promotion:

[0111] The following reaction systems were prepared at room temperature:

[0112] Reaction system 1: 3 mL of a mixed aqueous solution of 0.2 M NaH2PO4 and 50 mM TCEP, adjusted to pH 1.0 with 4 M NaOH solution;

[0113] Reaction system 2: a mixed aqueous solution of 3 mL 0.2 M NaH2PO4 and 50 mM TCEP, adjusted to pH 5.0 with 4 M NaOH solution;

[0114] Reaction system 3: a mixed aqueous solution of 3 mL 0.2 M NaH2PO4 and 50 mM TCEP, adjusted to pH 8.0 with 4 M NaOH solution;

[0115] Reaction system 4: a mixed aqueous solution of 3 mL 0.2 M NaH2PO4 and 50 mM TCEP, adjusted to pH 9.0 with 4 M NaOH solution;

[0116] The substrate peptide shown in SEQ ID NO: 1 was added to the above system to achieve a final concentration of 1 mM, followed by the addition of TiO2 (1.5 mg, 20 nm particle size). The solution was then placed in an ultrasonic bath at 37°C. o C reaction, detected after 4 hours.

[0117] The results of the crude product liquid chromatography spectrum show that:

[0118] Under pH=1 conditions, the target product accounts for 1%, the desulfurization product accounts for 3%, and the raw material accounts for 95%.

[0119] Under pH=5.0 conditions, the target product accounts for 3%, the desulfurization product accounts for 22%, and the raw material accounts for 63%.

[0120] Under pH=8.0 conditions, the target product accounts for 16%, the desulfurization product accounts for 11%, and the raw material accounts for 49%.

[0121] Under pH=9.0 conditions, the target product accounts for 39%, the desulfurization product accounts for 2%, and the raw material accounts for 19%.

[0122] It is evident that a higher pH (pH 8.0~9.0) is more conducive to the formation of the target product.

[0123] The UPLC chromatograms of different reaction systems at different pH values ​​are shown below. Figures 5-8 As shown.

[0124] Example 6

[0125] Under the aforementioned optimal conditions (the reaction system in Example 1), the reactivity of the universal peptide was investigated based on the difference in the penultimate amino acid X of the NC-terminal polypeptide. The amino acid sequence of the "universal peptide" is GKVFXC-OH (SEQ ID NO: 2), where X represents any other amino acid, including one of Ala, Pro, Asp, Asn, Lys, Met, Ser, Trp, Tyr, Arg, or His in this example. The reaction formula is shown below:

[0126] .

[0127] The proportion of products in the reaction system after the reaction was completed was detected, and the results are shown in Table 1.

[0128] Table 1

[0129]

[0130] Example 7

[0131] Penicillamine or selenocysteine ​​has similar properties to cysteine, so we tried to replace cysteine ​​with these two amino acids.

[0132] The reaction system in this embodiment is the same as that in Example 1. The proportion of products in the reaction system after the reaction is completed is detected, and the results are shown in Table 2.

[0133] Table 2

[0134]

[0135] Example 8

[0136] The amidation reaction described in this application can also occur when the C-terminus of the peptide used is an amide of cysteine ​​instead of a carboxylic acid. The results are shown in Table 3.

[0137] Table 3

[0138]

[0139] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: This application utilizes ultrasonic treatment at a pH of 8.0-9.0, preferably 9.0, and employs TCEP as a reducing agent to selectively and synergistically desulfurize and decarboxylate the C-terminal cysteine ​​of peptides, enabling the direct one-step acquisition of the C-terminal amidated product within a short time. Compared with previously used methods, this process route avoids enzyme catalysis and photocatalysis steps, has a wider substrate applicability, and is particularly suitable for peptides with low activity under enzymatic methods where the C-terminal cysteine ​​is adjacent to a proline. Obtaining the target product through a one-step reaction is simple, significantly improves reaction yield, reduces production costs, and is suitable for large-scale production. It provides a concise and efficient new method for synthesizing C-terminal amidated peptides, further providing technical support for the synthesis of peptide drugs.

[0140] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for ultrasound-assisted C-terminal amidation of a polypeptide, characterized in that, The method includes: The reaction system is obtained by mixing the substrate peptide, reducing agent and buffer solution; The reaction system was subjected to ultrasonic treatment to induce a reaction, thereby obtaining a product with C-terminal amidation of the polypeptide. The reducing agent includes one or more of tris(2-carbonylethyl)phosphine hydrochloride, sodium triphenylphosphine tri-m-sulfonate, or triphenylphosphine; The structure of the substrate polypeptide and the amino acid residues at the C-terminus of the substrate polypeptide is shown below: Formula I; The structural formulas of the product and the amino acid residues at the C-terminus of the product are shown below: Formula II; Among them, R 1 For H or Me; R 2 The sequence of amino acids in the substrate polypeptide, excluding the C-terminal amino acid; R is the sequence of amino acids in the product other than the C-terminal amino acid; X 1 It is SH or SeH; X 2 It can be OH or NH2.

2. The method according to claim 1, characterized in that, The frequency of the ultrasonic treatment is 20-100 kHz.

3. The method according to claim 2, characterized in that, The power density of the ultrasonic treatment is 0.5-2 W / mL.

4. The method according to claim 3, characterized in that, The pulse mode of the ultrasonic treatment has a duty cycle of 30-80%.

5. The method according to claim 1, characterized in that, The reducing agent includes tris(2-carbonylethyl)phosphohydrochloride.

6. The method according to claim 1, characterized in that, The concentration of the substrate polypeptide is 0.1~2 mM.

7. The method according to claim 1, characterized in that, The concentration of the reducing agent is 20~200 mM.

8. The method according to claim 1, characterized in that, The reaction temperature is 25~45℃.

9. The method according to claim 8, characterized in that, The reaction time is 2 to 15 hours.

10. The method according to claim 9, characterized in that, The reaction time is 2-5 hours.

11. The method according to claim 9, characterized in that, The pH of the reaction is 8.0 to 9.

0.

12. The method according to claim 1, characterized in that, The buffer solution includes NaH2PO4 or KH2PO4.

13. The method according to claim 12, characterized in that, The concentration of the buffer solution is 0.1~5 M.

14. The method according to claim 1, characterized in that, The method further includes: subjecting the reaction system containing the substrate peptide, the reducing agent, the buffer solution, and the auxiliary agent to ultrasonic treatment to obtain the product with C-terminal amidation of the peptide.

15. The method according to claim 14, characterized in that, The additives include TiO2.

16. The method according to claim 15, characterized in that, The concentration of the adjuvant is 0.1~2 mg / mL.

17. The use of the ultrasound-assisted C-terminal amidation method of any one of claims 1 to 16 in the preparation of polypeptide drugs.

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