A method for solid phase synthesis of N-methylated polypeptides

Abstract

The application discloses a solid-phase synthesis method of N-methylated polypeptide and belongs to the technical field of polypeptide synthesis. The application specifically relates to a solid-phase synthesis method of N-methylated polypeptide, which comprises the following steps: after N-methyl amino acid is coupled on a solid-phase synthesis, the next amino acid of the N-methyl amino acid is coupled under catalysis of a carbodiimide type coupling agent. The application provides a simple and practical technical route for preparation of N-methylated polypeptide and has a wide application in the technical field of polypeptide synthesis.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide synthesis, and specifically relates to a solid-phase synthesis method for N-methylated polypeptides. Background Technology

[0002] N-methyl amino acids are present in a variety of natural peptides. Their incorporation into bioactive peptides alters their chemical and physical properties, and may also lead to enhanced potency, new receptor subtype selectivity, and agonist-antagonist conversion. N-methylated peptides are also potential therapeutic agents, having been shown to improve important pharmacological parameters and are widely used in antibiotics, anticancer agents, antiviral agents, and immunosuppressive activities (Biron.E and Kissler.H. Convenient Synthesis of N-Methylamino Acids Compatible with Fmoc Solid-Phase Peptide Synthesis. J.Org.Chem. 2005, 70, 5183-5189).

[0003] However, N-methyl amino acids and their derivatives are relatively difficult to couple due to their large steric hindrance, making the synthesis of N-methylated peptides challenging (Ali.FE, Bennett.DB, Calva.RR, et al. Conformationally Constrained Peptides and Semipeptides Derived from RGD as Potent Inhibitors of the Platelet Fibrinogen Receptor and Platelet Aggregation. J. Med. Chem. 1994, 37, 769-780).

[0004] In 1999, Akaji's group successfully synthesized dolastatin 15, a depeptide isolated from Indian Ocean sea hare, using 2-chloro-1,3-dimethyl-2-methylimidazoline hexafluorophosphate (CIP) as an effective coupling agent. This resulted in the synthesis of dolastatin 15 containing an N-methyl fragment 2 and a pyrrolidone fragment 3. (Akaji.K, Hayashi.Y, Kiso.Y. Convergent Synthesis of Dolastatin 15 by Solid Phase Coupling of an N-Methylamino Acid. J. Org. Chem. 1999, 64, 405-411).

[0005] In 2005, Teixido's group discovered that PyAop / PyBop and HOAT are a good condensation system for coupling N-methyl amino acids (Teixido'.M, Albericio.F and Giralt E. Solid-phase synthesis and characterization of N-methyl-rich peptides.J.Peptide Res., 2005, 65, 153-166.).

[0006] In 2015, Adamska's group reported that the triazine-based coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine toluene-4-sulfonate could be used to synthesize N-methylated peptides with lower yields and fewer byproducts compared to standard coupling agents (such as TBTU or HATU) (Adamska.A.). B, Kluczyk. A et al. Synthesis of linear and cyclic opioid-based peptide analogs containing multiple N-methylated amino acid residues. J. Pept. Sci. 2015, 21, 807-810).

[0007] However, these methods share a common drawback: they are prone to racemization or the formation of byproducts such as diketopiperazine during the coupling reaction, resulting in low yields, complex post-processing, and relatively expensive coupling reagents. Summary of the Invention

[0008] This invention discloses a method for synthesizing N-methylated peptides, belonging to the field of peptide synthesis technology. These compounds are important synthetic intermediates and possess a wide range of biological activities. Under the catalysis of inexpensive and readily available carbodiimide-type coupling reagents, an amide coupling reaction occurs, yielding a series of high-yield, high-purity N-methylated peptides. This invention provides a simple and practical technical route for the preparation of N-methylated peptides, with wide applications in the field of peptide synthesis technology.

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for synthesizing N-methylated polypeptides, wherein after coupling an N-methyl amino acid in a solid-phase synthesis, the next amino acid of the N-methyl amino acid is coupled under the catalysis of a carbodiimide-type coupling agent.

[0010] In some embodiments of the present invention, the carbodiimide coupling agent is selected from DIC / HOAT, DCC / HOAT, or EDC / HOAT. In some specific embodiments of the present invention, the carbodiimide coupling agent is DIC / HOAT.

[0011] In some embodiments of the present invention, when coupling the next amino acid after the N-methyl amino acid, the amount of amino acid raw material added is 1 eq to 10 eq, preferably 5 eq; the concentration is 0.1 M to 1.5 M, preferably 0.5 M. In other specific embodiments, when coupling the next amino acid after the N-methyl amino acid, the amount of amino acid raw material added can be selected from 1 eq, 2 eq, 3 eq, 4 eq, 5 eq, 6 eq, 7 eq, 8 eq, 9 eq, or 10 eq; the concentration of the added amino acid raw material can be selected from 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, or 1.5 M. In some specific embodiments of the present invention, when coupling the next amino acid after the N-methyl amino acid, the amount of amino acid raw material added is 5 eq, and the concentration is 0.5 M.

[0012] In some embodiments of the present invention, when coupling the next amino acid after the N-methyl amino acid, the coupling reaction temperature is 20°C to 100°C, preferably 30°C. In other embodiments, the coupling reaction temperature can be selected from 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, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C. In one specific embodiment of the present invention, the coupling reaction temperature is 30°C.

[0013] In some embodiments of the present invention, the coupling reaction time is 1 h to 48 h, preferably 18 h, when coupling the amino acid following the N-methyl amino acid. In other embodiments, the coupling reaction time can be selected from 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, or 48 h. In some specific embodiments of the present invention, the coupling reaction time is 18 h.

[0014] In some embodiments of the present invention, the coupling agent for the coupling steps other than the amino acid following the N-methyl amino acid is selected from DIC / HOBT, DIC / HOAT, PyBOP / HOBT / DIEA, PyBOP / HOAT / DIEA, HATU / HOBT / DIEA, HATU / HOAT / DIEA, HBTU / HOBT / DIEA, or TBTU / HOBT / DIEA. In some specific embodiments of the present invention, the coupling agent for the other coupling steps is DIC / HOBT.

[0015] In some embodiments of the present invention, the solid-phase synthesis includes sequentially coupling N-methylated polypeptides onto a resin in the order of N-terminus to C-terminus to obtain an N-methylated polypeptide-resin.

[0016] In some embodiments of the present invention, after obtaining the N-methylated polypeptide-resin, a further step of cleavage with a cleavage reagent is included, wherein the cleavage reagent is a mixture of at least one solvent selected from EDT, phenol, anisole, and H2O with TFA. In some specific embodiments of the present invention, the cleavage reagent is a mixture of TFA, EDT, phenol, anisole, and H2O in a volume ratio of 87.5:5:2.5:2.5:2.5.

[0017] In some embodiments of the present invention, the cleavage time of the cleavage reagent for N-methylated peptide-resin is 1 h to 4 h, preferably 2.5 h. In other specific embodiments, the cleavage time can be selected from 1 h, 1.3 h, 1.5 h, 1.8 h, 2 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h, 3 h, 3.2 h, 3.4 h, 3.6 h, 3.8 h, or 4 h. In one specific embodiment of the present invention, the cleavage time is 2.5 h.

[0018] This invention also provides a solid-phase synthesis method for N-methylated peptides, comprising the following steps:

[0019] (1) After the N-terminal protecting group is removed from the resin, the corresponding amino acids are coupled sequentially from the N-terminus to the C-terminus according to the sequence of the N-methylated polypeptide until the N-methyl amino acid is attached.

[0020] (2) Add the next amino acid source of N-methyl amino acid and a carbodiimide-type coupling agent to the resin peptide obtained in step (1) and couple it under appropriate conditions.

[0021] (3) The resin peptide obtained in step (2) is further coupled with the remaining amino acids in sequence to obtain the target resin peptide;

[0022] (4) Add a cleavage reagent to lyse the resin peptide, filter and precipitate to obtain crude N-methylated peptide.

[0023] In some embodiments of the present invention, the carbodiimide coupling agent in step (2) above is selected from DIC / HOAT, DCC / HOAT, or EDC / HOAT. In some specific embodiments, the carbodiimide coupling agent is DIC / HOAT.

[0024] In some embodiments of the present invention, in step (2) above, the amount of the amino acid material input for the next amino acid after the N-methyl amino acid is 1 eq to 10 eq, preferably 5 eq; the concentration is 0.1 M to 1.5 M, preferably 0.5 M. In other specific embodiments, when coupling the next amino acid after the N-methyl amino acid, the amount of amino acid material input can be selected from 1 eq, 2 eq, 3 eq, 4 eq, 5 eq, 6 eq, 7 eq, 8 eq, 9 eq, or 10 eq; the concentration of the amino acid material input can be selected from 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, or 1.5 M. In some specific embodiments of the present invention, in step (2), the amount of the amino acid material input for coupling the next amino acid after the N-methyl amino acid is 5 eq, and the concentration is 0.5 M.

[0025] In some embodiments of the present invention, suitable conditions for step (2) include a reaction at a temperature of 20°C to 100°C for 1 to 48 hours. The preferred reaction temperature is 30°C, and other temperatures can be selected from 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, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 45°C, 50°C, 55°C, 56°C, 60°C, 65°C, 70°C, 75°C, 80°C, 80°C, 10 ... The reaction temperature can be 0℃, 85℃, 90℃, 95℃, or 100℃; the preferred reaction time is 18h, but other reaction times can be selected from 1h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, or 48h. In some specific embodiments of the present invention, a suitable condition for step (2) is a reaction at 30℃ for 18h.

[0026] To obtain a series of high-yield, high-purity N-methylated peptides, the specific technical solution adopted in this invention is as follows: The synthesis of N-methylated peptides is optimized by synthesizing the Ac-LQT{NME-LEU}RDIQR{NLE}L{L-2-NAL}-NH2 model peptide, including the following steps:

[0027] (1) Using Rink resin as the starting resin, amino acids with protecting groups were sequentially coupled using solid-phase synthesis. DIC / HOBT coupling reagent was used. After the reaction was complete, the mixture was washed, and Fmoc was removed using 20% ​​pip / DMF. After washing again, the next protected amino acid was coupled to obtain the raw resin peptide, as follows:

[0028]

[0029] (2) Add a certain concentration of protective amino acid Fmoc-Thr(tBu)-OH and coupling agent to the resin peptide in step (1), react at a certain temperature for a certain time, and after the reaction is complete, wash and remove Fmoc using 20% ​​pip / DMF.

[0030]

[0031] (3) The resin peptide from the previous step is coupled sequentially with amino acids having protecting groups using solid-phase synthesis. DIC / HOBT coupling reagent is used. After the reaction is complete, the peptide is washed and Fmoc is removed using 20% ​​pip / DMF. After washing again, the next protected amino acid is coupled until the last amino acid Fmoc-Leu-OH is coupled. Finally, the peptide is acetylated under the action of acetic anhydride and washed to obtain the target resin peptide, as shown below.

[0032]

[0033] (4) Add a cutting agent to lyse the resin. After a certain period of time, add diethyl ether to the filtered mother liquor to precipitate it. Centrifuge to obtain crude N-methyl amino acid polypeptide.

[0034] (5) Dissolve the crude polypeptide obtained in the previous step and separate and purify it using high performance liquid chromatography.

[0035] In steps (1) and (3), after adding an amino acid with a protecting group, the reaction is carried out at 25°C for 1 hour. The reaction is detected by ninhydrin reagent. If the result is negative, the reaction is complete.

[0036] In step (2), the coupling reagent is DIC / HOBT, DIC / HOAT, PyBOP / HOBT / DIEA, PyBOP / HOAT / DIEA, HATU / HOBT / DIEA, HATU / HOAT / DIEA, HBTU / HOBT / DIEA, TBTU / HOBT / DIEA, preferably DIC / HOAT; the specific concentration is 0.1M to 1.5M, preferably 0.5M; the specific temperature is 20 to 100℃, preferably 30℃; the specific time is 1 to 48h, preferably 18h.

[0037] In step (4), the cutting reagent is TFA, EDT, phenol, anisole sulfide and H2O in a ratio of 87.5:5:2.5:2.5:2.5; the time is 1 to 4 hours, preferably 2.5 hours.

[0038] This invention relates to peptide chains synthesized one-to-one by solid-phase synthesis. The amino acids added in steps (1) and (3) require protection and commonly used chemical reagents and condensation reagents, as shown in Table 1:

[0039] Table 1 Commonly Used Chemical Reagents

[0040]

[0041]

[0042] Terminology Explanation

[0043] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0044] In this invention, the term "polypeptide" or "peptide" has its common meaning in the art and can refer to an amide from two or more aminocarboxylic acid molecules (identical or different) that forms a covalent bond by the formal loss of water from the carbonyl carbon of one aminocarboxylic acid molecule to the nitrogen atom of another aminocarboxylic acid molecule. "Amino acid residue" also has its common meaning in the art and refers to an amino acid (as a single amino acid or as part of a peptide) in combination with a peptide, another amino acid, or another amino acid residue. Generally, when an amino acid combines with another amino acid or another amino acid residue, the water is removed, and the remaining amino acid is called an amino acid residue.

[0045] The "N-methylated polypeptide" described in this invention refers to a class of polypeptide molecules containing N-methyl amino acid residues in their polypeptide chains. N-methylation is an important method for modifying the peptide backbone. Many natural polypeptide products (extracted from plants, marine organisms, or microorganisms) contain N-methylated structures. N-methylated polypeptides convert the pure trans configuration of many peptide bonds into a configuration with a certain cis-trans ratio; increase the steric hindrance of peptide bonds; reduce the hydrogen bonds within the peptide chain; increase the basicity of the carbonyl group linked to the N-methylated peptide bond; and decrease its polarity. These structural changes all lead to significant alterations in the pharmacological activity of the modified polypeptide. However, the substitution of the active hydrogen of the amino group with a methyl group results in greater steric hindrance, reduced activity, and difficulty in coupling the next amino acid to the N-methyl amino acid, thus making the synthesis of N-methylated polypeptides challenging.

[0046] The term "amino acid" also has its common meaning in the art and can include protein amino acids and non-protein amino acids. The abbreviations for amino acid residues used in this invention are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0047] The term "protecting group" or "protecting base" as used in this invention has its common meaning in the art. A protecting group includes a reactive group (i.e., a protected group) attached to or configured to be attached to a molecule (e.g., a peptide) such that the protecting group prevents or otherwise inhibits the participation of the protected group in a reaction. Protection can be achieved by attaching a protecting group to a molecule. Deprotection can occur when the protecting group is removed from the molecule, for example, by a chemical transformation that removes the protecting group.

[0048] The "solid-phase synthesis" described in this invention refers to a synthetic method in which reactants are attached to an insoluble solid support. Solid-phase synthesis of peptides involves first attaching one amino acid to the insoluble solid support, and then attaching other amino acids one at a time to the amino acids already attached to the solid support. In solid-phase peptide synthesis, resins are typically used as the solid support. The process usually involves covalently attaching the C-terminus of the first amino acid of the target peptide to the solid support, then using the N-terminus of that amino acid as the starting point for synthesis, reacting by removing the amino protecting group and adding an excess of activated second amino acid to lengthen the peptide chain. This process is repeated until the desired peptide chain length is achieved. Finally, the peptide chain is cleaved from the resin, separated, and purified to obtain the target peptide. As is known in the art, resins used for solid-phase peptide synthesis include, but are not limited to, polystyrene-ethylene crosslinking resins, polyacrylamide resins, polyethylene glycol resins, and their derivatives. These resins, after introducing reactive groups, can directly attach the (first) amino acid. Depending on the introduced reactive groups, these resins and resin derivatives can be classified as chloromethyl resins, carboxyl resins, amino resins, or hydrazide-type resins. Currently, commonly used resins in this field include, but are not limited to, PAM resin, MBHA resin, Wang resin, Rink resin, and TRT resin. In some embodiments of the present invention, the resin used is a resin derivative obtained by coupling the first amino acid at the N-terminus of the target N-methylated polypeptide with the resin, such as Fmoc-{L-2-NAL}-Rink resin and Fmoc-Lys-2-Cl-TRT resin.

[0049] In this invention, "coupling" refers to the process of adding a new amino acid to a bound amino acid or peptide. Peptide synthesis includes solid-phase synthesis, liquid-phase synthesis, and a combination of solid-phase and liquid-phase methods have also been reported. In this art, coupling can be performed by sequentially coupling one amino acid at a time, or by segmental coupling of multiple peptide fragments according to the peptide sequence. In this embodiment of the invention, the peptide preparation uses a solid-phase method, and the coupling is performed sequentially.

[0050] The "coupling reagent," "condensation reagent," or "coupling agent" mentioned in this invention refers to reagents used for amino acid coupling in peptide solid-phase synthesis. In peptide solid-phase synthesis, different coupling methods and / or different coupling reagents can be selected based on different resins, different amino acid sequences, and / or different protecting groups. Coupling reagents include, but are not limited to, carbodiimide-type and onium salt-type reagents. Carbodiimide-type reagents mainly include DCC, DIC, EDC, etc. Carbodiimide-type coupling reagents can be used alone or in combination with other condensation reagents. When carbodiimide-type condensation reagents are used in combination with HOBT, HOAT, etc., side reactions can be controlled to a very low level. Onium salt-type condensation reagents mainly include HBTU, HATU, PyBOP, etc. During use, organic bases for activating amino acids, such as DIPEA, can be added to onium salt-type condensation reagents.

[0051] In this invention, when multiple condensing agents are used, these condensing agents can be used in combination. In this invention, the coupling agent can be DIC, DIC / HOAT, DIC / HOBT, HATU / DIPEA, HATU / HOBT / DIPEA, HBTU / HOBT / DIPEA, or PyBOP / HOBT / DIPEA. Wherein, "DIC / HOAT" represents a combination of DIC and HOAT, "DIC / HOBT" represents a combination of DIC and HOBT, "HATU / DIPEA" represents a combination of HATU and DIPEA, "HATU / HOBT / DIPEA" represents a combination of HATU, HOBT, and DIPEA, "HBTU / HOBT / DIPEA" represents a combination of HBTU, HOBT, and DIPEA, and "PyBOP / HOBT / DIPEA" represents a combination of PyBOP, HOBT, and DIPEA. Those skilled in the art will understand that using two or more condensing agents in combination means adding the two or more condensing agents in a single coupling reaction. The combination of two or more condensing agents is within the knowledge of those skilled in the art. Those skilled in the art know how to add the two or more condensing agents in a single coupling reaction, including the amount, timing, and order of addition. In this invention, multiple combinations of coupling agents can be used during coupling with a single raw material (which may involve coupling one amino acid, coupling a short peptide with two or more amino acids, or coupling part or all of a side chain). For example, in this invention, the coupling agents used can be two or more combinations of DIC, DIC / HOAT, DIC / HOBT, HATU / DIPEA, HATU / HOBT / DIPEA, HBTU / HOBT / DIPEA, and PyBOP / HOBT / DIPEA. When using two or more combinations of condensing agents, as those skilled in the art know, multiple coupling reactions can be performed sequentially, with each coupling reaction using one of the condensing agent combinations.

[0052] Beneficial effects: This invention provides a new, efficient, and inexpensive condensation system for synthesizing N-methylated peptides. Under the DIC / HOAT condensation system, the reaction is carried out at 30°C for 18 hours to obtain crude peptides with high yield and high purity. This method is simple to operate, highly efficient, and low-cost for synthesizing N-methylated peptides. Attached Figure Description

[0053] Figure 1 Here is the 1MS spectrum of the compound;

[0054] Figure 2 The HPLC chromatogram of compound 1 is shown below.

[0055] Figure 3The MS spectrum of compound 2;

[0056] Figure 4 The HPLC chromatogram of compound 2 is shown below.

[0057] Figure 5 The MS spectrum of compound 3 is shown below.

[0058] Figure 6 This is the HPLC chromatogram of compound 3. Detailed Implementation

[0059] The present invention will be further described below with reference to specific embodiments. The following non-limiting embodiments 1 to 3 are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims are within the scope of protection of the present invention.

[0060] Example 1 Solid-phase synthesis of N-methylated polypeptide (compound 1)

[0061] This embodiment focuses on the synthesis and purification of Ac-LQT{NME-LEU}RDIQR{NLE}L{L-2-NAL}-NH2. These amino acids were synthesized using solid-phase synthesis with Fmoc protection of the α-amino group. The specific synthesis steps are as follows:

[0062] 1. Weigh 1g of Fmoc-{L-2-NAL}-Rink resin and perform [Operation A], i.e., add 20% Pip / DMF to remove the N-terminal Fmoc protecting group, react at 25℃ for 20min, wash 5 times with DMF after the reaction, and detect with ninhydrin reagent. A positive result indicates that the reaction is complete. Then perform [Operation B], i.e., add a mixed solution containing 1.5mmol DIC, 1.5mmol HOBT, and 1.5mmol of an amino acid with a protecting group, at a concentration of 0.5M, react at 25℃ for 1 hour. After the reaction is complete, a negative result with ninhydrin indicates that the reaction is complete, and then wash 3 times with industrial DMF. Subsequent steps are performed alternately with [Operation A] and [Operation B]. As the synthesis proceeds, only the corresponding amino acid added in [Operation B] is changed. The reaction continues until it is linked to {NME-LEU}. The reaction is complete when the ninhydrin solution is colorless. The above reaction yields the intermediate peptide chain. The molecular weight is confirmed by MS to obtain the model resin peptide, as shown in the figure.

[0063]

[0064] 2. Optimization of the condensation scheme for Fmoc-Thr(tBu)-OH in N-methylated peptides

[0065] 2.1 Based on the resin peptide obtained in step 1, 100 mg was taken and [Operation A] was performed again, followed by [Operation B]. In this operation, a mixed solution containing 5 eq DIC, 5 eq HOAT, and 5 eq Fmoc-Thr(tBu)-OH with a protecting group was added at a concentration of 0.5 M. The reaction was carried out at 25 °C for 12 hours. After the reaction was completed, [Operation A] was performed again. After washing with DMF 5 times and drying, the resin peptide was obtained. It was then lysed with reagent K (i.e., TFA∶EDT∶phenol∶anisole∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide was obtained. The structure was identified by MS and HPLC, the molecular weight was correct, and the purity of the crude product was 62%.

[0066] 2.2 Based on the resin peptide obtained in (1), take 100 mg and perform [Operation A] again, and then perform [Operation B]. In this operation, add a mixed solution containing 5 eq DIC, 5 eq HOBT, and 5 eq Fmoc-Thr(tBu)-OH with a protecting group, with a concentration of 0.5 M. React at 25 °C for 12 hours. After the reaction is completed, perform [Operation A], wash with DMF 5 times, dry, and then lyse with K reagent (i.e., TFA∶EDT∶phenol∶anisyl thioether∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure is identified by MS and HPLC, the molecular weight is correct, and the purity of the crude product is 31%.

[0067] 2.3 Based on the resin peptide obtained in (1), take 100 mg and perform [Operation A] again, and then perform [Operation B]. In this operation, add a mixed solution containing 5 eq PyBOP, 5 eq HOBT, 10 eq DIPEA and 5 eq amino acids with protecting groups Fmoc-Thr(tBu)-OH, with a concentration of 0.5 M. React at 25 °C for 12 hours. After the reaction is completed, perform [Operation A], wash with DMF 5 times, dry, and then lyse with K reagent (i.e., TFA∶EDT∶phenol∶anisole thioether∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure is identified by MS and HPLC, the molecular weight is correct, and the purity of the crude product is 43%.

[0068] 2.4 Based on the resin peptide obtained in (1), take 100 mg and perform [Operation A] again, and then perform [Operation B]. In this operation, add a mixed solution containing 5 eq PyBOP, 5 eq HOAT, 10 eq DIPEA and 5 eq Fmoc-Thr(tBu)-OH with a protecting group, with a concentration of 0.5 M. React at 25 °C for 12 hours. After the reaction is completed, perform [Operation A], wash with DMF 5 times, dry, and then lyse with K reagent (i.e., TFA∶EDT∶phenol∶anisyl thioether∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure is identified by MS and HPLC, the molecular weight is correct, and the purity of the crude product is 50%.

[0069] 2.5 Based on the resin peptide obtained in (1), 100 mg was taken and [Operation A] was performed again, followed by [Operation B]. In this operation, a mixed solution containing 5 eq HATU, 5 eq HOBT, 10 eq DIPEA, and 5 eq Fmoc-Thr(tBu)-OH with a protecting group was added at a concentration of 0.5 M. The reaction was carried out at 25 °C for 12 hours. After the reaction was completed, [Operation A] was performed again. After washing 5 times with DMF and drying, the peptide was lysed with reagent K (i.e., TFA∶EDT∶phenol∶anisole∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. The crude peptide was obtained by precipitation with ice-cold ether. The structure and molecular weight were identified by MS and HPLC. The purity of the crude peptide was 44%.

[0070] 2.6 Based on the resin peptide obtained in (1), take 100 mg and repeat [Operation A], then proceed to [Operation B]. In this operation, add a mixed solution containing 5 eq HATU, 5 eq HOAT, 10 eq DIPEA and 5 eq Fmoc-Thr(tBu)-OH with a protecting group, with a concentration of 0.5 M. React at 25 °C for 12 hours. After the reaction is complete, repeat [Operation A]. Wash with DMF 5 times, dry the product, and then lyse it with reagent K (i.e., TFA∶EDT∶phenol∶anisole∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure and molecular weight are correct, and the purity of the crude product is 55%, as identified by MS and HPLC.

[0071] 2.7 Based on the resin peptide obtained in (1), take 100 mg and repeat [Operation A], then proceed to [Operation B]. In this operation, add a mixed solution containing 5 eq HBTU, 5 eq HOBT, 10 eq DIPEA and 5 eq Fmoc-Thr(tBu)-OH with a protecting group, with a concentration of 0.5 M. React at 25 °C for 12 hours. After the reaction is complete, repeat [Operation A]. Wash with DMF 5 times, dry the product, and then lyse it with reagent K (i.e., TFA∶EDT∶phenol∶anisole∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure and molecular weight are correct, and the purity of the crude product is 46%, as identified by MS and HPLC.

[0072] 2.8 Based on the resin peptide obtained in (1), take 100 mg and repeat [Operation A], then proceed to [Operation B]. In this operation, add a mixed solution containing 5 eq TBTU, 5 eq HOBT, 10 eq DIPEA and 5 eq Fmoc-Thr(tBu)-OH with a protecting group, with a concentration of 0.5 M. React at 25 °C for 12 hours. After the reaction is complete, repeat [Operation A]. Wash with DMF 5 times, dry the product, and then lyse it with reagent K (i.e., TFA∶EDT∶phenol∶anisole∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure and molecular weight are correct, and the purity of the crude product is 45%, as identified by MS and HPLC.

[0073] Table 2 Optimization of condensation schemes

[0074]

[0075] 3. Optimization of the reaction concentration of Fmoc-Thr(tBu)-OH in N-methylated peptides

[0076] 3.1 Based on the resin peptide obtained in (1), take 100 mg and repeat [Operation A], then proceed to [Operation B]. In this operation, add a mixed solution containing 5 eq DIC, 5 eq HOAT, and 5 eq Fmoc-Thr(tBu)-OH with a protecting group, with a concentration of 0.2 M. React at 25 °C for 12 hours. After the reaction is complete, repeat [Operation A]. Wash with DMF 5 times, dry the product, and then lyse it with reagent K (i.e., TFA∶EDT∶phenol∶anisole∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure and molecular weight are correct as determined by MS and HPLC. The purity of the crude product is 36%.

[0077] 3.2 Based on the resin peptide obtained in (1), take 100 mg and perform [Operation A] again, and then perform [Operation B]. In this operation, add a mixed solution containing 5 eq DIC, 5 eq HOAT, and 5 eq Fmoc-Thr(tBu)-OH with a protecting group, with a concentration of 0.7 M. React at 25 °C for 12 hours. After the reaction is completed, perform [Operation A], wash with DMF 5 times, dry, and then lyse with K reagent (i.e., TFA∶EDT∶phenol∶anisyl thioether∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure is identified by MS and HPLC, the molecular weight is correct, and the purity of the crude product is 60%.

[0078] Table 3 Optimization of reactant concentrations

[0079]

[0080]

[0081] 4. Optimization of reaction temperature for Fmoc-Thr(tBu)-OH in N-methylated peptides

[0082] 4.1 Based on the resin peptide obtained in (1), take 100 mg and perform [Operation A] again, and then perform [Operation B]. In this operation, add a mixed solution containing 5 eq DIC, 5 eq HOAT, and 5 eq Fmoc-Thr(tBu)-OH with a protecting group, with a concentration of 0.5 M. React at 30 °C for 12 hours. After the reaction is completed, perform [Operation A], wash with DMF 5 times, dry, and then lyse with K reagent (i.e., TFA∶EDT∶phenol∶anisyl thioether∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure and molecular weight are correct and the purity of the crude product is 65% by MS and HPLC.

[0083] 4.2 Based on the resin peptide obtained in (1), take 100 mg and perform [Operation A] again, and then perform [Operation B]. In this operation, add a mixed solution containing 5 eq DIC, 5 eq HOAT, and 5 eq amino acids with protecting groups Fmoc-Thr(tBu)-OH, with a concentration of 0.5 M. React at 50 °C for 12 hours. After the reaction is completed, perform [Operation A], wash with DMF 5 times, dry, and then lyse with K reagent (i.e., TFA∶EDT∶phenol∶anisyl thioether∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure and molecular weight are correct and the purity of the crude product is 50% by MS and HPLC.

[0084] Table 4 Optimization of reaction temperature

[0085]

[0086] 5. Optimization of reaction time for Fmoc-Thr(tBu)-OH in N-methylated peptides

[0087] 5.1 Based on the resin peptide obtained in (1), take 100 mg and perform [Operation A] again, and then perform [Operation B]. In this operation, add a mixed solution containing 5 eq DIC, 5 eq HOAT, and 5 eq amino acids with protecting groups Fmoc-Thr(tBu)-OH, with a concentration of 0.5 M. React at 30 °C for 18 hours. After the reaction is completed, perform [Operation A], wash with DMF 5 times, dry, and then lyse with K reagent (i.e., TFA∶EDT∶phenol∶anisyl thioether∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure and molecular weight are correct and the purity of the crude product is 68% by MS and HPLC.

[0088] 5.2 Based on the resin peptide obtained in (1), take 100 mg and perform [Operation A] again, and then perform [Operation B]. In this operation, add a mixed solution containing 5 eq DIC, 5 eq HOAT, and 5 eq amino acids with protecting groups Fmoc-Thr(tBu)-OH, with a concentration of 0.5 M. React at 30 °C for 36 hours. After the reaction is completed, perform [Operation A], wash with DMF 5 times, dry, and then lyse with K reagent (i.e., TFA∶EDT∶phenol∶anisyl thioether∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) for 2.5 h. After precipitation with ice-cold ether, the crude peptide is obtained. The structure is identified by MS and HPLC, the molecular weight is correct, and the purity of the crude product is 60%.

[0089] Table 5 Optimization of reaction time

[0090]

[0091] 6. The N-methyl resin peptide containing Fmoc-Thr(tBu)-OH synthesized in Example 2.1 is continued by alternating steps [A] and [B]. As the synthesis proceeds, only the corresponding amino acid is added in [B]. This continues until the last amino acid is added, then [A] is performed again. Finally, the peptide is acetylated in the presence of acetic anhydride, washed, and the above reaction yields the N-methylated resin peptide.

[0092] 7. Then, the N-methyl amino acid resin peptide was lysed for 2.5 hours using 10 ml of K reagent (i.e., TFA∶EDT∶phenol∶anisole∶water = 87.5∶5∶2.5∶2.5∶2.5, volume ratio) to detach the peptide chain from the resin and remove all protecting groups. The mother liquor was then added to 100 ml of icy diethyl ether to precipitate the peptide. The peptide was centrifuged twice at 3000 rpm for 2 min to obtain the crude peptide.

[0093] 8. Dissolve the crude product in a mixture of water and acetonitrile, and purify it using high-performance liquid chromatography (HPLC). The mobile phase is H₂O / 0.1% TFA, ACN / 0.1% TFA. A gradient elution chromatographic system is used for separation and purification on a C18 preparative column, and the target fraction is collected. The purity of the collected target peak is determined by analytical HPLC. Qualified samples are lyophilized in liquid nitrogen and then freeze-dried in a vacuum freeze dryer to obtain 97% pure N-methylated polypeptide compound 1Ac-LQT{NME LEU}RDIQR{NLE}L{L-2-NAL}-NH₂. The theoretical molecular weight of N-methylated polypeptide compound 1 is 1620.96, and the actual detected molecular weight is 1621.0. Detection was performed at 220 nm using a C18 (4.6*250 mm) 5 μm column with a linear gradient from 5% to 65% over 25 minutes at a rate of 1 mL / min in water (0.065% TFA) and acetonitrile (0.05% TFA). R =22.5min, Ms and HPLC chromatograms are as follows Figure 1 and Figure 2 As shown.

[0094] Example 2 Solid-phase synthesis of N-methylated polypeptide (compound 2)

[0095] This embodiment selects the optimal conditions from Example 1 for the reaction, focusing on the synthesis and purification of Ac-SL{NME-GLN}TLRDIQ{D-ARG}{NLE}L{L-2-NAL}-NH2 (compound 2). These amino acids were synthesized using Fmoc-protected α-amino groups in a solid-phase manner. The specific synthesis steps are as follows:

[0096] Weigh 1g of Fmoc-{L-2-NAL}-Rink resin and perform [Operation A], which is to add 20% Pip / DMF to remove the N-terminal Fmoc protecting group, react at 25°C for 20min, wash 5 times with DMF after the reaction, and detect the reaction with ninhydrin test reagent. If the result is positive, the reaction is complete.

[0097] Then perform [Operation B], which involves adding a mixed solution containing 1.5 mmol DIC, 1.5 mmol HOBT, and 1.5 mmol of an amino acid with a protecting group at a concentration of 0.5 M. React at 25 °C for 1 hour. After the reaction is complete, a negative result is obtained by testing with ninhydrin, indicating that the reaction is complete. Then wash three times with industrial DMF.

[0098] The subsequent steps were performed alternately as [Operation A] and [Operation B]. As the synthesis proceeded, only the corresponding amino acids were added in [Operation B]. The reaction continued until the peptide was ligated to {NME-GLN}. A colorless test with ninhydrin solution indicated the reaction was complete. This process yielded the intermediate peptide chain, and its molecular weight was confirmed by MS.

[0099] Based on the peptide chain obtained above, [Operation A] was performed again, followed by [Operation B]. In this operation, a mixed solution containing 1.5 mmol DIC, 1.5 mmol HOAT, and 1.5 mmol of the amino acid Fmoc-Leu-OH with a protecting group was added at a concentration of 0.5 M. The reaction was carried out at 30 °C for 18 hours. After the reaction was completed, the mixture was washed three times with industrial DMF.

[0100] The process is then repeated alternating between [Operation A] and [Operation B]. As the synthesis proceeds, only the corresponding amino acid is added in [Operation B]. This continues until the last amino acid is added, at which point [Operation A] is repeated. Finally, acetylation is performed in the presence of acetic anhydride. After washing, the N-methylated resin peptide is obtained.

[0101] Then, the resin was lysed for 2.5 hours using 10 ml of a solution of TFA:EDT:phenol:water (92.5%:2.5%:2.5%:2.5%) to detach the peptide chains from the resin and remove all protecting groups. The mother liquor was then added to 100 ml of ice-cold ether to precipitate the peptides. The crude peptides were obtained by centrifugation at 3000 rpm for 2 minutes, repeated twice.

[0102] The crude product was dissolved in a mixture of water and acetonitrile, and then purified by high-performance liquid chromatography (HPLC). The mobile phase was H₂O / 0.1% TFA, ACN / 0.1% TFA, and a C18 preparative column was used for gradient elution chromatography. The target fraction was collected. The purity of the collected target peak was determined by analytical HPLC. Qualified samples were lyophilized in liquid nitrogen and then freeze-dried in a vacuum freeze dryer to obtain 95% pure N-methylated polypeptide compound 2, Ac-SL{NME-GLN}TLRDIQ{D-ARG}{NLE}L{L-2-NAL}-NH₂. The theoretical molecular weight of compound 2 is 1707.99, and the actual detected molecular weight is 1708. Detection was performed at 220 nm using a C18 (4.6*250 mm) 5 μm column with a linear gradient from 5% to 65% over 25 minutes at a rate of 1 mL / min in water (0.065% TFA) to acetonitrile (0.05% TFA). R =20.2 min, MS and HPLC chromatograms are as follows Figure 3 and Figure 4 As shown.

[0103] Example 3 Solid-phase synthesis of N-methylated polypeptide (compound 3)

[0104] This embodiment focuses on the synthesis and purification of RCG{NME-GLY}L. These amino acids were synthesized using solid-phase synthesis with Fmoc protection of the α-amino group. The specific synthesis steps are as follows:

[0105] Weigh 1g of Fmoc-Lys-2-Cl-TRT resin and perform [Operation A], which is to add 20% Pip / DMF to remove the N-terminal Fmoc protecting group, react at 30℃ for 20min, wash 5 times with DMF after the reaction, and detect with ninhydrin test reagent. If the result is positive, the reaction is complete.

[0106] Then perform [Operation B], which involves adding a mixed solution containing 1.5 mmol DIC, 1.5 mmol HOBT, and 1.5 mmol of an amino acid with a protecting group at a concentration of 0.5 M. React at 30 °C for 1 hour. After the reaction is complete, a negative result is obtained by testing with ninhydrin, indicating that the reaction is complete. Then wash three times with industrial DMF.

[0107] The subsequent steps were performed alternately as [Operation A] and [Operation B]. As the synthesis proceeded, only the corresponding amino acids were added in [Operation B]. The reaction continued until {NME-GLY} was ligated. A colorless result using ninhydrin solution indicated the reaction was complete. This process yielded the intermediate peptide chain, and its molecular weight was confirmed by MS.

[0108] Based on the resin peptide obtained above, [Operation A] was performed again, followed by [Operation B]. In this operation, a mixed solution containing 1.5 mmol DIC, 1.5 mmol HOAT, and 1.5 mmol of the amino acid Fmoc-Gly-OH with a protecting group was added at a concentration of 0.5 M. The reaction was carried out at 30 °C for 18 hours. After the reaction was completed, the solution was washed three times with industrial DMF.

[0109] The process is then repeated alternately with steps [A] and [B]. As the synthesis proceeds, only the corresponding amino acid is added in step [B]. This continues until the last amino acid is added, at which point step [A] is executed again. This reaction yields the N-methylated resin peptide.

[0110] Then, the resin was lysed for 2.5 hours using 10 ml of a solution of TFA:EDT:phenol:water (92.5%:2.5%:2.5%:2.5%) to detach the peptide chains from the resin and remove all protecting groups. The mother liquor was then added to 100 ml of ice-cold ether to precipitate the peptides. The crude peptides were obtained by centrifugation at 3000 rpm for 2 minutes, repeated twice.

[0111] The crude product was dissolved in a mixture of water and acetonitrile, and purified by high-performance liquid chromatography (HPLC). The mobile phase was H₂O / 0.1% TFA, ACN / 0.1% TFA. A C18 preparative column was used for gradient elution chromatography, and the target fraction was collected. The purity of the collected target peak was determined by analytical HPLC. Qualified samples were lyophilized in liquid nitrogen and then lyophilized in a vacuum freeze dryer to obtain 95% pure N-methylated polypeptide compound 3, RCG{NME-GLY}L. The theoretical molecular weight of compound 3 is 518.63, and the actual detected molecular weight is 518.2. Detection was performed at 220 nm using a C18 (4.6*250 mm) 5 μm column with a linear gradient from 5% to 65% over 25 minutes at a rate of 1 mL / min in water (0.065% TFA) and acetonitrile (0.05% TFA). R =12.6 min, MS and HPLC chromatograms are as follows Figure 5 and Figure 6 As shown.

Claims

1. A solid-phase synthesis method for N-methylated polypeptides, characterized in that, After coupling an N-methyl amino acid to the N-methyl amino acid in solid-phase synthesis, the next amino acid is coupled under the catalysis of a carbodiimide-type coupling agent. The carbodiimide coupling agent is DIC / HOAT. In the coupling of the next amino acid to the N-methyl amino acid, the amount of amino acid raw material is 1 eq to 10 eq, the concentration is 0.1 M to 1.5 M, the coupling reaction temperature is 20℃ to 100℃, and the coupling reaction time is 1 h to 48 h. Among them, except for the amino acid following the N-methyl amino acid, the coupling agents for other coupling steps are selected from DIC / HOBT, DIC / HOAT, PyBOP / HOBT / DIEA, PyBOP / HOAT / DIEA, HATU / HOBT / DIEA, HATU / HOAT / DIEA, HBTU / HOBT / DIEA, or TBTU / HOBT / DIEA.

2. The synthesis method according to claim 1, characterized in that, When coupling the next amino acid to an N-methyl amino acid, the amount of amino acid raw material used is 5 eq; the concentration is 0.5 M.

3. The synthesis method according to claim 1, characterized in that, The coupling reaction temperature is 30°C when coupling the next amino acid after the N-methyl amino acid.

4. The synthesis method according to claim 1, characterized in that, When coupling the next amino acid after the N-methyl amino acid, the coupling reaction time is 18 h.

5. The synthesis method according to claim 1, characterized in that, Except for the amino acid following the N-methyl amino acid, the coupling agent for other coupling steps is DIC / HOBT.

6. The synthesis method according to any one of claims 1 to 5, wherein the solid-phase synthesis comprises sequentially coupling N-methylated polypeptide-resin onto a resin in the order from the N-terminus to the C-terminus of the N-methylated polypeptide sequence.

7. The synthesis method according to claim 6, characterized in that, After obtaining the N-methylated polypeptide-resin, the process further includes a step of cleavage with a cleavage reagent, wherein the cleavage reagent is a mixture of at least one solvent selected from EDT, phenol, anisole thioether and H2O with TFA.

8. The synthesis method according to claim 7, characterized in that, The cutting reagent is a mixture of TFA, EDT, phenol, anisole sulfide and H2O in a volume ratio of 87.5:5:2.5:2.5:2.

5.

9. The synthesis method according to claim 7, characterized in that, The lysis time of the cleavage reagent is 1h to 4h.

10. The synthesis method according to claim 9, characterized in that, The cleavage time of the cleavage reagent was 2.5 h.

11. A solid-phase synthesis method for N-methylated polypeptides, characterized in that, Includes the following steps: (1) After the resin removes the N-terminal protecting group, the corresponding amino acids are coupled sequentially from the N-terminus to the C-terminus according to the sequence of the N-methylated polypeptide until the N-methyl amino acid is attached; (2) Add the next amino acid source of N-methyl amino acid and a carbodiimide-type coupling agent to the resin peptide obtained in step (1) and couple it under appropriate conditions. (3) The resin peptide obtained in step (2) is further coupled with the remaining amino acids in sequence to obtain the target resin peptide; (4) Add a cleavage reagent to lyse the resin peptide, filter and precipitate to obtain crude N-methylated peptide. Wherein, the carbodiimide coupling agent in step (2) is DIC / HOAT; In step (2), the amount of the next amino acid raw material after the N-methyl amino acid is 1 eq to 10 eq; the concentration is 0.1 M to 1.5 M. The suitable reaction conditions for step (2) include a temperature of 20℃~100℃ and a reaction time of 1h~48h.

12. The synthesis method according to claim 11, characterized in that, In step (2), the amount of the next amino acid raw material after N-methyl amino acid is 5 eq; the concentration is 0.5M.

13. The synthesis method according to claim 11 or 12, wherein suitable reaction conditions for step (2) include a reaction at 30°C for 18 hours.

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