CN111153983B

CN111153983B - Semisynthesis preparation method of somaglutide

Info

Publication number: CN111153983B
Application number: CN202010165273.7A
Authority: CN
Inventors: 吴志猛; 戴士杰; 周志昉
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2022-07-01
Anticipated expiration: 2040-03-11
Also published as: CN111153983A

Abstract

The invention relates to a polypeptide synthesis technology, and belongs to the technical field of pharmaceutical chemistry. The invention provides a preparation method of somagluteptide, which is based on a natural chemical linking method (STL) of serine and threonine, so that the somagluteptide has high purity and yield, few reaction steps and simple operation, and is beneficial to realizing large-scale preparation of the somagluteptide.

Description

Semisynthesis preparation method of somaglutide

Technical Field

The invention relates to a polypeptide synthesis technology, and belongs to the technical field of pharmaceutical chemistry.

Background

Compared with other hypoglycemic agents on the market, the somaglutide has better clinical treatment effect in controlling and maintaining blood sugar level, is expected to become a long-acting hypoglycemic agent which dominates the diabetes drug market in a new generation, and therefore, the efficient and economic synthesis process of the somaglutide is especially important.

The solid phase synthesis (SPPS) method is mainly adopted for the synthesis method of the somaltulipide reported at present, and the advantages of the solid phase synthesis are mainly shown in that the initial reactants and the products are connected on a solid phase carrier, so that all reactions can be carried out in one reaction container, the automation operation is convenient, the products with high yield can be obtained by adding excessive reactants, and the products can be easily separated. However, the use of protected amino acids is combined with the continuous use of excess reactants and, in addition, each amino acid extension in solid phase polypeptide synthesis requires substantial washing operations, making the process relatively expensive. In addition, the formation of secondary structures during the synthesis of polypeptides by SPPS often results in a reduction in the efficiency of the individual synthetic steps. Impurities are often deletion peptides that have lost one or more amino acids in the final sequence. These impurities, due to their similar polarity to the target peptide, may be difficult to achieve good separation from the target peptide, resulting in contamination of the product with the missing peptide.

The currently reported method for synthesizing the somaglutide needs to gradually couple 2- (2- (2-aminoethoxy) ethoxy) acetic acid, Glu and octadecanedioic acid in the process of synthesizing the fatty chain side chain, so that the problems of strong steric hindrance effect, difficult coupling reaction and low product yield are easily caused due to the long side chain; in addition, although the reported method for synthesizing the somaglutide by sequential coupling and fragment simplifies the step of connecting the side chain of the fatty chain, the side chain Lys at the 26-position of the somaglutide needs to be protected by special protecting agent groups (Mmt, Alloc) before connecting the side chain of the fatty chain, so that special deprotection reagents (Pd (PPh) are also needed to be used for deprotection₃)₄) Or repeated deprotection treatments, resulting in large yieldsThe production cost is increased.

Therefore, intensive research into a preparation method of somaglutide, which is efficient and economical, is required at present.

Disclosure of Invention

In order to achieve the above object, the present invention provides the following technical solutions:

a process for the semi-synthetic preparation of somaglutide, comprising the steps of:

step 1, coupling and acid hydrolysis are carried out on a compound I with the following formula and Arg34GLP-1(11-37) (SEQ NO:1) through chemical linking reaction to obtain a compound II Aib8 and Arg34GLP-1(7-37) (SEQ NO: 2);

step 2, carrying out activated ester reaction on the compound 17- ((S) -1-tert-butoxycarbonyl-3- {2- [2- ({2- [2- (carbonylmethoxy) ethoxy ] ethylcarbamoyl } methoxy) ethoxy ] ethylcarbamoyl } propylcarbamoyl) heptadecane tert-butyl ester to obtain the following compound III;

step 3, connecting the compound III to a side chain amino of 26-Lys in a compound II sequence, and reacting to obtain a compound IV;

and 4, deprotecting the compound IV, precipitating with glacial ethyl ether, purifying, and freeze-drying to finally obtain the somaglutide.

Chemoselective reaction of a peptide fragment having a salicylaldehyde ester (SAL) at the C-terminus with an unprotected peptide fragment of the other side chain having an N-terminal serine or threonine residue (carrying a 1, 2-hydroxylamine bifunctional group) to provide an isolatable intermediate having an N, O-benzylidene acetal structure at the ligation site. Without purification, the N, O-benzylidene acetal intermediate can be converted by simple acid hydrolysis to the target full-length polypeptide product with a natural peptide bond at the ligation site. Therefore, 2 peptides used in the step are peptide fragments with unprotected side chains, and can be specifically coupled to generate a target compound without generating side reactants.

In one embodiment, the step of synthesizing compound i in step 1 is:

step 1-1, coupling Boc-His (Trt) -Aib-Glu (otbu) -Gly-OH and salicylaldehyde dimethyl acetal at the reaction temperature of PyBOP and DIEA at 0 ℃ to obtain an intermediate Boc-His (Trt) -Aib-Glu (otbu) -Gly-O-salicylaldehyde dimethyl acetal, namely a compound 0;

and 1-2, carrying out full deprotection, glacial ethyl ether precipitation and freeze-drying on the compound 0 to obtain a compound I.

In one embodiment, Arg34GLP-1(11-37) (SEQ NO:1) at step 1 is derived from a microbial fermentation.

In one embodiment, the reaction of step 1 is carried out under a buffer system of pyridine acetate, and the molar ratio of pyridine to acetic acid is 1: 0-120, preferably 1: 120 of a solvent; the reaction temperature is 20-55 ℃, preferably 40 ℃.

In one embodiment, the reaction of step 3 is carried out in a boric acid buffer system, and the pH is 8-11, preferably 9.5; the concentration of the boric acid in the buffer system is 0.01 mol/L-0.1 mol/L, and preferably 0.05 mol/L.

In one embodiment, compound III described in step 3 is added dropwise at a uniform rate to compound II.

In one embodiment, the compound III in the step 3 is uniformly dripped into the compound II within 0.1-1 hour, and the preferable dripping time is 0.5 hour.

When the dripping speed is too high, the side chain amino groups of N-terminal and 26-position Lys are easily generated and are simultaneously connected with the side chain of the fatty chain, which is not beneficial to the conversion of the reaction; when the dropping speed is too slow, the dropping time is increased, resulting in prolonged production period and increased production cost.

In one embodiment, the deprotection method of step 4 is treatment with a cleavage reagent, which is TFA, H₂One group of O and TIPS or TFA, TIS, H₂O and DODT.

In one embodiment, the cleavage reagents TFA, H₂The volume ratio of O to TIPS is 95:2.5: 2.5; the cleavage reagents TFA, TIS, H₂The volume ratio of O to DODT was 92.5:2.5:2.5: 2.5.

Advantageous effects

Experimental results prove that the soma peptide prepared by the semi-synthesis process route provided by the invention has the advantages of high purity, high yield, few reaction steps and simplicity in operation, and is beneficial to realizing large-scale preparation of the soma peptide.

1. The invention provides a natural chemical linking method (STL) based on serine and threonine, which comprises the steps of carrying out chemical selective linking on tetrapeptide of which the C end is Salicylaldehyde (SAL) and a peptide skeleton Arg34GLP-1(11-37) of which the N end is a threonine residue to obtain an intermediate of which the linking position has an N, O-benzylidene acetal structure, and converting the intermediate into a target full-length polypeptide product [ Aib8, Arg34GLP-1(7-37) ] with natural peptide bonds at the linking position through simple acidolysis. The two steps are carried out in the same container system, no intermediate is required to be purified, and the conversion rate of the product of the two steps can reach 85% under the optimal reaction condition. The method is characterized in that it uses peptide fragments with unprotected side chains, does not cause epimerization of amino acids during the reaction, is easy to handle, is rapidly converted, and forms natural peptide bonds in the form of Xaa-Ser and Xaa-Thr at the ligation site. The reaction process can not generate deletion peptide losing one or more amino acids, and the problem that the product is polluted by the deletion peptide is solved.

2. In the invention, the side chain activated ester is chemically and selectively coupled to the side chain amino group of Lys at position 26 of the amino acid sequence in a peptide skeleton [ Aib8, Arg34GLP-1(7-37) ] (SEQ NO:2) under 0.05mol/L boric acid buffer solution with the pH value of 9.5. The reaction time was 0.5 hours and the product conversion was 95%. The step does not need special chemical reagent protection and repeated deprotection treatment on the amino group of the Lys side chain, and solves the problems of strong steric hindrance effect, difficult coupling reaction and low product yield caused by gradual coupling of each group on the side chain. The method greatly simplifies the process flow, shortens the synthesis period, reduces the generation of waste liquid, has few byproducts, high product yield and reduced production cost, and is beneficial to large-scale industrial production of the somaglutide.

3. The strategy of the invention can promote the chemical synthesis of polypeptide protein drugs. Serine and threonine account for 12.7% of the protein in total, and many polypeptide protein drugs also contain abundant serine and threonine residues inside. The small peptide fragments (less than 20 amino acids) are prepared by using a recombinant gene technology or a linear SPPS technology, then the chemical ligation (STL) strategy of serine and threonine is applied to the coupling of 2 or more peptide fragments, the synthesis steps are few, and the product conversion rate is high, so that the production cost of polypeptide protein medicines with medium size (20-100 amino acids) can be remarkably reduced, and the large-scale production of the polypeptide protein medicines is facilitated.

Drawings

FIG. 1 is a scheme of the process for the semi-synthesis of the Somaltulip of the present invention;

FIG. 2 MAIDI-TOF mass spectrum of Compound I prepared in example 2;

FIG. 3 an analytical liquid phase diagram of Compound I prepared in example 2;

FIG. 4 is an analytical liquid phase diagram of acidolysis of the reaction mixture in example 3;

FIG. 5 MAIDI-TOF mass spectrum of Compound II prepared in example 4;

FIG. 6 analytical liquid phase diagram of Compound II prepared in example 4;

FIG. 7 is an analytical liquid phase diagram of the reaction solution in example 6;

FIG. 8 MAIDI-TOF mass spectrum of Compound IV prepared in example 7;

FIG. 9 an analytical liquid phase diagram of Compound IV prepared in example 7;

figure 10 MAIDI-TOF mass spectrum of the somaglutide prepared in example 9;

figure 11 an analytical liquid phase diagram of the somaglutide prepared in example 9;

Detailed Description

Some of the abbreviations commonly used in the present invention have the following meanings:

(1) NCL: native chemical ligation

(2) STL serine/threonine ligation reaction

(3) Pyridine, Pyridine

(4) AcOH acetic acid

(5) mol to mol

(6) TFA trifluoroacetic acid

(7) MeCN acetonitrile

(8)H₂O is water

(9) TIPS triisopropylsilane

(10) Boc tert-butyloxycarbonyl

(11) Trt is trityl, triphenylmethyl

(12) Tert-butyl ester of Otbu

(13) PyBOP Benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate

(14) DIEA/DIPEA N, N-diisopropylethylamine

(15) Aib 2-methylalanine

(16) SAL salicylaldehyde

(17) GLP-1 glucagon-like peptides

(18) Lys lysine

(19) Arg: arginine

(20) Mmt: terephthalic acid monomethyl ester

(21) And (3) Alloc: allyl carboxyl group

(22) Pd (PPh 3): tetrakis (triphenylphosphine) palladium

(23) DMF: n, N-dimethylformamide

(24) DCM: methylene dichloride

(25) HSTU: n, N, N ', N' -tetramethylurea-O- (N-succinimidyl) hexafluorophosphate

(26) THF: tetrahydrofuran (THF)

Illustrating according to what is contained in the claims

In order to make those skilled in the art better understand the technical solution of the present invention, the present invention is further illustrated below with reference to the following embodiments.

FIG. 1 shows a route for the semi-synthesis of the somaglutide of the invention.

EXAMPLE 1 preparation of crude Compound I

Boc-His (Trt) -Aib-Glu (Otbu) -Gly-OH (commercially available: 1g, 1.213mmol from Gill Biochemical (Shanghai)), salicylaldehyde dimethyl acetal (407.8mg, 2.426mmol), PyBOP (946.8mg, 1.8195mmol) was weighed, dissolved with 50mL of DMF, and 600uLDIEA (3.639mmol) was added under ice-water bath, and magnetically stirred at 0 ℃ for 20 hours. The complete conversion of the reaction was checked by liquid phase. After the reaction is finished, concentrating the reaction solution under reduced pressure, adding DCM for redissolving, washing with saturated saline for three times, putting the collected DCM solution into a certain amount of anhydrous sodium sulfate for drying, standing for 0.5 hour, performing suction filtration, collecting filtrate, and draining to obtain 1.89g of a compound 0 crude product for later use.

A sample of all 0 of the above compound was taken and 50mL of cleavage reagent (TFA/H) was added₂0/TIPS, 95:2.5:2.5, v/v/v), magnetically stirred at room temperature for 2 hours. After the reaction, the reaction solution was concentrated to a volume of 15mL, slowly added dropwise to 100mL of diethyl ether for precipitation, centrifuged, washed with anhydrous diethyl ether for 2 times, and dried under vacuum to obtain 1.12g of a solid, i.e., Compound I.

EXAMPLE 2 purification of crude Compound I

500mg of the crude compound I prepared in example 1 are weighed, dissolved in 15mL of distilled Water and purified by semipreparative purification using the Water1525 system at a wavelength of 214nm on a 20X 250mm reverse phase C column₁₈Column, column temperature 37 ℃, mobile phase of water containing 0.1% TFA (phase a) and acetonitrile containing 0.1% TFA (phase B), flow rate 8ml/min, gradient: b%: 20-60% for 30min, collecting the target components, and collecting the liquidConcentrating under reduced pressure.

The detection is carried out by using MAIDI-TOFMS, the result is shown in figure 2, and the calculation of C is carried out by using MAIDI-TOFMS₂₄H₃₀N₆O₈Theoretical molecular weight [ M + H]⁺m/z＝531.220,[M+Na]⁺553.202, 531.098, 553.081 was observed. The above concentrate was lyophilized to give 195mg of Compound I in the form of a white solid powder with a yield of 80%. The pure product was analyzed by an analytical liquid phase analysis (using liquid phase condition 1), and the purity was 90% as shown in fig. 3.

EXAMPLE 3 Synthesis of Compound II

Compound I (50mg, 0.094mmol) and Arg34GLP-1(11-37) (150mg, 0.05mmol) were weighed, dissolved in 100ul pyridine acetate buffer (acetic acid: pyridine, mol: mol, 120: 1), and reacted at 35 ℃ with magnetic stirring for 20 hours. The solvent was removed by blow drying, and the residue was then subjected to acid hydrolysis using 10mL of acid hydrolysis solution (TFA/water, 75/25, v/v) and magnetically stirred at room temperature for 2 hours. The solvent was removed by blow drying, and lyophilized to obtain 230mg of a reaction mixture. The reaction mixture was subjected to analytical liquid phase analysis (using liquid phase conditions: liquid phase analysis using Agilent 1260 system, wavelength 214nm, reverse phase C18 column of 4.6X 250mm, column temperature 35 ℃, mobile phase 0.1% water (phase A) and 0.1% acetonitrile (phase B), flow rate 1mL/min, gradient: B%: 30% -45%, 0-20 min.), and as a result, as shown in FIG. 4, the retention time of the product in the reaction mixture was 14.936min and the product conversion was 85%. The product is the crude product of the compound II [ Aib8, Arg34GLP-1(7-37) ].

EXAMPLE 4 purification of crude Compound II [ Aib8, Arg34GLP-1(7-37) ], preparation of

40mg of crude linear peptide [ Aib8, Arg34GLP-1(7-37) ] compound II (SEQ NO:2) prepared in example 3 was weighed, dissolved in a mixed solution of acetonitrile and Water (1/1, v/v), and semi-preparative purified by a Water1525 system at a wavelength of 214nm on a 20X 250mm reversed phase C18 column at 37 ℃ in a mobile phase of 0.1% Water (phase A) and 0.1% acetonitrile (phase B) at a flow rate of 8ml/min, and gradient: b%: 36 to 54 percent, collecting target components in 30min, and carrying out reduced pressure concentration on the collected liquid.

The detection is carried out by using MAIDI-TOFMS, the result is shown in figure 5, and the calculation of C is carried out by using MAIDI-TOFMS₁₅₂H₂₃₀N₄₂O₄₇Theoretical molecular weight [ M + H]⁺m/z 3,396.698, 3,397.006 was observed. The concentrate was lyophilized to give 14mg of Compound II as a white solid powder in a yield of 45%. The pure product was analyzed by an analytical liquid phase analysis (using a liquid phase condition: liquid phase analysis using Agilent 1260 system, wavelength 214nm, reverse phase C18 column of 4.6X 250mm, column temperature 35 ℃, mobile phase 0.1% water (phase A) and 0.1% acetonitrile (phase B), flow rate 1mL/min, gradient B%: 20-55% -90% -90% -20% -20%, 0-10-20-25-26-35 min.) and, as shown in FIG. 6, the purity was 95% by analysis.

EXAMPLE 5 preparation of Compound III

Tert-butyl 17- ((S) -1-tert-butoxycarbonyl-3- {2- [2- ({2- [2- (carbonylmethoxy) ethoxy ] ethylcarbamoyl } methoxy) ethoxy ] ethylcarbamoyl } propylcarbamoyl) heptadecanoate (3.88g, 4.58mmol), HSTU (3.30g, 9.17mmol) were each weighed, dissolved in 350mL of THF, added with 1.5mL of LDIPEA (1.18mg, 9.17mmol), and magnetically stirred at room temperature for 4 hours. The complete conversion of the reaction was checked by analytical liquid phase. After the reaction, the reaction solution was concentrated under reduced pressure, DCM was added to redissolve, washed with saturated brine three times, the collected DCM solution was dried over anhydrous sodium sulfate, left to stand for 0.5 hour, and filtered with suction to obtain 4.7147g of sample with a yield of 121%. The product is compound III

EXAMPLE 6 Synthesis of Compound IV

Weighing the linear peptide [ Aib8, Arg34GLP-1(7-37)]Compound II (SEQ NO:2) (60mg, 17.1umol) was dissolved in 6mLH₃BO₃A boric acid solution (0.05mol, pH 9.5) and 3mL of acetonitrile were magnetically stirred at room temperature, and 3mL of an acetonitrile solution (10umol/mL) containing the compound iii prepared in example 8 was uniformly dropped to the above solution over 0.5 hour by means of a syringe pump. The complete conversion of the reaction was checked by liquid phase. After the reaction is finished, the conversion condition of the reaction product is detected by analyzing a liquid phase (liquid phase condition: performing liquid phase analysis by adopting an Agilent 1260 system, the wavelength is 214nm, a chromatographic column is a reversed phase C18 column with 4.6 multiplied by 250mm, the column temperature is 35 ℃, a mobile phase is 0.1 percent of water (phase A) and 0.1 percent of acetonitrile (phase B), the flow rate is 1mL/min, the gradient is B percent, 20-55-90-20 percent and 0-10-20-25-26-35 min.), the result is shown in figure 7, the retention time of the product is 15.623min, and the conversion rate of the product is 95 percent. The reaction solution was then concentrated under reduced pressure and lyophilized to give 120mg of crude product, which was then ready for purification.

EXAMPLE 7 purification of Compound IV

12mg of the crude product prepared in example 6 above was weighed, dissolved in 1mL of a mixed solvent of acetonitrile and water (3/1, v/v), and semi-preparative purified using a Waters1525 system at a wavelength of 214nm on a 10X 250mm reverse phase C18 column at 37 ℃ in a mobile phase of 0.1% water (phase A) and 0.1% acetonitrile (phase B) at a flow rate of 8mL/min, gradient: b%: 55-70%, 30min, collecting the target component, and concentrating under reduced pressure.

The detection is carried out by using MAIDI-TOFMS, the result is shown in figure 8, and the theoretical molecular weight C is calculated by using MAIDI-TOFMS₁₉₅H₃₀₇N₄₅O₅₉[M+H]⁺m/z 4,224.248, 4,224.476 was observed. The above concentrate was lyophilized to give 3mg of compound IV in 50% yield. The pure compound IV was analyzed by an analytical liquid phase analysis (using a liquid phase condition: a reverse phase C18 column with a wavelength of 214nm, a chromatographic column of 4.6X 250mm, a column temperature of 35 ℃, a mobile phase of 0.1% water (phase A) and 0.1% acetonitrile (phase B), a flow rate of 1mL/min, a gradient of B%: 20-55% -90% -90% -20% -20%, 0-10-20-25-26-35 min.) and, as a result, the purity was 95% by analysis as shown in FIG. 9. The product is the compound IV.

EXAMPLE 8 deprotection of Compound IV

3mg of Compound IV (0.7umol) prepared in example 7 above was weighed, 0.5mL of a cleavage reagent (TFA/water/TIPS, 95:2.5:2.5, v/v/v) was added, and magnetic stirring was performed at room temperature for 2 hours. After the reaction is finished, slowly dripping the reaction solution into 20ml of diethyl ether for precipitation, centrifuging, washing for 2 times by anhydrous diethyl ether, drying in vacuum to obtain a solid, and waiting for purification.

Example 9 purification of Somaltulip

The crude product prepared in example 8 was dissolved in 1mL of a mixed solvent of 50% acetonitrile and 50% water, and subjected to semipreparative purification using a Waters1525 system at a wavelength of 214nm on a 10X 250mm reverse phase C18 column at a column temperature of 37 ℃ and a mobile phase of 0.1% water (phase A) and 0.1% acetonitrile (phase B) at a flow rate of 4mL/min, gradient: b%: 42-57 percent, collecting target components for 30min, and concentrating under reduced pressure.

The detection is carried out by using MAIDI-TOFMS, the result is shown in figure 10, and the theoretical molecular weight C is calculated by using MAIDI-TOFMS₁₈₇H₂₉₁N₄₅O₅₉[M+H]⁺m/z＝4,111.123,[M+2H]²⁺m/z 2,056.566, 4,112.857; 2,056.969. The above concentrate was lyophilized to obtain 2mg of somaglutide in 67% yield. The results of analytical liquid phase analysis (using liquid phase conditions: liquid phase analysis using Agilent 1260 system, wavelength 214nm, chromatographic column of 4.6X 250mm reversed phase C18 column, column temperature 35 ℃, mobile phase of 0.1% water (phase A) and 0.1% acetonitrile (phase B), flow rate 1mL/min, gradient B%: 20-55% -90% -90% -20% -20%, 0-10-20-25-26-35 min.) were obtained for the pure product of Somalobutate, and the purity was 98% by analysis, as shown in FIG. 11.

Sequence listing

<120> semisynthetic preparation method of somaglutide

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys

1 5 10 15

Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

20 25

Claims

1. A preparation method of somaglutide, which is characterized by comprising the following steps:

step 1, coupling and acid hydrolysis are carried out on a compound I shown in the following formula and Arg34GLP-1(11-37) shown in SEQ NO:1 through chemical linking reaction to obtain a compound II Aib8 shown in SEQ NO:2 and Arg34GLP-1 (7-37);

step 2, carrying out activated ester reaction on a compound 17- ((S) -1-tert-butoxycarbonyl-3- {2- [2- ({2- [2- (carboxymethoxy) ethoxy ] ethylcarbamoyl } methoxy) ethoxy ] ethylcarbamoyl } propylcarbamoyl) heptadecane tert-butyl ester to obtain a following compound III;

and 4, carrying out deprotection, diethyl ether precipitation, purification and freeze-drying on the compound IV to finally obtain the somatomedin.

2. The method for preparing the somaglutide according to claim 1, wherein the step of synthesizing the compound i in step 1 comprises:

and (1) carrying out full deprotection, glacial ethyl ether precipitation and freeze-drying on the compound 0 after purification to obtain a compound I.

3. The method for preparing somaglutide according to claim 1, wherein Arg34GLP-1(11-37) shown in SEQ No. 1 in step 1 is derived from microbial fermentation.

4. The preparation method of somaglutide according to claim 1, wherein the reaction of step 1 is performed under a buffer system of pyridine acetate, and the molar ratio of pyridine to acetic acid is 1: 0 to 120 parts; the temperature of the reaction is between 20 and 55 ℃.

5. The preparation method of the somaglutide according to claim 1, wherein the reaction of step 3 is performed in a boric acid buffer system, and the pH is 8-11; the concentration of boric acid in the buffer system is 0.01-0.1 mol/L.

6. The method for preparing somaglutide according to claim 1, wherein compound iii is added dropwise to compound ii at a constant rate during the reaction in step 3.

7. The method for preparing somaglutide according to claim 1 or 6, wherein compound III is added dropwise to compound II at constant speed within 0.1-1 hour in the reaction of step 3.

8. The method for preparing the somaglutide according to claim 1, wherein the deprotection method in step 4 is a treatment with a cleavage reagent, and the cleavage reagent is TFA or H₂One group of O and TIPS or TFA, TIS, H₂O and DODT.

9. The method for preparing somaglutide according to claim 8, wherein the cleavage reagents TFA, H₂The volume ratio of O to TIPS is 95:2.5: 2.5; the cleavage reagents TFA, TIS, H₂The volume ratio of O to DODT was 92.5:2.5:2.5: 2.5.

10. The method for preparing somaglutide according to claim 1 or 4, wherein the reaction of step 1 is performed under a buffer system of pyridine acetate, and the molar ratio of pyridine to acetic acid is 1: 120 of a solvent; the temperature of the reaction was carried out at 35 ℃.

11. The process for the preparation of somaglutide according to claim 1 or 5, wherein the reaction of step 3 is carried out in a borate buffered system, with a pH range of 9; the concentration of boric acid in the buffer system is 0.05 mol/L.

12. The method for preparing somaglutide according to claim 1 or 6, wherein compound iii is added dropwise to compound ii at constant speed within 0.5 hour in the reaction of step 3.