Method for synthesizing semaglutide

Abstract

Provided is a method for synthesizing semaglutide. The method mainly comprises: coupling a resin with a protected amino acid and a polypeptide fragment by means of a solid phase synthesis method to obtain a semaglutide peptide resin, and performing cleavage and purification on the semaglutide peptide resin to obtain the semaglutide, wherein the fragment comprises Gly29-Arg30, R1-Lys[AEEA-AEEA-γGlu(OR2)-C18-R3]-OH, Glu15-Gly16, Thr5-Phe6, and His1-Aib2-Glu3-Gly4. The method effectively avoids the occurrence of DKP side reactions, increases the resin weight gain rate to 98% or more, ameliorates the problem of difficult coupling caused by β-sheets, and effectively avoids the generation of impurities. In the crude peptide, the content of [D-His1] semaglutide does not exceed 0.4% and may even be 0%, the content of [D-Glu3] semaglutide does not exceed 0.19%, the contents of [Plus-Gly4] semaglutide, [D-Thr5] semaglutide, and [Plus-Gly16] semaglutide are all 0%, the content of [D-Phe6] semaglutide does not exceed 0.24%, the content of [D-γGlu15] semaglutide does not exceed 0.7%, and the content of [D-γGlu20-3] semaglutide does not exceed 0.29%; the synthesis yield reaches 70% or more; and the maximum single impurity content does not exceed 0.09%, and the total yield is 58% or more. The method greatly reduces costs and facilitates industrial production.

Description

A method for synthesizing smegglutinin Technical Field

[0001] This invention belongs to the field of polypeptide drug preparation, specifically a method for synthesizing a GLP-1 analog, smegglutinin. Background Technology

[0002] Semaglutide, developed by Novo Nordisk, is a long-acting GLP-1 analog primarily used to lower blood sugar in patients with type 2 diabetes and to control weight in overweight patients. Compared to similar hypoglycemic drugs such as liraglutide and dulaglutide, semaglutide has a more significant effect on lowering blood sugar and can achieve better weight loss results. The sequence structure of semaglutide is as follows:

[0003] His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-L ys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0004] The original research company, Novo Nordisk, chose a preparation method combining bio-fermentation and chemical synthesis (CN101910193). The reaction of unprotected linear peptides and side-chain OSu esters can easily lead to reactions at unexpected sites, forming a large number of misjoined peptide impurities, which is not conducive to subsequent separation and purification, and the yield is also relatively low.

[0005] Existing synthetic processes for semaglutide include traditional methods such as stepwise solid-phase peptide coupling and fragment condensation. However, due to the long sequence and numerous hydrophobic amino acids in semaglutide, secondary structures like β-sheets are easily formed during peptide resin synthesis, leading to coupling difficulties, the generation of numerous impurities, and very low condensation efficiency. While fragment condensation solves the coupling difficulties at some sites, the selected fragments cannot avoid DKP side reactions, resulting in low resin weight gain, small synthesis scale, and low yield. Furthermore, the synthesized crude peptides contain many impurities, such as epipeptides and intercalated peptides. These impurities have similar physicochemical properties to semaglutide, significantly increasing the difficulty of separating and purifying the product, leading to a substantial decrease in yield. Small synthesis scale and high impurity content reduce the purity and content of the target peptide, resulting in low yield, significantly increasing costs, and hindering industrial production.

[0006] Impurity control is of great significance for drug quality control. The types and amounts of impurities in a drug are collectively referred to as the impurity profile. Any substance that affects the purity of a drug is collectively called an impurity. Since adverse reactions caused by drugs in clinical use are sometimes related to impurities present in the drug, in addition to the pharmacological activity of the drug itself, the core issue of drug impurity profile control is to standardize impurity research and control them within a safe and reasonable range.

[0007] Therefore, there is an urgent need for a method to prepare smegglutide with fewer types of impurities, lower impurity content, lower synthesis cost, and higher product purity and yield. Summary of the Invention

[0008] In view of the shortcomings of the existing methods for synthesizing semaglutide, in order to overcome the problems of DKP side reactions, coupling difficulties, and resin polycondensation that occur during the synthesis of semaglutide, fundamentally avoid DKP side reactions to the greatest extent, solve the difficulties of stepwise coupling, reduce the generation of impurities that are difficult to remove in the purification process, and ultimately achieve the goal of improving yield and reducing cost, this invention provides a technical solution for synthesizing semaglutide, which mainly includes the following steps:

[0009] 1) By solid-phase synthesis, resin is coupled with protected amino acids and polypeptide fragments to obtain Smeglu peptide resin.

[0010] Among them, bits 29-30 use Gly 29 -Arg 30 Dipeptide fragment,

[0011] The 20th position uses R1-Lys[AEEA-AEEA-γGlu(OR2)-C18-R3]-OH.

[0012] 15-16 bits use Glu 15 -Gly 16 Dipeptide fragment,

[0013] 5-6 bits use Thr 5 -Phe 6 Dipeptide fragment,

[0014] 1-4 use His 1 -Aib 2 -Glu 3 -Gly 4 Tetrapeptide fragment,

[0015] R1, R2, and R3 are protecting groups, respectively.

[0016] 2) Peptide resin cleavage yielded crude smegglutinin peptide;

[0017] 3) Purify to obtain smeglucopyrithione peptide.

[0018] In an embodiment of the present invention, the resin in step 1) is a resin containing benzyl ester bonds.

[0019] In an embodiment of the present invention, the resin in step 1) is a hydroxyl resin.

[0020] Preferably, the resin in step 1) is selected from Wang Resin, DEG-Wang Resin, HMPA-MBHA resin, and HMPA-AM resin.

[0021] In an embodiment of the present invention, in step 1), Gly 29 -Arg 30 The dipeptide fragment is Fmoc-Gly-Arg(Pbf)-OH.

[0022] When hydroxyl resins such as Wang Resin are used as solid-phase reaction supports, they are sequentially coupled to Fmoc-Arg(Pbf)-Gly-Wang Resin. After deprotection, the α-amino group of Arg30 residue is in a free state. This freed α-amino group immediately undergoes intramolecular aminolysis of the benzyl ester bond in the Wang Resin and other hydroxyl resins, generating a six-membered ring diketopiperazine derivative, which is simultaneously released from the resin, leading to reaction termination (DKPS). This reaction is very rapid, which significantly reduces the yield and increases production costs, resulting in decreased economic efficiency.

[0023] During peptide chain elongation, H-Arg(Pbf)-Gly-Wang Resin exhibits a significant tendency for diketopiperazine cyclization side reactions. The Gly of this invention... 29 -Arg 30 Using the dipeptide fragment Fmoc-Gly-Arg(Pbf)-OH can bypass the DKPS sensitive site, avoid the occurrence of the six-membered ring diketopiperazine side reaction, and significantly improve the synthesis yield.

[0024] In an embodiment of the present invention, in step 1), the 20th position adopts Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH.

[0025] Using this fragment directly avoids the risks of introducing palladium (a heavy gold salt) during Lys(Alloc) deprotection or the introduction of genotoxic hydrazine during Lys(IVDde) deprotection, while also avoiding the risks associated with the derivatized peptide [D-γGlu]. 20-3 The formation of impurities such as smegglutinin. The structures of these impurities are as follows:

[0026] [D-γGlu 20-3 Smegglutinin:

[0027] His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gl y16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-D-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0028] In an embodiment of the present invention, Glu in step 1) 15 -Gly 16 The dipeptide fragment is Fmoc-Glu(OtBu)-Gly-OH.

[0029] Using this fragment effectively avoids the insertion of the peptide [Plus-Gly]. 16 Smegglutinin, D-γGlu 15 The generation of smegglutinin greatly reduces the difficulty of purification. The structure of the impurity is as follows:

[0030] [Plus-Gly 16 Smegglutinin:

[0031] His1-Aib2-Glu3-Gly4-Thr5-D-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gl y-Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0032] [D-γGlu 15 Smegglutinin:

[0033] His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-D-γGlu15- Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0034] In an embodiment of the present invention, Thr in step 1) 5 -Phe 6 The dipeptide fragment is Fmoc-Thr(tBu)-Phe-OH.

[0035] Using this fragment reduces the amount of the differential peptide [D-Thr] 5 Smegglutinin, D-Phe 6 The production of smegglutinin.

[0036] [D-Thr 5 Smegglutinin:

[0037] His1-Aib2-Glu3-Gly4-D-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15- Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0038] [D-Phe 6 Smegglutinin:

[0039] His1-Aib2-Glu3-Gly4-Thr5-D-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15- Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0040] In an embodiment of the present invention, His in step 1) 1 -Aib 2 -Glu 3 -Gly 4 The tetrapeptide fragment is either Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH or Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-OH.

[0041] Differential peptide [D-His] 1 The physicochemical properties of smegglutinin and its target peptide are similar, greatly increasing the difficulty of separating and purifying smegglutinin products, resulting in a significant decrease in product yield. Using a tetrapeptide fragment effectively avoids the [D-His] difference. 1 The formation of smegglutinin impurities also reduced the amount of the differential peptide [D-Glu]. 3 Smegglutinin, an intercalation peptide [Plus-Gly] 4 [Smigratide, intramolecular cyclization side reaction impurities. It also solves the problem of difficult coupling of Aib and His, avoiding the formation of deletion peptide impurities. The impurity structure is as follows:]

[0042] [D-His 1 Smegglutinin:

[0043] D-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15- Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0044] [D-Glu 3 Smegglutinin:

[0045] His1-Aib2-D-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15- Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0046] [Plus-Gly 4 Smegglutinin:

[0047] His1-Aib2-Glu3-Gly-Gly4-Thr5-D-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu1 5-Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala 24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH

[0048] In an embodiment of the present invention, the degree of resin substitution in step 1) is 0.3 to 0.5 mmol / g.

[0049] In an embodiment of the present invention, the condensing agent used in step 1) for coupling is selected from HOBt, DIC, DMAP, Oxyma, HOPO, HBTU, DIPEA, and PyBOP.

[0050] In the embodiments of the present invention, the condensing agent used for coupling in step 1) is selected from the combination of HOBt, DIC, and DMAP; the combination of Oxyma and DIC; the combination of HOPO and DIC; the combination of HOBt, DIPEA, and TBTU; the combination of HBTU and DIPEA; the combination of HOBt, DIPEA, and HBTU; and the combination of PyBOP, HOBt, and DIPEA.

[0051] Furthermore, the amount of the condensing agent is 0.9 to 3.0 times the molar amount of the amino acid, and the reaction time is 2 to 6 hours.

[0052] In an embodiment of the present invention, the reaction solvent used for fragment coupling in step 1) is selected from 1 to 2 of DMF and DCM.

[0053] Furthermore, the volume percentage of DCM in the reaction solvent is 10% to 90%.

[0054] The coupling process uses a mixed solvent of DMF and DCM, which not only increases the solubility of the fragments but also effectively reduces the formation of diffusive peptides, avoids the formation of difficult-to-purify impurities, and greatly improves the purity and purification yield of the final crude peptide.

[0055] In an embodiment of the present invention, the pyrolysis reagent in step 2) is selected from trifluoroacetic acid and other components, and the other components are selected from 1 to 4 of purified water, phenol, 3-mercaptopropionic acid, triisopropylsilane, and m-cresol.

[0056] Furthermore, the lysis reagent contains more than 85% trifluoroacetic acid, with other components each accounting for 1% to 5%.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 1. The method of the present invention improves the problem of difficult coupling caused by β-sheet, effectively avoids the generation of missing peptide impurities, and greatly improves the purity and synthesis yield of the final crude peptide. The purity of the crude peptide is over 70%, and the synthesis yield reaches over 70%.

[0059] 2. The method of the present invention effectively avoids the occurrence of DKP side reactions, improves the resin weight gain rate and the total product yield, with the resin weight gain rate reaching over 98% and the total yield over 58%.

[0060] 3. The method of this invention improves the yield of smegglutinin while effectively inhibiting or reducing [D-His] 1 Smegglutinin, D-Glu 3 Smegglutinin, [Plus-Gly] 4 Smegglutinin, [D-Thr] 5 Smegglutinin, D-Phe 6 Smegglutinin, [D-γGlu] 15 Smegglutinin, [Plus-Gly] 16 Smegglutinin, [D-γGlu] 20-3 The formation of eight impurities, including smegglutinin. In the crude peptide: [D-His...] 1 The content of smegglutinin does not exceed 0.4%, and may even be 0%; [D-Glu] 3 The content of smegglutinin does not exceed 0.19%, [Plus-Gly] 4 The content of smegglutinin is 0%, [D-Thr] 5 The content of smegglutinin is 0, [D-Phe 6 The content of smegglutinin does not exceed 0.24%, [D-γGlu] 15The content of smegglutinin does not exceed 0.7%, [Plus-Gly] 16 The content of smegglutinin is 0%, and [D-γGlu] 20-3 The content of smegglutinin does not exceed 0.29%. This significantly reduces the difficulty of crude peptide purification, greatly improving the purity and yield of smegglutinin. After simple purification steps, these impurities in smegglutinin are basically removed, resulting in a final peptide purity of over 99.5%; the maximum single impurity content does not exceed 0.09%, which is almost negligible; and the overall yield is over 58%. The method of this invention has advantages such as short synthesis cycle, good synthesis effect, high purity, few impurities, high yield, and low cost, reducing synthesis costs and facilitating large-scale industrial production. Attached Figure Description

[0061] Figure 1 is the HPLC chromatogram of crude smegglutinin peptide from Example 1 of the present invention;

[0062] Figure 2 is the HPLC chromatogram of the smegglutinin peptide from Example 1 of the present invention;

[0063] Figure 3 is the HPLC chromatogram of crude smegglutinin from Example 2 of the present invention;

[0064] Figure 4 is the HPLC chromatogram of the smegglutinin peptide from Example 2 of the present invention.

[0065] Figure 5 is the HPLC chromatogram of the crude smegglutinin peptide from Example 3 of the present invention.

[0066] Figure 6 is the HPLC chromatogram of the smegglutinin peptide from Example 3 of the present invention.

[0067] Figure 7 is the HPLC chromatogram of the crude smegglutinin peptide from Example 4 of the present invention.

[0068] Figure 8 is the HPLC chromatogram of the smegglutinin peptide from Example 4 of the present invention.

[0069] Figure 9 is the HPLC chromatogram of the crude smegglutinin peptide from Example 5 of the present invention.

[0070] Figure 10 is the HPLC chromatogram of the smegglutinin peptide from Example 5 of the present invention.

[0071] Figure 11 shows the HPLC chromatogram of crude smegglutinin from Comparative Example 2.

[0072] Figure 12 shows the HPLC chromatogram of Smeglucopyranoside ester peptide of Comparative Example 2.

[0073] Figure 13 shows the HPLC chromatogram of crude smegglutinin from Comparative Example 3.

[0074] Figure 14 shows the HPLC chromatogram of Smeglucopyranoside ester peptide of Comparative Example 3.

[0075] Figure 15 shows the HPLC chromatogram of crude smegglutinin from Comparative Example 4.

[0076] Figure 16 shows the HPLC chromatogram of Smeglucopyranoside ester peptide from Comparative Example 4.

[0077] Figure 17 shows the HPLC chromatogram of crude smegglutinin from Comparative Example 5.

[0078] Figure 18 shows the HPLC chromatogram of Smeglucopyranoside, a comparative example of peptide 5. Detailed Implementation

[0079] The present invention will be further described in detail below through embodiments, which are intended to illustrate the invention and not limit it. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0080] Example 1: Synthesis of Smegglutinin

[0081] Synthesis of crude smegglutinin:

[0082] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh 11.89 g of Fmoc-Gly-OH (2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.26 mmol / g, and the synthesis scale was 14.0 mmol.

[0083] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (19.76 g, 2.0 eq.) and HOBT (4.16 g, 2.2 eq.) were weighed and dissolved in 100 ml DMF. DIC (5.30 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0084] Repeat the above steps, sequentially coupling Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, and Fmoc-Gln(Trt)-O H, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH were used to synthesize the peptide resin. After washing with DMF four times, DCM four times, and MeOH three times, 132.45 g of Smeglu peptide resin was obtained after drying, with a resin weight gain of 98.83%.

[0085] Weigh 132.45 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1324.5 ml of lysis reagent [(TFA / TIS / MPR / / H2O / m-cresol=90.0 / 2.5 / 2.5 / 2.5 / 2.5)], and carry out the lysis reaction at 25±5℃ for 1–4 h. After the reaction is complete, filter and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 58.68 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 70.88% and a synthesis yield of 70.1%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 1 and Table 1, respectively.

[0086] Table 1: Related substances statistics of crude peptide in Example 1 by liquid chromatography-HPLC

[0087] Refining of crude smegglutinin peptides:

[0088] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0089] The weight of smegglutinin was 33.80 g, with a total yield of 58.7%, HPLC purity of 99.65%, and a maximum single impurity of 0.09%. The chromatogram and data of smegglutinin are shown in Figure 2 and Table 2, respectively.

[0090] Table 2: Related substances statistics of peptide liquid chromatography-HPLC in Example 1

[0091] Example 2: Synthesis of Smegglutinin

[0092] Synthesis of crude smegglutinin:

[0093] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh 11.89 g of Fmoc-Gly-OH (2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.26 mmol / g, and the synthesis scale was 14.0 mmol.

[0094] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (19.76 g, 2.0 eq.) and HOBT (4.54 g, 2.4 eq.) were weighed and dissolved in 100 ml DMF. DIC (4.24 g, 2.4 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0095] Repeat the above steps, sequentially coupling Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, and Fmoc-Gln(Trt)-O H, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH were used to synthesize the peptide resin. After washing four times with DMF, four times with DCM, and three times with MeOH, 133.01 g of Smeglu peptide resin was obtained after drying, with a resin weight gain of 99.51%.

[0096] Weigh 133.01 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1330.0 ml of lysis reagent [(TFA / TIS / MPR / / H2O / m-cresol=90.0 / 2.5 / 2.5 / 2.5 / 2.5)]. Incubate the lysis reaction at 25±5℃ for 1–4 h. After the reaction is complete, filter the solution and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 59.02 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 70.37% and a synthesis yield of 70.7%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 3 and Table 3, respectively.

[0097] Table 3: Statistical analysis of related substances in crude peptide liquid chromatography-HPLC in Example 2

[0098] Refining of crude smegglutinin peptides:

[0099] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0100] The weight of smegglutide was 34.03 g, with a total yield of 59.1%, HPLC purity of 99.59%, and a maximum single impurity of 0.07%. The chromatogram and data of smegglutide are shown in Figure 4 and Table 4, respectively.

[0101] Table 4: Related substances statistics of peptide liquid chromatography-HPLC in Example 2

[0102] Example 3: Synthesis of Smegglutinin

[0103] Synthesis of crude smegglutinin:

[0104] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh 11.89 g of Fmoc-Gly-OH (2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.26 mmol / g, and the synthesis scale was 14.0 mmol.

[0105] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (19.76 g, 2.0 eq.) and HOBT (4.16 g, 2.2 eq.) were weighed and dissolved in 100 ml of 50% DCM / DMF mixed solvent. DIC (5.30 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction, which was then added to the synthesis column. The reaction was carried out at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The sample was dried and washed 4 times with DMF.

[0106] Repeat the above steps, sequentially coupling Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, and Fmoc-Gln(Trt)-O H, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH were used to synthesize the peptide resin. After washing with DMF four times, DCM four times, and MeOH three times, 133.82 g of Smeglu peptide resin was obtained after drying, with a resin weight gain of 100.48%.

[0107] Weigh 133.82 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1338.0 ml of lysis reagent [(TFA / TIS / MPR / / H2O / m-cresol=90.0 / 2.5 / 2.5 / 2.5 / 2.5)]. Incubate the lysis reaction at 25±5℃ for 1–4 h. After the reaction is complete, filter the solution and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 58.75 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 70.86% and a synthesis yield of 70.8%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 5 and Table 5, respectively.

[0108] Table 5: Statistical analysis of related substances in crude peptide liquid chromatography-HPLC of Example 3

[0109] Refining of crude smegglutinin peptides:

[0110] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0111] The weight of smegglutinin was 34.63 g, with a total yield of 60.1%, HPLC purity of 99.57%, and a maximum single impurity of 0.09%. The chromatogram and data of smegglutinin are shown in Figure 6 and Table 6, respectively.

[0112] Table 6: Statistical analysis of related substances in the liquid HPLC of the peptide in Example 3

[0113] Example 4: Synthesis of Smegglutinin

[0114] Synthesis of crude smegglutinin:

[0115] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh Fmoc-Gly-OH (11.89 g, 2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.27 mmol / g, and the synthesis scale was 14.6 mmol.

[0116] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (20.61 g, 2.0 eq.) and HOBT (4.34 g, 2.2 eq.) were weighed and dissolved in 100 ml DMF. DIC (5.53 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0117] Repeat the above steps, sequentially coupling Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, and Fmoc-Gln(Trt)-O H, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH were used to synthesize the peptide resin. After washing four times with DMF, four times with DCM, and three times with MeOH, 135.70 g of Smeglu peptide resin was obtained after drying, with a resin weight gain of 98.50%.

[0118] Weigh 135.70 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1357.0 ml of lysis reagent [(TFA / TIS / MPR / H2O / phenol = 89.0 / 1.0 / 2.5 / 2.5 / 5.0)], and carry out the lysis reaction at 25±5℃ for 1-4 h. After the reaction is complete, filter and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 60.52 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 70.57% and a synthesis yield of 69.7%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 7 and Table 7, respectively.

[0119] Table 7: Related substances statistics of crude peptide in Example 4 by liquid chromatography-HPLC

[0120] Refining of crude smegglutinin peptides:

[0121] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0122] The weight of smegglutinin was 35.42 g, with a total yield of 59.0%, HPLC purity of 99.61%, and a maximum single impurity of 0.08%. The chromatogram and data of smegglutinin are shown in Figure 8 and Table 8, respectively.

[0123] Table 8: Related substances statistics of peptide liquid chromatography-HPLC in Example 4

[0124] Example 5: Synthesis of Smegglutinin

[0125] Synthesis of crude smegglutinin:

[0126] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh Fmoc-Gly-OH (11.89 g, 2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.27 mmol / g, and the synthesis scale was 14.6 mmol.

[0127] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (20.61 g, 2.0 eq.) and HOPO (5.28 g, 2.2 eq.) were weighed and dissolved in 100 ml DMF. DIC (5.53 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0128] Repeat the above steps, sequentially coupling Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, and Fmoc-Gln(Trt)-O H, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH were used to synthesize the peptide resin. After washing with DMF four times, DCM four times, and MeOH three times, 137.06 g of Smeglu peptide resin was obtained after drying, with a resin weight gain of 98.50%.

[0129] Weigh 137.06 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1370.0 ml of lysis reagent [(TFA / TIS / MPR / / H2O / m-cresol=90.0 / 2.5 / 2.5 / 2.5 / 2.5)]. Incubate the lysis reaction at 25±5℃ for 1–4 h. After the reaction is complete, filter the solution and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 61.73 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 70.44% and a synthesis yield of 71.3%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 9 and Table 9, respectively.

[0130] Table 9: Statistical analysis of related substances in crude peptide liquid chromatography-HPLC of Example 5

[0131] Refining of crude smegglutinin peptides:

[0132] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0133] The weight of smegglutinin was 36.13 g, with a total yield of 60.2%, HPLC purity of 99.63%, and a maximum single impurity of 0.09%. The chromatogram and data of smegglutinin are shown in Figure 10 and Table 10, respectively.

[0134] Table 10: Related substances statistics of peptide liquid chromatography-HPLC in Example 5

[0135] Note: [Plus-Gly] 4 Smegglutinin, [D-Thr] 5 Smegglutinin was not found in the crude peptide but appeared in the refined peptide. The possible reason is that the crude peptide contained a larger number of impurity peaks. [Plus-Gly] 4 Smegglutinin, [D-Thr] 5 Smegglutinin has a relatively low content in crude peptides, and its absence can be caused by peak tailing or peak forward extension. This phenomenon is quite common in peptide preparation and analysis.

[0136] Comparative Example 1: Synthesis of Smegglutinin

[0137] Synthesis of crude smegglutinin peptide

[0138] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh 11.89 g of Fmoc-Gly-OH (2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.28 mmol / g, and the synthesis scale was 15.2 mmol.

[0139] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Arg(Pbf)-OH (19.72 g, 2.0 eq.) and HOBT (4.52 g, 2.2 eq.) were weighed and dissolved in 100 ml DMF. DIC (5.76 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0140] Repeat the above steps to couple Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln( The peptide resins were synthesized using the following formulas: Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH. The resin was then washed four times with DMF, four times with DCM, and three times with MeOH. After drying, 117.39 g of Smeglu peptide resin was obtained, with a resin weight gain of 74.47%. The peptide resin weight gain was significantly low, and peptide resin cleavage was not performed.

[0141] Comparative Example 2: Synthesis of Smegglutinin

[0142] Synthesis of crude smegglutinin peptide

[0143] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh 11.89 g of Fmoc-Gly-OH (2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.28 mmol / g, and the synthesis scale was 15.2 mmol.

[0144] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (21.46 g, 2.0 eq.) and HOBT (4.52 g, 2.2 eq.) were weighed and dissolved in 100 ml DMF. DIC (5.76 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0145] Repeat the above steps, sequentially coupling Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, and Fmoc-Glu(OtBu)-OH. The linear peptide resins were synthesized using styr(tBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH. The S20-position alloc protecting group was removed using phenylsilane and tetrakis(triphenylphosphine)palladium, followed by sequential coupling with Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and tert-butyl octadecanoate. After washing with DMF four times, DCM four times, and MeOH three times, and then drying, 139.63g of Smeglu peptide resin was obtained, with a resin weight gain of 99.04%.

[0146] Weigh 139.63 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1396.0 ml of lysis reagent [(TFA / TIS / MPR / / H2O / m-cresol=90.0 / 2.5 / 2.5 / 2.5 / 2.5)], and carry out the lysis reaction at 25±5℃ for 1–4 h. After the reaction is complete, filter and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 61.22 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 67.8% and a synthesis yield of 66.4%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 11 and Table 11, respectively.

[0147] Table 11: Statistical analysis of related substances in crude peptides of Comparative Example 2 by liquid chromatography-HPLC

[0148] Refining of crude smegglutinin peptides:

[0149] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0150] The weight of smegglutinin was 34.55 g, with a total yield of 55.3%, HPLC purity of 99.28%, and a maximum single impurity of 0.34%. The chromatogram and data of smegglutinin are shown in Figure 12 and Table 12, respectively.

[0151] Table 12: Statistical analysis of related substances in comparative example 2 (HPLC) of arginine peptide

[0152] Comparative Example 3: Synthesis of Smegglutinin

[0153] Synthesis of crude smegglutinin:

[0154] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh 11.89 g of Fmoc-Gly-OH (2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.26 mmol / g, and the synthesis scale was 14.0 mmol.

[0155] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (19.76 g, 2.0 eq.) and HOBT (4.16 g, 2.2 eq.) were weighed and dissolved in 100 ml DMF. DIC (5.30 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0156] Repeat the above steps, sequentially coupling Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, and Fm The following peptide resins were synthesized: oc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH. The resin was then washed four times with DMF, four times with DCM, and three times with MeOH. After drying, 133.05 g of Smeglu peptide resin was obtained, with a resin weight gain of 99.04%.

[0157] Weigh 133.05 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1330.5 ml of lysis reagent [(TFA / TIS / MPR / / H2O / m-cresol=90.0 / 2.5 / 2.5 / 2.5 / 2.5)]. Incubate the lysis reaction at 25±5℃ for 1–4 h. After the reaction is complete, filter the solution and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 58.35 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 67.5% and a synthesis yield of 68.4%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 13 and Table 13, respectively.

[0158] Table 13: Statistical analysis of related substances in crude peptides of Comparative Example 3 by liquid chromatography-HPLC

[0159] Refining of crude smegglutinin peptides:

[0160] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0161] The weight of smegglutinin was 31.05 g, with a total yield of 53.9%, an HPLC purity of 98.80%, and a maximum single impurity of 0.46%. The chromatogram and data of smegglutinin are shown in Figure 14 and Table 14, respectively.

[0162] Table 14: Statistical analysis of related substances in comparative example 3 (pure peptide) by liquid HPLC

[0163] Comparative Example 4: Synthesis of Smegglutinin

[0164] Synthesis of crude smegglutinin:

[0165] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh Fmoc-Gly-OH (11.89 g, 2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.27 mmol / g, and the synthesis scale was 14.6 mmol.

[0166] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (20.61 g, 2.0 eq.) and HOBT (4.34 g, 2.2 eq.) were weighed and dissolved in 100 ml DMF. DIC (5.53 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0167] Repeat the above steps, sequentially coupling Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, and Fm The following peptide resins were synthesized: oc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, and Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH. The resin was then washed four times with DMF, four times with DCM, and three times with MeOH. After drying, 135.66 g of Smeglu peptide resin was obtained, with a resin weight gain of 98.46%.

[0168] Weigh 135.66 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1357.0 ml of lysis reagent [(TFA / TIS / MPR / / H2O / m-cresol=90.0 / 2.5 / 2.5 / 2.5 / 2.5)]. Incubate the lysis reaction at 25±5℃ for 1–4 h. After the reaction is complete, filter the solution and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 59.47 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 66.3% and a synthesis yield of 65.6%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 15 and Table 15, respectively.

[0169] Table 15: Statistical analysis of related substances in crude peptide HPLC of Comparative Example 4

[0170] Refining of crude smegglutinin peptides:

[0171] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0172] The weight of smegglutinin was 31.20 g, with a total yield of 51.9%, an HPLC purity of 98.81%, and a maximum single impurity of 0.43%. The chromatogram and data of smegglutinin are shown in Figure 16 and Table 16, respectively.

[0173] Table 16: Statistical analysis of related substances in comparative example 4 (pure peptide) by liquid HPLC

[0174] Comparative Example 5: Synthesis of Smegglutinin

[0175] Synthesis of crude smegglutinin:

[0176] Weigh 50.00 g of Wang resin (20 mmol, substitution value 0.40 mmol / g) and add it to the peptide synthesis column. Add 500 ml of DMF to swell for 30 min, then dry under vacuum. Wash twice with DMF and dry under vacuum. Weigh Fmoc-Gly-OH (11.89 g, 2.0 eq.), HOBT (5.94 g, 2.2 eq.), and DMAP (0.49 g, 0.2 eq.), dissolve them in 500 ml of DMF, and activate with DIC (7.57 g, 3.0 eq.) for 4 min at 0–10 °C. Add this to the synthesis column and react at 25 ± 5 °C under nitrogen protection for 4–8 h. Dry under vacuum and wash four times with DMF. The amino acid resin substitution value was determined to be 0.27 mmol / g, and the synthesis scale was 14.6 mmol.

[0177] Fmoc protection was removed twice with 20% PIPE / DMF for 10 min + 10 min respectively, followed by 7 washes with DMF. Fmoc-Gly-Arg(Pbf)-OH (20.61 g, 2.0 eq.) and HOBT (4.34 g, 2.2 eq.) were weighed and dissolved in 100 ml DMF. DIC (5.53 g, 3.0 eq.) was added at 0–10 °C for 4 min to activate the reaction. The solution was then added to the synthesis column and reacted at 25 ± 5 °C under nitrogen protection for 2–4 h. The ninhydrin test result was negative. The solution was dried and washed 4 times with DMF.

[0178] Repeat the above steps to couple Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Gl in sequence. u(OtBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gl The N-terminal Fmoc protecting group was removed by removing γ-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-Phe-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, and Fmoc-His(Trt)--OH. The peptide resin synthesis was completed, and after washing with DMF four times, DCM four times, and MeOH three times, 134.86 g of Smeglu peptide resin was obtained after drying, with a resin weight gain of 98.83%.

[0179] Weigh 134.86 g of the above-mentioned Smeglucopyrepeptide resin and add it to 1349.0 ml of lysis reagent [(TFA / TIS / MPR / / H2O / m-cresol=90.0 / 2.5 / 2.5 / 2.5 / 2.5)]. Incubate the lysis reaction at 25±5℃ for 1–4 h. After the reaction is complete, filter the solution and wash the resin with 150 ml of TFA. The filtrate was added to 15 L of frozen isopropyl ether for precipitation. The precipitated solid was centrifuged and washed to obtain 59.13 g of crude smegglutinin: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a purity of 64.5% and a synthesis yield of 63.5%. The HPLC chromatogram and data of the crude smegglutinin are shown in Figure 17 and Table 17, respectively.

[0180] Table 17: Statistical analysis of related substances in crude peptide HPLC of Comparative Example 5

[0181] Refining of crude smegglutinin peptides:

[0182] The crude smegglutinin solution was filtered using an organic membrane and purified using a reverse-phase packing material. The purification process involved a first step using ammonium salt and a second step using sodium phosphate. Samples with a purity >99% were collected, concentrated, and lyophilized to obtain refined smegglutinin peptides.

[0183] The weight of smegglutinin was 29.78 g, with a total yield of 49.6%, an HPLC purity of 98.28%, and a maximum single impurity of 0.51%. The chromatogram and data of smegglutinin are shown in Figure 18 and Table 18, respectively.

[0184] Table 18: Statistical analysis of related substances in comparative example 5 (pure peptide) by liquid HPLC

[0185] As can be seen from the HPLC chromatograms and data in Examples 1-5 and the comparative examples, the synthesis method of the present invention greatly and simultaneously inhibits / reduces [D-His] 1 Smegglutinin, D-Glu 3 Smegglutinin, [Plus-Gly] 4 Smegglutinin, [D-Thr] 5 Smegglutinin, D-Phe 6 Smegglutinin, [D-γGlu] 15 Smegglutinin, [Plus-Gly] 16 Smegglutinin, [D-γGlu] 20-3 The formation of eight impurities, including smegglutinin. In Examples 1-5, [D-His...] 1 The content of smegglutinin does not exceed 0.4%, and may even be 0%; [D-Glu] 3 The content of smegglutinin does not exceed 0.19%, [Plus-Gly] 4 The content of smegglutinin is 0%, [D-Thr] 5 The content of smegglutinin is 0, [D-Phe 6 The content of smegglutinin does not exceed 0.24%, [D-γGlu] 15 The content of smegglutinin does not exceed 0.7%, [Plus-Gly] 16 The content of smegglutinin is 0%, and [D-γGlu] 20-3 The content of smegglutinin does not exceed 0.29%.

[0186] Comparative Example 2, however, did not employ R1-Lys[AEEA-AEEA-γGlu(OR2)-C18-R3]-OH, [D-γGlu] 20-3The content of smegglutinin was as high as 1.26%, and after purification, the content was 0.34%, even higher than the content in the crude peptide of this invention; Comparative Example 3 did not use Glu 15 -Gly 16 Dipeptide fragment, [D-γGlu] 15 The content of smegglutinin is as high as 1.45%, [Plus-Gly] 16 The content of smegglutinin is as high as 2.54%, and after purification, [Plus-Gly] 16 The content of smegglutinin was 0.46%, even higher than the content in the crude peptide of this invention; Comparative Example 4 did not use Thr. 5 -Phe 6 Dipeptide fragment, [D-Thr] 5 The content of smegglutinin is as high as 1.25%, [D-Phe 6 The content of smegglutinin was as high as 0.91%, and after purification, the contents were 0.42% and 0.43%, respectively, which were even higher than the contents in the crude peptide of this invention; Comparative Example 5 did not use His... 1 -Aib 2 -Glu 3 -Gly 4 Tetrapeptide fragment, [D-His] 1 The content of smegglutinin is as high as 1.02%, [Plus-Gly] 4 The content of smegglutinin is as high as 2.56%, [D-Glu] 3 The content of smeglucopyne is as high as 0.80%, and after purification, the contents are 0.42% and 0.43%, respectively, which are even higher than the contents in the crude peptide of this invention.

[0187] As can be seen from the HPLC chromatograms and data in Examples 1-5 and the Comparative Example, the synthesis method of the present invention effectively avoids the occurrence of DKP side reactions, improves the resin weight gain and total product yield, with a resin weight gain of over 98% and a total yield of over 58%. However, Comparative Example 1 did not use Gly at positions 29-30. 29 -Arg 30 The dipeptide fragment resulted in a resin weight gain of only 74.47%, which is far lower than the method of this invention.

[0188] As can be seen from the HPLC chromatograms and data in Examples 1-5 and the comparative examples, the synthesis method of the present invention improves the problem of difficult coupling caused by β-sheet, effectively avoids the generation of missing peptide impurities, and greatly improves the purity and synthesis yield of the final crude peptide. The purity of the crude peptide is over 70%, and the synthesis yield reaches over 70%.

[0189] As can be seen from the HPLC chromatograms and data in Examples 1-5 and the comparative examples, the synthesis method of the present invention significantly reduces the difficulty of crude peptide purification and greatly improves the purity and yield of smegglutinin. After simple purification steps, these impurities in smegglutinin are basically removed, and the final purity of the refined peptide is above 99.5%; the maximum single impurity content does not exceed 0.09%, which is almost negligible; and the total yield is above 58%. In contrast, the maximum single impurity content in the comparative examples is not less than 0.3%.

[0190] In conclusion, the method of the present invention has the advantages of short synthesis cycle, good synthesis effect, high purity, few impurities, high yield and low cost, which reduces the synthesis cost and is conducive to large-scale industrial production.

Claims

1. A method for synthesizing smegglutinin, characterized in that, The main steps include: 1) By solid-phase synthesis, resin is coupled with protected amino acids and polypeptide fragments to obtain Smeglu peptide resin. Among them, bits 29-30 use Gly 29 -Arg 30 Dipeptide fragment, The 20th position uses R1-Lys[AEEA-AEEA-γGlu(OR2)-C18-R3]-OH. 15-16 bits use Glu 15 -Gly 16 Dipeptide fragment, 5-6 bits use Thr 5 -Phe 6 Dipeptide fragment, 1-4 use His 1 -Aib 2 -Glu 3 -Gly 4 Tetrapeptide fragment, R1, R2, and R3 are protecting groups, respectively. 2) Peptide resin cleavage yielded crude smegglutinin peptide. 3) Purify to obtain smeglucopyrithione peptide.

2. The method for synthesizing smegglutinin according to claim 1, characterized in that, The resin in step 1) is a resin containing benzyl ester bonds.

3. The method for synthesizing smegglutinin according to claim 1, characterized in that, The resin used in step 1) is a hydroxyl resin.

4. The method for synthesizing smegglutinin according to any one of claims 1, characterized in that, In step 1), the resin is selected from Wang Resin, DEG-Wang Resin, HMPA-MBHA resin, and HMPA-AM resin.

5. The method for synthesizing smegglutinin according to claim 1, characterized in that, In step 1), Gly 29 -Arg 30 The dipeptide fragment is Fmoc-Gly-Arg(Pbf)-OH.

6. The method for synthesizing smegglutinin according to claim 1, characterized in that, In step 1), the 20th position uses Fmoc-Lys[AEEA-AEEA-γGlu(OtBu)-C18-OtBu]-OH.

7. The method for synthesizing smegglutinin according to claim 1, characterized in that... In step 1), Glu 15 -Gly 16 The dipeptide fragment is Fmoc-Glu(OtBu)-Gly-OH.

8. The method for synthesizing smegglutinin according to claim 1, characterized in that, In step 1), Thr 5 -Phe 6 The dipeptide fragment is Fmoc-Thr(tBu)-Phe-OH.

9. The method for synthesizing smegglutinin according to claim 1, characterized in that, In step 1), His 1 -Aib 2 -Glu 3 -Gly 4 The tetrapeptide fragment is either Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH or Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-OH.

10. The method for synthesizing smegglutinin according to claim 1, characterized in that, In step 1), the degree of resin substitution is 0.3-0.5 mmol / g.

11. The method for synthesizing smegglutinin according to claim 1, characterized in that, The condensing agent used in step 1) for coupling is selected from HOBt, DIC, DMAP, Oxyma, HOPO, HBTU, DIPEA, and PyBOP.

12. The method for synthesizing smegglutinin according to claim 1, characterized in that, The condensing agent used in the coupling in step 1) is selected from the combination of HOBt, DIC, and DMAP; the combination of Oxyma and DIC; the combination of HOPO and DIC; the combination of HOBt, DIPEA, and TBTU; the combination of HBTU and DIPEA; the combination of HOBt, DIPEA, and HBTU; and the combination of PyBOP, HOBt, and DIPEA.

13. The method for synthesizing smegglutinin according to any one of claims 11-12, characterized in that, The amount of the condensing agent is 0.9 to 3.0 times the molar amount of the amino acids, and the reaction time is 2 to 6 hours.

14. The method for synthesizing smegglutinin according to claim 1, characterized in that, In step 1), the reaction solvent used for fragment coupling is selected from 1 to 2 of DMF and DCM.

15. The method for synthesizing smegglutinin according to claim 14, characterized in that, The volume percentage of DCM in the reaction solvent is 10% to 90%.

16. The method for synthesizing smegglutinin according to claim 1, characterized in that: In step 2), the lysis reagent is selected from trifluoroacetic acid and other components, and the other components are selected from 1 to 4 of purified water, phenol, 3-mercaptopropionic acid, triisopropylsilane, and m-cresol.

17. The method for synthesizing smegglutinin according to claim 16, characterized in that: The lysis reagent contains more than 85% trifluoroacetic acid, with other components each accounting for 1% to 5%.

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