CN112028986A

CN112028986A - Synthesis method of semaglutide

Info

Publication number: CN112028986A
Application number: CN202010954234.5A
Authority: CN
Inventors: 冷国庆; 张琪; 谢俊; 侯继元; 杨桦; 于通皓; 杨振博; 王艳; 张凤莲; 景文岩; 黄天一; 孙宝伟; 汪莹莹; 关伟萍; 付文君
Original assignee: Harbin Jixianglong Biological Technology Co ltd
Current assignee: Harbin Jixianglong Biological Technology Co ltd
Priority date: 2020-09-11
Filing date: 2020-09-11
Publication date: 2020-12-04

Abstract

The invention discloses a method for synthesizing semaglutide, which comprises the following steps: (1) taking Fmoc-Gly-Wang resin as a solid phase carrier, removing Fmoc protecting groups, gradually coupling amino acids from a C end to an N end in sequence, and coupling to 20 th amino acid R-Lys (Fmoc) -OH at the N end of a main chain; (2) removing the Fmoc protecting group of the side chain of the 20 th amino acid at the N end of the main chain, and sequentially coupling the side chain amino acids; (3) removing the protecting group of the 20 th amino acid R at the N end of the main chain, and sequentially coupling the rest amino acids of the main chain according to a peptide sequence to obtain the semaglutide full-protection peptide resin; wherein, the 18 th-19 th amino acid at the N end and the 1 st-2 th amino acid at the N end are dipeptide fragments; (4) and cracking and precipitating the semaglutide full-protection peptide resin through a cracking solution to obtain the semaglutide crude peptide. The method is simple and convenient, and the prepared semaglutide has high purity and is beneficial to industrial production.

Description

Synthesis method of semaglutide

Technical Field

The invention relates to the technical field of polypeptide synthesis, in particular to a synthetic method of semaglutide.

Background

Semaglutide (Semaglutide) is a novel long-acting GLP-1 analogue, and the peptide sequence of the Semaglutide is as follows: his (His)¹-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-Ala²⁴-Trp²⁵-Leu²⁶-Val²⁷-Arg²⁸-Gly²⁹-Arg³⁰-Gly³¹-OH. From the peptide sequence, 2-position amino acid is non-natural alpha-aminoisobutyric acid (Aib), and a polyethylene glycol (PEG) -modified octadecanoic acid fatty chain is arranged in a lysine side chain of 20-position amino acid of the peptide sequence, so that the hydrophilicity of the whole peptide is greatly enhanced, the whole peptide can be tightly combined with albumin, can resist the degradation of dipeptidyl peptidase 4(DPP-4), can reduce renal excretion, remarkably prolong the half-life period, is suitable for subcutaneous injection once a week, and achieves the effect of long circulation.

At present, for the synthesis of semaglutide, the extension of a peptide chain is mainly realized by gradually coupling each amino acid unit, but more missing peptide impurities with the properties close to those of main components are generated in the synthesis process. For example, the deletion peptide impurities generated by the deletion of amino acids with simple structure and small molecular weight of alanine (Ala) or glycine (Gly) are difficult to remove in the purification process, and the yield is seriously influenced, especially, the amino acids at the 18 th position and the 19 th position of the peptide chain are two continuous Ala, and the deletion of single Ala is more easy to generate impurities to influence the purification.

In addition, the large steric hindrance amino acid Aib is introduced in the synthesis process of the semaglutide²Later, the N-terminal amino acid His is caused¹Difficult coupling, producing large amounts of [ des-His ]¹]If multiple repeated administrations are adopted, racemic impurities [ D-His ] are caused¹]Is not favorable for subsequent purification.

Therefore, the problem to be solved by the technical personnel in the field is how to provide a simple, effective and practical method for synthesizing semaglutide, which is beneficial to the practical industrial production.

Disclosure of Invention

In view of this, the invention provides a simple and high-purity method for synthesizing semaglutide.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for synthesizing semaglutide comprises the following steps:

(1) taking Fmoc-Gly-Wang resin as a solid phase carrier, removing Fmoc protecting groups, gradually coupling amino acids from the C end to the N end according to the sequence of the amino acid sequence of the backbone of the semaglutide, and coupling to the 20 th amino acid R-Lys (Fmoc) -OH at the N end of the backbone;

(2) removing the Fmoc protecting group of the side chain of the 20 th amino acid at the N end of the main chain, and sequentially coupling the side chain amino acids;

(3) removing the protecting group of the 20 th amino acid R at the N end of the main chain, and sequentially coupling the rest amino acids of the main chain according to a peptide sequence to obtain the semaglutide full-protection peptide resin; wherein, the 18 th-19 th amino acid at the N end and the 1 st-2 th amino acid at the N end are dipeptide fragments;

(4) and cracking and precipitating the semaglutide full-protection peptide resin through a cracking solution to obtain the semaglutide crude peptide.

The side chain amino acids are coupled in sequence, so that the problems of low coupling efficiency and low yield caused by steric hindrance effect when the whole coupling is introduced can be solved; the 18 th-19 th amino acid at the N end is a dipeptide fragment, so that the problem of high purification difficulty caused by deletion of single amino acid Ala to generate deleted peptide is solved, and the purity of the crude peptide is improved; the 1 st-2 th amino acid at the N end adopts a dipeptide fragment, so that the problem of large steric hindrance of the amino acid Aib is solved²Upper coupling His¹The problem of great difficulty is solved, and His is avoided¹While avoiding the problem of racemization due to repeated coupling to His.

Preferably, in the step (1),

the substitution degree of Fmoc-Gly-Wang resin is 0.3-0.6 mmol/g;

the deprotection reagent used for removing the Fmoc protecting group is a solution of piperidine and DMF with the volume ratio of 1:1-1: 5.

Preferably, in the step (1),

the solvent used for coupling the amino acid is DMF and/or DCM;

the condensing agent used when coupling the amino acids is any one of the following combinations:

DIC/A, TBTU/B, TBTU/A/B, HATU/A/B, HATU/B, HBTU/A/B, PyBOP/A/B, PyAOP/A/B, PyAOP/B; wherein, A is HOBt or HOAt, B is DIEA or TEA;

the feeding amount of the amino acid is 2-5 times of the mole number of Fmoc-Gly-Wangprein during amino acid coupling;

r is Cbz, Alloc or Dde protecting group.

Preferably, Fmoc-Arg (pbf) -OH is dissolved by a solvent, added into the resin after being activated by adding a condensing agent, and reacted for 2h under nitrogen bubbling at room temperature; after the reaction is finished, washing with a solvent to obtain peptide resin; repeating the steps of Fmoc protection removal and coupling, and sequentially coupling 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 and Dde-Lys (Fmoc) -OH according to peptide sequence.

Preferably, in the step (2),

the deprotection reagent used for removing the Fmoc protecting group of the side chain of the 20 th amino acid at the N end of the main chain is solution of piperidine and DMF with the volume ratio of 1:1-1: 5.

Preferably, in the step (2),

the side chain amino acids coupled in sequence are Fmoc-AEEA, Fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester;

the solvent used for coupling the side chain amino acid is DMF and/or DCM;

the condensing agent used when coupling the side chain amino acid is any one of the following combinations:

TBTU/B, TBTU/A/B, HATU/A/B, PyBOP/A/B, PyAOP/A/B; wherein, A is HOBt or HOAt, B is DIEA or TEA;

the dosage of the side chain amino acid is 2 to 5 times of the mole number of Fmoc-Gly-Wang resin.

Preferably, Fmoc-AEEA is dissolved by using a solvent, added into the peptide resin after being activated by adding a condensing agent, and subjected to nitrogen gas blowing reaction for 1h at room temperature; after the reaction is finished, washing by using a solvent; and repeating the steps of Fmoc protection removal and coupling, and sequentially coupling Fmoc-AEEA, Fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester according to the peptide sequence.

Preferably, in the step (3),

the deprotection reagent used for removing the protecting group of the 20 th amino acid R at the N end of the main chain is DMF solution containing hydrazine hydrate, and the volume fraction of the hydrazine hydrate is 1.5-3%.

Preferably, in the step (3),

the 18 th-19 th amino acid at the N end adopts a dipeptide fragment of Fmoc-Ala-Ala-OH;

the 1 st-2 nd amino acid at the N end selects dipeptide segment Boc-His (Trt) -Aib-OH.

Preferably, in step (4)

The lysis solution is a mixed solution of TFA, A and B, wherein A is EDT or TIS, and B is phenol or TA or water;

the cracking time is 1-3h, and the cracking temperature is 10-40 ℃.

Preferably, the ratio of TFA: a: the volume ratio of B is (90-95): (5-2.5): (5-2.5), the using amount of the lysis solution is 6-10mL/g of the semaglutide full-protection peptide resin.

Preferably, in step (4)

The reagent used for precipitation is diethyl ether or methyl tert-butyl ether.

According to the technical scheme, the problems of high coupling difficulty, low purity, high cost and complex operation in the synthetic process of the semaglutide are effectively solved, so that the synthetic method is more beneficial to industrial amplification and meets the actual production requirement.

Drawings

FIG. 1 shows an HPLC chromatogram of a crude peptide of S.metreuterin of example 1;

FIG. 2 shows HPLC chromatogram of the semaglutide protamine of example 1;

FIG. 3 shows the mass spectrum of the semaglutide of example 1;

FIG. 4 shows an HPLC chromatogram of crude S.metreuptake peptide of comparative example 1;

FIG. 5 shows an HPLC chromatogram of a crude peptide of S.metreuterin of example 2;

FIG. 6 shows an HPLC chromatogram of a crude peptide of S.metreuptake of example 3;

FIG. 7 shows the HPLC chromatogram of the crude peptide of S.metreuterin of example 4.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The abbreviations used in the present invention have the meanings given in Table 1.

TABLE 1

The starting materials used in the examples are all commercially available starting materials.

Example 1

A method for synthesizing semaglutide comprises the following steps:

1. swelling of the resin

12.84g of Fmoc-Gly-Wang resin with the substitution degree of 0.39mmol/g are weighed and poured into a solid phase synthesis reactor, nitrogen is blown, 100mL of DMF is added for swelling for 1h, and then the solvent is pumped out.

2. DeFmoc protection

To the resin was added 50mL of piperidine: reacting a deprotection reagent 1 of DMF (V/V)1:3 for 5min, draining, adding 50mL of the deprotection reagent 1 again, reacting for 15min, and draining.

3. Washing machine

And after deprotection is finished, adding 50ml of DMF (dimethyl formamide) washing resin into the resin, draining, adding DMF again for washing, repeating the washing for 6 times in total, and taking the resin ninhydrin for positive detection.

4. Coupled amino acids

Fmoc-Arg (pbf) -OH and 2.04g HOBT, which are 3 times the molar number of Fmoc-Gly-Wang resin, were weighed, dissolved in 50mL DMF, and 2.4mL DIC was added thereto under stirring, followed by activation for 2min under stirring. And pouring the activated amino acid solution into resin, carrying out nitrogen gas blowing reaction at room temperature for 2h, taking resin ninhydrin for detection, carrying out colorless and transparent detection, and adding 50mL of DMF for washing for 2 times after the coupling reaction is finished to obtain Fmoc-Arg (pbf) -Gly-Wang resin.

Repeating the Fmoc protection-coupling removal operation, and sequentially coupling the amino acids Fmoc-G according to the peptide sequence of the main chainly-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, Dde-Lys (Fmoc) -OH to give the following peptide resins: Dde-Lys²⁰(Fmoc)-Glu²¹(OtBu)-Phe²²-Ile²³-Ala²⁴-Trp²⁵(Boc)-Leu²⁶-Val²⁷-Arg²⁸(pbf)-Gly²⁹-Arg³⁰(pbf)-Gly³¹-wangrein. The dosage of each amino acid is 3 times of the mole number of Fmoc-Gly-Wangprein.

5. DeFmoc protection

Adding 50mL of deprotection reagent 1 into the peptide resin, reacting for 5min, then pumping, adding 50mL of deprotection reagent 1 again, reacting for 15min, and then pumping.

6. Coupling side chain amino acids

Fmoc-AEEA with 3 times mole number of Fmoc-Gly-Wang resin and 4.82g TBTU were weighed, dissolved in 100mL DMF, stirred, added with 2.5mL DIEA, and stirred and activated for 2 min. And pouring the activated amino acid solution into peptide resin, carrying out nitrogen gas blowing reaction for 1h at room temperature, taking resin ninhydrin for detection, carrying out colorless and transparent detection, finishing the coupling reaction, and adding 100mL of DMF (dimethyl formamide) for washing for 2 times.

Repeating Fmoc protection removal and coupling operation, and sequentially coupling amino acid Fmoc-AEEA, Fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester according to a side chain peptide sequence to obtain the following peptide resin:

Dde-Lys²⁰(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic)-Glu²¹(OtBu)-Phe²²-Ile²³-Ala²⁴-Trp²⁵(Boc)-Leu²⁶-Val²⁷-Arg²⁸(pbf)-Gly²⁹-Arg³⁰(pbf)-Gly³¹-Wang resin. The feeding amount of each amino acid is 3 times of the mole number of Fmoc-Gly-Wang resin.

7. De Dde protection

Adding 100mL of deprotection reagent 2 (2% hydrazine hydrate-containing DMF solution) into the peptide resin, reacting for 10min, draining, adding 100mL of deprotection reagent 2 into the reaction column again, reacting for 5min, draining, after the reaction is finished, adding 100mL of DMF, washing for 6 times, and detecting positive ninhydrin to obtain the following peptide resin:

H-Lys²⁰(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic)-Glu²¹(OtBu)-Phe²²-Ile²³-Ala²⁴-Trp²⁵(Boc)-Leu²⁶-Val²⁷-Arg²⁸(pbf)-Gly²⁹-Arg³⁰(pbf)-Gly³¹-Wang resin。

8. coupling of backbone residual amino acids

Using HOBT/DIC as a condensing agent and DMF as a solvent, repeatedly coupling and deprotecting according to the methods of steps 4 and 5, sequentially connecting protected amino acids Fmoc-Ala-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH and 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, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Boc-His (Trt) -Aib-OH, and the charged amount of each amino acid is 3 times of the molar number of Fmoc-Gly-Wang resin.

Washing with DMF after coupling is finished, washing and shrinking with methanol, and drying by blowing with nitrogen to obtain the semaglutide full-protection peptide resin:

Boc-His¹(Trt)-Aib²-Glu³(OtBu)-Gly⁴-Thr⁵(tBu)-Phe⁶-Thr⁷(tBu)-Ser⁸(tBu)-Asp⁹(OtBu)-Val¹⁰-Ser¹¹(tBu)-Ser¹²(tBu)-Tyr¹³(tBu)-Leu¹⁴-Glu¹⁵(OtBu)-Gly¹⁶-Gln¹⁷(Trt)-Ala¹⁸-Ala¹⁹--Lys²⁰(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic)-Glu²¹(OtBu)-Phe²²-Ile²³-Ala²⁴-Trp²⁵(Boc)-Leu²⁶-Val²⁷-Arg²⁸(pbf)-Gly²⁹-Arg³⁰(pbf)-Gly³¹-Wang resin；

9. cleavage peptide resin

The ratio of TFA: EDT (electro-thermal transfer coating): water (V/V) ═ 95:2.5:2.5 as a lysate, adding the lysate into the semaglutide full-protection peptide resin according to the proportion of 10mL/g, reacting for 2 hours at room temperature, precipitating the filtrate by methyl tert-butyl ether to obtain a crude peptide solution, and performing suction filtration, washing and drying to obtain the semaglutide crude peptide, wherein the method comprises the following steps:

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-Ala²⁴-Trp²⁵-Leu²⁶-Val²⁷-Arg²⁸-Gly²⁹-Arg³⁰-Gly³¹-OH。

HPLC detection of the crude semaglutide peptide resulted in the calculation of 73.570% purity and 5.612% maximum single impurity, as shown in FIG. 1.

Further, the crude semaglutide peptide was dissolved in purified water, and then the pH was adjusted to neutral with aqueous ammonia. Taking 0.1% TFA/purified water and 0.1% TFA/acetonitrile as mobile phases, adopting an HPLC semi-preparative liquid chromatography system and a C18 reversed-phase preparative column, carrying out gradient elution and purification on a crude peptide solution, and collecting target peak components to obtain a collecting solution with the HPLC purity of more than 98.0%; and then, taking purified water and acetonitrile as mobile phases, performing salt conversion refining on the TFA system collected liquid by adopting an HPLC semi-preparative liquid chromatography system and a C18 reversed-phase preparation column, collecting target peak components to obtain refined liquid with the HPLC purity of more than 99.5%, and performing reduced pressure distillation and freeze drying to obtain the Semetreutide refined peptide. HPLC detection is carried out on the semaglutide protamine, the result is shown in figure 2, and the retention time of the semaglutide protamine is 7.008 min. The mass spectrometric detection of semaglutide protamine is shown in fig. 3.

Comparative example 1

(1) The following peptide resins were prepared according to the procedure of example 1:

(2) coupling and deprotection were repeated according to the methods of example 1, steps 4 and 5, using HOBT/DIC as a condensing agent and DMF as a solvent, and protected amino acids Fmoc-Ala-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (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, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH and Fmoc-Aib-OH were sequentially connected, each amino acid charge was 3 times the molar number of Fmoc-Gly-Wangrestin, the following peptide resins were obtained:

Fmoc-Aib²-Glu³(OtBu)-Gly⁴-Thr⁵(tBu)-Phe⁶-Thr⁷(tBu)-Ser⁸(tBu)-Asp⁹(OtBu)-Val¹⁰-Ser¹¹(tBu)-Ser¹²(tBu)-Tyr¹³(tBu)-Leu¹⁴-Glu¹⁵(OtBu)-Gly¹⁶-Gln¹⁷(Trt)-Ala¹⁸-Ala¹⁹--Lys²⁰(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic)-Glu²¹(OtBu)-Phe²²-Ile²³-Ala²⁴-Trp²⁵(Boc)-Leu²⁶-Val²⁷-Arg²⁸(pbf)-Gly²⁹-Arg³⁰(pbf)-Gly³¹-Wang resin。

(3) pouring six parts of the peptide resin prepared in the step (2) into a solid phase reactor respectively, removing Fmoc protection according to the method of the embodiment 1, and washing;

taking six parts of Boc-His (Trt) -OH, wherein the dosage of the Boc-His (Trt) -OH is 3 times of the mole number of Fmoc-Gly-Wangprein, and after DMF is dissolved, respectively using the following six condensing agents for activation: DIC/HOBT, HOBT/TBTU/DIEA, HOBT/HBTU/DIEA, HOBT/PyBop/DIEA, HATU/DIEA, PyAOP/DIEA; and respectively pouring the activated amino acid mixed solution into peptide resin, and reacting for 2h at room temperature.

After the reaction, the crude peptide of semaglutide is obtained by treating the crude peptide according to the method in the example 1.

Coupled His under the condition of detecting N-terminal non-dipeptide fragment by using chloranil chromogenic method and HPLC method¹The results obtained for the crude peptide were as follows:

by DIC/HOBT, IVThe crude peptide obtained by chloranil chromogenic method is 53.694% pure, and the [ des-His ] is positive¹]The deletion peptide impurity was 27.082% (fig. 4);

the crude peptide obtained by positive color development with HOBT/TBTU/DIEA and chloranil has purity of 42.484%, [ des-His ]¹]The deletion peptide impurity was 38.901%;

positive by HOBT/HBTU/DIEA and chloranil-chloranil color development method, the purity of the obtained crude peptide is 51.475%, [ des-His ]¹]The deletion peptide impurity was 31.418%;

the crude peptide obtained by positive color development with HOBT/PyBop/DIEA and chloranil has purity of 6.630%, [ des-His ]¹]The deletion peptide impurity was 31.418%;

adopting HATU/DIEA, and chloranil to show positive color development;

positive by adopting PyAOP/DIEA and chloranil color development method.

From the results, His was identified¹To Aib²The coupling difficulty of (A) is high, and a large amount of deletion peptide impurities [ des-His ] are generated¹]If repeated feeding is adopted, the measures of prolonging the time and the like tend to waste materials and generate more byproducts.

In contrast, the crude semaglutide peptide obtained by coupling the dipeptide fragments in the embodiment 1 of the invention has high purity and small maximum single impurity, and is suitable for industrial production.

Example 2

On the basis of example 1, the condensing agent used in coupling Boc-His (Trt) -Aib-OH was replaced by TBTU/DIEA, and the HPLC detection result of the obtained crude semaglutide peptide is shown in FIG. 5, wherein the purity of the semaglutide is 72.088% and the maximum single hetero 6.843%.

Example 3

On the basis of example 1, the condensing agent used in coupling Boc-His (Trt) -Aib-OH was replaced by HATU/DIEA, and the HPLC detection result of the obtained crude semaglutide peptide is shown in FIG. 6, wherein the purity of the semaglutide is 71.369% and the maximum single hetero 4.609%.

Example 4

On the basis of example 1, the condensing agent used in coupling Boc-His (Trt) -Aib-OH was replaced by PyAOP/DIEA, and HPLC detection results of crude semaglutide peptide are shown in FIG. 7, wherein the purity of semaglutide is 72.596% and the maximum single hetero 5.276%.

Example 5

Validation of Lys removal from DMF solutions of hydrazine hydrate at different concentrations²⁰Effect of Dde protection above:

on the basis of the embodiment 1, the following peptide resins are selected as raw materials:

Dde-Lys²⁰(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic)-Glu²¹(OtBu)-Phe²²-Ile²³-Ala²⁴-Trp²⁵(Boc)-Leu²⁶-Val²⁷-Arg²⁸(pbf)-Gly²⁹-Arg³⁰(pbf)-Gly³¹and-Wang resin, namely weighing five parts of 2g of peptide resin in a solid phase reactor, adding 20mL of DMF (dimethyl formamide) solutions of hydrazine hydrate with different concentrations into the solid phase reactor respectively, reacting for 10min, draining, adding 20mL of DMF solutions of hydrazine hydrate with different concentrations into the solid phase reactor respectively, reacting for 5min, draining, adding 20mL of DMF (dimethyl formamide) washing peptide resin, washing for 6 times in total, and adding 20mL of methanol for washing to obtain the peptide resin without the Dde protection. With TFA: EDT-water-95: 2.5:2.5 lysate, 10mL/g, was cleaved at room temperature for 2h to obtain crude intermediate peptide, which was detected by HPLC as follows:

removing Dde by using a DMF solution containing 1.5% hydrazine hydrate to obtain an intermediate with the purity of 86.34% and the maximum single impurity of 7.309%;

removing Dde by using a DMF solution containing 2% hydrazine hydrate to obtain an intermediate with the purity of 96.59% and the maximum single impurity of 1.97%;

removing Dde by using a DMF solution containing 3% hydrazine hydrate to obtain an intermediate with the purity of 85.32% and the maximum single impurity of 11.86%;

removing Dde by using a DMF solution containing 4% hydrazine hydrate to obtain an intermediate with the purity of 82.01% and the maximum single impurity of 17.49%;

the Dde was removed with 6% hydrazine hydrate in DMF to give an intermediate with a purity of 65.93% and a maximum single impurity of 30.37%.

In conclusion, the DMF solution containing 2% hydrazine hydrate has the best effect of removing Dde.

Example 6

The reaction end point was judged by ninhydrin color method using TBTU/DIEA and DIC/HOAt as condensing agents used for coupling side chain amino acids based on example 1, and the test results were as follows:

when Fmoc-AEEA is coupled, DIC/HOAt combination is adopted, and the coupling ninhydrin color development method is positive; the TBTU/DIEA combination is adopted for replacement, and the coupling ninhydrin color development method is negative;

when Fmoc-Glu-OtBu is coupled, DIC/HOAt combination is adopted, and the positive is obtained by a coupling ninhydrin color development method; the TBTU/DIEA combination is adopted for replacement, and the coupling ninhydrin color development method is negative;

when coupling octadecanedioic acid mono-tert-butyl ester, adopting DIC/HOAt combination, and coupling ninhydrin coloration method to make it be positive; the replacement was performed by TBTU/DIEA combination, and the color development of ninhydrin coupling was negative.

The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for synthesizing semaglutide, which is characterized by comprising the following steps:

2. The method of claim 1, wherein the S-Metroglucide is synthesized,

in the step (1), the step (c),

the substitution degree of Fmoc-Gly-Wang resin is 0.3-0.6 mmol/g;

3. The method of claim 1, wherein the S-Metroglucide is synthesized,

in the step (1), the step (c),

the solvent used for coupling the amino acid is DMF and/or DCM;

the feeding amount of the amino acid is 2-5 times of the mole number of Fmoc-Gly-Wang resin when the amino acid is coupled;

r is Cbz, Alloc or Dde protecting group.

4. The method of claim 1, wherein the S-Metroglucide is synthesized,

in the step (2),

5. The method of claim 1, wherein the S-Metroglucide is synthesized,

in the step (2),

the solvent used for coupling the side chain amino acid is DMF and/or DCM;

6. The method of claim 1, wherein the S-Metroglucide is synthesized,

in the step (3), the step (c),

7. The method of claim 1, wherein the S-Metroglucide is synthesized,

in the step (3), the step (c),

8. The method of claim 1, wherein the S-Metroglucide is synthesized,

in the step (4)

the cracking time is 1-3h, and the cracking temperature is 10-40 ℃.

9. The method of claim 8, wherein the S-Metroglucide is synthesized,

TFA in lysate: a: the volume ratio of B is (90-95): (5-2.5): (5-2.5), the using amount of the lysis solution is 6-10mL/g of the semaglutide full-protection peptide resin.

10. The method of claim 1, wherein the S-Metroglucide is synthesized,

in the step (4)

The reagent used for precipitation is diethyl ether or methyl tert-butyl ether.