阅读说明：本技术 一种索玛鲁肽的合成方法 (Synthetic method of somaglutide ) 是由 袁慧星 黄嘉诚 汪伟 尹传龙 于 2021-09-30 设计创作，主要内容包括：本发明涉及多肽合成技术领域,尤其涉及一种索马鲁肽的合成方法。本发明首先固相合成片段I(第1～19位),液相合成侧链单体(第20位氨基酸及其侧链),然后固相合成偶联侧链单体和其余氨基酸的片段II(第21～31位),最后将片段I和片段II缩合,精制,获得索马鲁肽。本发明采用特殊的合成策略,提高了粗肽纯度,收率大幅度提高,同时缩短了生产周期,降低了成本、且片段和侧链合成工艺更有利于规模化大生产,同时解决了三废处理中对钯残留的难题。(The invention relates to the technical field of polypeptide synthesis, in particular to a synthetic method of Somaloutide. According to the invention, firstly, a fragment I (1 st to 19 th positions) is synthesized in a solid phase, a side chain monomer (20 th position amino acid and a side chain thereof) is synthesized in a liquid phase, then, a fragment II (21 st to 31 th positions) of the side chain monomer and other amino acids is synthesized in a solid phase, and finally, the fragment I and the fragment II are condensed and refined to obtain the Somaloude. The method adopts a special synthesis strategy, improves the purity of the crude peptide, greatly improves the yield, shortens the production period, reduces the cost, is more favorable for large-scale production by the fragment and side chain synthesis process, and solves the problem of palladium residue in the three-waste treatment.)

1. A method for synthesizing Somalutide is characterized by comprising the following steps:

a) synthesizing a segment I with protecting groups coupled on the N end of an amino acid sequence shown in SEQ ID NO. 1, the His side chain, the Glu side chain, the Thr side chain, the Ser side chain, the Asp side chain, the Tyr side chain and the Gln side chain;

b) synthesizing a side chain monomer Fmoc-Lys (AEEA-AEEA-gamma-Glu-octaneedioICAcid) -OH;

c) coupling protective amino acids one by one on a solid phase carrier according to the sequence from the C end to the N end of an amino acid sequence shown in SEQ ID NO. 2, coupling the side chain monomer synthesized in the step b) to obtain peptide resin, and cracking to obtain a fragment II;

d) condensing the fragment I and the fragment II according to the amino acid sequence of the Somaloutide, and refining to obtain the Somaloutide.

2. The synthesis method according to claim 1, wherein step a) is specifically:

Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -Phe-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Boc-His (trt) -Aib-OH are coupled one by one on a solid support in the order from the C-terminal to the N-terminal of the amino acid sequence shown in SEQ ID NO. 1, and fragment I is obtained by cleavage and purification.

3. The method of synthesis according to claim 2, wherein in step a), the solid support is 2-CTC Resin, the degree of substitution of Fmoc-Ala-2 CTCs is 0.2-0.6 mmol/g;

the coupling agent is DIC/HOBt, and the coupling time is 1-3 hours.

4. The synthesis method of claim 2, wherein in the step a), the cracked lysate consists of TFA and DCM in a volume ratio of 1: 99-5: 95, or consists of TFE and DCM in a volume ratio of 1: 3-1: 4; the cracking temperature is room temperature, and the time is 2 hours;

the purification is as follows: concentrating the crude product of the fragment I after cracking to dryness, adding ethyl acetate for dissolving, washing by adopting a saturated sodium chloride aqueous solution and a citric acid aqueous solution, collecting an organic phase, and concentrating.

5. The synthesis method according to claim 1, wherein step b) comprises:

b1) mixing Fmoc-Lys-OH and Boc-AEEA-OSu, adding DIPEA, reacting at room temperature for 2 hours, concentrating, adding HCl/EA saturated solution, removing Boc protecting group, and concentrating to dryness; repeating the above steps, coupling a second Boc-AEEA-OSu, and refining the coupled product to obtain product 1: Fmoc-Lys (AEEA-AEEA) -OH;

b2) taking tert-butyl octadecanoate monoester, adding THF, Boc anhydride and anhydrous pyridine, reacting for 0.5 hour at room temperature, then adding NH2-Glu (OtBu) -OH, stirring for reacting for 2 hours, adding diethyl ether, and separating out a solid to obtain a product 2: γ -Glu (OtBu) -OctadecaneedioICAcid;

b3) and (3) carrying out condensation reaction on the product 1 and the product 2 for 2 hours under the action of DCC and DCM to obtain a side chain monomer Fmoc-Lys (AEEA-AEEA-gamma-Glu-octanedioic acid) -OH.

6. The synthesis method according to claim 1, characterized in that the refining is: and (3) adding the coupling product into ethyl acetate to dissolve, dropwise adding petroleum ether to separate out a precipitate, and filtering.

7. The synthesis method according to claim 1, wherein step c) is specifically:

after 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 and Fmoc-Glu (OtBu) -OH are coupled one by one on a solid phase carrier according to the sequence from C end to N end of an amino acid sequence shown in SEQ ID NO. 2, side chain monomers Fmoc-Lys (AEEA-AEEA-gamma-Glu-Octadecanoacid) -OH are coupled, and fragment II is obtained after cracking and purification.

8. The method of synthesis according to claim 1, wherein in step c) the solid support is 2-CTC Resin, the degree of substitution of Fmoc-Ala-2 CTCs is 0.2-0.6 mmol/g;

the solvent of the coupling reaction is dichloromethane and/or DMF; the coupling agent is a combination of DIPCDI and a condensing agent A or a combination of DIPEA and a condensing agent B, wherein the condensing agent A is HOBt or HOAt, and the condensing agent B is PyBOP, PyAOP, HATU, HBTU or TBTU.

9. The method of claim 1, wherein in step c),

the cleaved lysate consists of TFA, TIS and DCM in a molar ratio of 1.2: 1.1; the cracking temperature is room temperature, and the time is 3 hours;

the purification is that the n-hexane of the fragment II after cracking is pulped, stirred, filtered and dried under reduced pressure.

10. The synthesis process according to claim 1, wherein in step d), the condensation reaction is carried out in DMF; condensing agents for the condensation include HOSu and DIC, and the temperature for the condensation is room temperature and the time is 2 hours; and the condensation reaction also comprises the steps of filtering and collecting filtrate after the condensation reaction is finished.

Technical Field

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

Background

Somarumtide (Semaglutide) is a long-acting GLP-1 analogue developed by Nove Nordisk. 12/2017, 5/FDA approved, marketed in the united states.

Its structure (peptide sequence):

H-His⁷-Aib⁸-Glu⁹-Gly¹⁰-Thr¹¹-Phe¹²-Thr¹³-Ser¹⁴-Asp¹⁵-Val¹⁶-Ser¹⁷-Ser¹⁸-Tyr¹⁹-Leu²⁰-Glu²¹-Gly²²-Gln²³-Ala²⁴-Ala²⁵-Lys²⁶(AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu²⁷-Phe²⁸-Ile²⁹-Ala³⁰-Trp³¹-Leu³²-Val³³-Arg³⁴-Gly³⁵-Arg³⁶-Gly³⁷-OH

AEEA is a special amino acid, 2- (2- (2-aminoethoxy) ethoxy) acetic acid.

The concrete structure is as follows:

the existing synthesis processes of the somaglutide are roughly divided into two main types:

the first type is a gene recombination mode: (1) synthesizing a main chain [ Arg34] GLP-1- (9-37) peptide by a gene recombination method; (2) solid-phase synthesis of a side chain fragment, namely, 17- ((S) -1-tert-butoxycarbonyl-3- { 2- [ 2- ({ 2- [ 2- (2, 5-dioxopyrrolidin-1-yloxycarbonylmethoxy) ethoxy ] ethylcarbamoyl } methoxy) ethoxy ] ethylcarbamoyl } propylcarbamoyl) heptadecanoic acid tert-butyl ester; (3) Boc-His (Boc) -Aib-OH. Then coupling the side chain segment and Boc-His (Boc) -Aib-OH in sequence, and finally obtaining the crude peptide of the Somalutide by trifluoroacetic acid cleavage. Such as US9732137B2, CN 105154498.

The second type is the Fmoc solid phase synthesis format. The synthesis methods are reported more frequently, and the standard Fmoc solid phase and the Lys deprotection group are sequentially carried out to carry out the side chain continuous coupling synthesis, and the fragment condensation is also carried out.

Patents CN104017062A and US8129343 are the preparation of crude [ Aib8, Arg34] GLP-1- (7-37) peptide by standard Fmoc solid phase peptide synthesis and purification by preparative HPLC to obtain pure unprotected [ Aib8, Arg34] GLP-1- (7-37). [ Aib8, Arg34] GLP-1- (7-37) and 17- ((S) -1-tert-butoxycarbonyl-3- { 2- [ 2- ({ 2- [ 2- (2, 5-dioxopyrrolidin-1-yloxycarbonylmethoxy) ethoxy ] ethylcarbamoyl } methoxy) ethoxy ] ethylcarbamoyl } propylcarbamoyl) heptadecanoic acid tert-butyl ester were reacted in 2:1 acetonitrile/water and purified by preparative HPLC to give the title compound.

Patent CN201210501406 adopts Fmoc solid phase peptide synthesis to prepare [ Aib8, Arg34] GLP-1- (7-37) full-protection peptide resin, removes Lys26 epsilon amino protecting group, couples 17- ((S) -1-tert-butoxycarbonyl-3- { 2- [ 2- ({ 2- [ 2- (2, 5-dioxopyrrolidin-1-yloxycarbonylmethoxy) ethoxy ] ethylcarbamoyl } methoxy) ethoxy ] ethylcarbamoyl } propylcarbamoyl) heptadecanoic acid tert-butyl ester to Lys26 epsilon amino, and obtains the product after cracking and purification.

In patents CN109456401B and CN108059666B, six fragments (1-4, 5-9, 10-16, 17-22, 23-27 and 28-31) are synthesized by a liquid phase or solid phase method, and then the fragments are coupled by a solid phase method to obtain a fully-protected peptide resin, and are cracked to obtain crude somalutide peptide.

Among the above methods, the patents US9732137B2 and CN105154498 require the main chain synthesis by gene recombination, and the limitations of the gene recombination technology and the uncertainty of impurities in such synthesis limit the process. Patent CN201210501406 adopts successive coupling, Alloc is usually used at Lys26 epsilon amino protecting group, tetratriphenylphosphine palladium is needed to be removed, then side chain modification is carried out, palladium residue is introduced in the process, the price is high, the production cost is increased, meanwhile, the site is modified, and as the peptide chain is long, the steric hindrance is large, the modification difficulty is large, multiple feeding is needed, and the cost is further increased. The patents CN109456401B and CN108059666B adopt fragment condensation, do not avoid using a protecting group of a Lys site, and also adopt palladium tetratriphenylphosphine for removing, and then perform side chain modification. Although the steric hindrance is reduced, the division into 6 segments naturally increases the cost of the raw material and at the same time complicates the process.

Disclosure of Invention

In view of the above, the present invention provides a method for synthesizing somaglutide. The method obviously improves the purity of the somaglutide and the purity of the yield, reduces the production cost, shortens the production period and is environment-friendly.

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

the invention provides a method for synthesizing Somalutide, which comprises the following steps:

a) synthesizing a segment I with protecting groups coupled on the N end of an amino acid sequence shown in SEQ ID NO. 1, the His side chain, the Glu side chain, the Thr side chain, the Ser side chain, the Asp side chain, the Tyr side chain and the Gln side chain;

b) synthesizing a side chain monomer Fmoc-Lys (AEEA-AEEA-gamma-Glu-octaneedioic Acid) -OH;

c) coupling protective amino acids one by one on a solid phase carrier according to the sequence from the C end to the N end of an amino acid sequence shown in SEQ ID NO. 2, coupling the side chain monomer synthesized in the step b) to obtain peptide resin, and cracking to obtain a fragment II;

d) condensing the fragment I and the fragment II according to the amino acid sequence of the Somaloutide to obtain the Somaloutide.

In some embodiments, step a) is specifically:

Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -Phe-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Boc-His (trt) -Aib-OH are coupled one by one on a solid support in the order from the C-terminal to the N-terminal of the amino acid sequence shown in SEQ ID NO. 1, and fragment I is obtained by cleavage and purification.

In some embodiments, step a):

the solid phase carrier is 2-CTC Resin, and the replacement degree of Fmoc-Ala-2CTC is 0.2-0.6 mmol/g.

The coupling agent is DIC/HOBt, and the coupling time is 1-3 hours.

The cracking solution for cracking consists of TFA and DCM in a volume ratio of 1: 99-5: 95, or

The catalyst consists of TFE and DCM in a volume ratio of 1: 3-1: 4;

the cracking temperature is room temperature, and the time is 2 hours;

the purification is as follows: concentrating the crude product of the fragment I after cracking to dryness, adding ethyl acetate for dissolving, washing by adopting a saturated sodium chloride aqueous solution and a citric acid aqueous solution, collecting an organic phase, and concentrating.

In some embodiments, step b) specifically comprises:

b1) mixing Fmoc-Lys-OH and Boc-AEEA-OSu, adding DIPEA, reacting at room temperature for 2 hours, concentrating, adding HCl/EA saturated solution, removing Boc protecting group, and concentrating to dryness; repeating the above steps, coupling a second Boc-AEEA-OSu, and refining the coupled product to obtain product 1: Fmoc-Lys (AEEA-AEEA) -OH;

the refining is as follows: adding ethyl acetate to the coupling product for dissolving, dripping petroleum ether to separate out a precipitate, and filtering;

b2) taking tert-butyl octadecanoate monoester, adding THF, Boc anhydride and anhydrous pyridine, reacting for 0.5 hour at room temperature, then adding NH2-Glu (OtBu) -OH, stirring for reacting for 2 hours, adding diethyl ether, and separating out a solid to obtain a product 2: γ -Glu (OtBu) -Octadecaneedioic Acid;

b3) and (3) carrying out condensation reaction on the product 1 and the product 2 for 2 hours under the action of DCC and DCM to obtain a side chain monomer Fmoc-Lys (AEEA-AEEA-gamma-Glu-octanedioic Acid) -OH.

In some embodiments, step c) is specifically:

after 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 and Fmoc-Glu (OtBu) -OH are coupled one by one on a solid phase carrier according to the sequence from C end to N end of an amino Acid sequence shown in SEQ ID NO. 2, side chain monomers Fmoc-Lys (AEEA-AEEA-gamma-Glu-Octadecandioic Acid) -OH are coupled, and fragment II is obtained after cracking and purification.

In some embodiments, in step c),

the solvent of the coupling reaction is dichloromethane and/or DMF; the coupling agent is a combination of DIPCDI and a condensing agent A, or a combination of DIPEA and a condensing agent B, wherein the condensing agent A is HOBt or HOAt, and the condensing agent B is PyBOP, PyAOP, HATU, HBTU or TBTU; the cleaved lysate consists of TFA, TIS and DCM in a molar ratio of 1.2: 1.1;

in some embodiments, in step c),

the cracking temperature is room temperature, and the time is 3 hours; the purification is that the n-hexane of the fragment II after cracking is pulped, stirred, filtered and dried under reduced pressure.

In some embodiments, in step d), the condensation reaction is carried out in DMF; condensing agents for the condensation include HOSu and DIC, and the temperature for the condensation is room temperature and the time is 2 hours; and the condensation reaction also comprises the steps of filtering and collecting filtrate after the condensation reaction is finished.

According to the invention, firstly, a fragment I (1 st to 19 th positions) is synthesized in a solid phase, a side chain monomer (20 th position amino acid and a side chain thereof) is synthesized in a liquid phase, then, a fragment II (21 st to 31 th positions) of the side chain monomer and other amino acids is synthesized in a solid phase, and finally, the fragment I and the fragment II are condensed and refined to obtain the Somaloude. The invention adopts a special synthesis strategy, and obviously improves the purity and yield of the somaglutide. The invention has at least one of the following beneficial effects:

(1) the invention obviously improves the purity and yield of the Somalutide and is beneficial to large-scale production;

(2) the method completely avoids using noble metals such as palladium tetratriphenylphosphine and the like, solves the problem of palladium residue in the three-waste treatment, and has the advantages of low cost, safety, environmental protection and simpler process;

(3) the synthesis strategy of the invention can obviously reduce steric hindrance and improve reaction yield.

Drawings

FIG. 1 shows a mass spectrum MS plot;

FIG. 2 shows a mass spectrum MS plot;

FIG. 3 shows crude peptide HPLC (70.57%);

FIG. 4 shows crude peptide HPLC (71.92%);

FIG. 5 shows protamine HPLC (99.88%);

FIG. 6 shows protamine HPLC (99.95%).

Detailed Description

The invention provides a method for synthesizing Somalutide. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

TABLE 1

The synthetic route of the invention comprises the following steps:

(1) synthesis of fragment I:

Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(Boc)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-OH

(2) synthesis of fragment II

Liquid phase synthesis of side chains

(3) Solid phase Synthesis of fragment II

(4) Condensing the segment I and the segment II to synthesize the Somalutide

The invention relates to a method for synthesizing Somalutide, which comprises the following steps:

a) synthesis of fragment I:

Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(Boc)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-OH；

b) liquid phase synthesis of side chain monomer Fmoc-Lys (AEEA-AEEA-gamma-Glu-octaneedioICAcid) -OH, and the specific structure is as follows:

c) synthesis of fragment II:

NH₂-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OH.

d) condensing and de-protecting the fragment I and the fragment II in a liquid phase to finally obtain the crude peptide of the Somalutide; and purifying the crude peptide by a high performance liquid chromatography method to prepare the somaglutide.

In the step a), the carrier Resin is 2-CTC Resin, and the replacement degree of Fmoc-Ala-2CTC is 0.2-0.6mmol/g, preferably 0.3-0.5 mmol/g; .

In the step a), DIC/HOBt is preferably used as a coupling reagent, and the following amino acid raw materials are sequentially coupled: Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -Phe-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Boc-His (Trt) -Aib-OH, coupling time of 1-3 hours, preferably 2 hours.

In the step a), a cracking reagent used for cracking is TFA, DCM is 1: 99-5: 95, or TFE: DCM 1:3 to 1:4, preferably TFE: DCM 1:3 (volume ratio); and (3) cracking for 2 hours at room temperature, then concentrating to dryness, adding ethyl acetate to dissolve, washing for 3 times by adopting a saturated sodium chloride aqueous solution and a citric acid aqueous solution, collecting an organic phase, and concentrating to obtain a fragment I.

The step b) of synthesizing the side chain monomer specifically comprises the following steps:

starting materials adopt Fmoc-Lys-OH and Boc-AEEA-OSu or Boc-AEEA-ONb, preferably Boc-AEEA-OSu; the alkali is selected from DEA, DIPEA, pyridine, etc., preferably DIPEA, and the solvent is THF, DCM, DMF, preferably DCM. Reacting for 2 hours at room temperature under the condition of dissolution; wherein BocAEEA-OSu is prepared by condensation of BocAEEA-OH and HOSu under DCC condition. The refining method in the step comprises the following steps: after concentration, ethyl acetate was added: petroleum ether is 1: and 3, recrystallizing. ② adopting HCl/EA saturated liquid to remove Boc group, 2 hours, 10ml/g dosage. After the reaction is finished, concentrating to be dry. Thirdly, repeating the first step, coupling a second Boc-AEEA-OSu, and repeating the second step to obtain a compound Fmoc-Lys (AEEA-AEEA) -OH; fourthly, taking tert-butyl octadecanoic Acid monoester as a raw material, adding THF, Boc anhydride and anhydrous pyridine, reacting for 0.5 hour at room temperature, then adding NH2-Glu (OtBu) -OH, stirring and reacting for 2 hours, adding diethyl ether, and separating out solids to obtain gamma-Glu (OtBu) -octanedioic Acid; and fifthly, carrying out condensation reaction on the product of the third step and the product of the fourth step for 2 hours under the conditions of DCC and DCM to obtain a side chain monomer Fmoc-Lys (AEEA-AEEA-gamma-Glu-octanedioic Acid) -OH.

Step c) Synthesis of fragment II:

NH₂-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OH。

the resin in the step c) adopts 2CTCResin resin, and the amino acids which are sequentially coupled according to a classical Fmoc strategy coupling method are 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-gamma-Glu-Octadecanoic Acid) -OH.

In the step c), the solvent for the coupling reaction is dichloromethane and/DMF, preferably DMF; the Fmoc removal solvent was 20% piperidine DMF solution and finally the resin was shrunk with methanol.

In the step c), the coupling agent is a combination of DIPCDI and a condensing agent A, or a combination of DIPEA and a condensing agent B, wherein the condensing agent A is HOBt or HOAt, and the condensing agent B is PyBOP, PyAOP, HATU, HBTU or TBTU. Further, the ratio of each component in the coupling agent is, in terms of molar ratio, DIPCDI: a: 1.2:1.1, DIPEA: a: B: 2.0:1.1:1.0, Fmoc-Lys (AEEA- γ -Glu-octaneedioic Acid) -OH, HOAt/DIC system is used, and HOBt/DIC system is used, preferably, the reaction multiple is 1.2 times equivalent of amino Acid. The step 3) is carried out, and the used deprotection reagent is TFA: and (3) TIS: DCM ═ 2: 3: 95 (V: V); or TFE: DCM ═ 1: 3(V: V) -1: 4(V: V), preferably TFE: DCM ═ 1: 3(V: V).

The crude product purification method in the step c) comprises the following steps: after suction filtration, the filtrate was concentrated, dissolved in ethyl acetate, washed 3 times with 5% aqueous citric acid solution and saturated aqueous sodium chloride solution, respectively, and the organic layer was collected and concentrated.

In the synthesis method provided by the invention, in the steps a) -c), when amino acids are coupled, because each amino acid has a protecting group, the N-terminal protecting group needs to be removed for coupling, and a removing agent used for removing the N-terminal Fmoc protecting group is preferably piperidine solution, more preferably 20% piperidine DMF solution, including but not limited to.

In some embodiments, step d) is specifically:

dissolving the segment I in a DCM solution, adding HONb and DIC, stirring and reacting for 2 hours, after the reaction is finished, adding a 5% citric acid aqueous solution and a saturated sodium chloride aqueous solution, washing for 2 times, collecting an organic layer, drying anhydrous sodium sulfide, adding a DCM solution of the segment II, stirring and clarifying, adding DIPEA, and stirring and reacting for 2 hours. After the reaction is finished, concentrating, adding 5mL/g of lysate, cracking, and precipitating by diethyl ether to obtain the crude peptide.

The lysate in the step d) is TFA, TIS and H₂O, PhOMe, respectively; the preferred ratio is TFA: and (3) TIS: h₂O, PhOMe ═ 85:5:5:5(V: V). The amount of the lysis solution can beIs 4mL/g to 10mL/g, preferably 5mL/g, and the reaction is carried out for 2 to 3 hours, preferably 2 hours.

In the step d), the reaction solvent may be an organic solvent such as DCM, DMF, THF, etc., preferably DCM.

In step d), the coupling reagent may be a compound of the same type as HOSu or HONb, preferably HONb.

The precipitation solvent in step d): diethyl ether, methyl tertiary ether and the like, preferably diethyl ether.

Preparing refined peptide: the purification is carried out by reversed phase high performance liquid chromatography and freeze-drying.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 Synthesis of fragment I

Selecting 10g of Fmoc-Ala-CTC Resin amino acid Resin with the substitution degree of 0.3mmol/g, adding the Fmoc-Ala-CTC Resin into a reaction column, adding DMF for washing for 1 time, adding DCM for swelling for 30 minutes, draining, adding 20% piperidine/DMF solution for removing Fmoc twice (5 minutes +7 minutes), after removal, washing the DMF for 5 times, weighing Fmoc-Ala-OH (1.868g, 6mmol), HOBt (0.973g, 7.2mmol), adding the mixture into the reaction column, dissolving the DMF, adding DIC (1.22mL,7.8mmol), reacting at room temperature for 1 hour, detecting ninhydrin for complete reaction, performing suction filtration, washing the DMF for 5 times with a proper amount, performing Fmoc-Gln (trt) -OH (3.664g, 6mmol), Fmoc-Gly-OH (1.784g, 6mmol), Fmoc-Glu (OtBu) -OH (2.553g, 6mmol) and Fmoc-Leu-OH (2.120g, 6mmol), Fmoc-Tyr (Boc) -OH (2.757g, 6mmol), Fmoc-Ser (tBu) -OH (2.301g, 6mmol), Fmoc-Ser (tBu) -OH (2.301g, 6mmol), Fmoc-Val-OH (2.036g,6mmol), Fmoc-Asp (OtBu) -OH (2.469g, 6mmol), Fmoc-Ser (tBu) -OH (2.301g, 6mmol), Fmoc-Thr (tBu) -OH (2.385g, 6mmol), Fmoc-Thr (tBu) -Phe-OH (3.162g, 6mmol), Fmoc-Gly-OH (1.784g, 6mmol), Fmoc-Glu (OtBu) -OH (2.553g, 6mmol), Boc-His Trt) -Aib-OH (3.496g, 6mmol), wherein Fmoc-Thr-OH, Boc-t-Phe-His are coupled for a period of 2 hours (2 hours), the other amino acids were all 1 hour. Finally obtaining the peptide resin

Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(Boc)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-2CTC Resin

A cracking step: the cracking reagent used was TFE: DCM 1:3 to 1:4, preferably TFE: DCM 1:3 (volume ratio); splitting at room temperature for 2 hr, concentrating to dryness, dissolving in ethyl acetate, washing with saturated aqueous sodium chloride solution and citric acid solution for 3 times, collecting organic phase, and concentrating to obtain compound I (fragment I)

Example 2 fragment I Synthesis

Selecting 10g of Fmoc-Ala-CTC Resin amino acid Resin with the substitution degree of 0.3mmol/g, adding the Fmoc-Ala-CTC Resin into a reaction column, adding DMF for washing for 1 time, adding DCM for swelling for 30 minutes, draining, adding 20% piperidine/DMF solution for removing Fmoc twice (5 minutes +7 minutes), after removal, washing the DMF for 5 times, weighing Fmoc-Ala-OH (1.868g, 6mmol), HOBt (0.973g, 7.2mmol), adding the mixture into the reaction column, dissolving the DMF, adding DIC (1.22mL,7.8mmol), reacting at room temperature for 2 hours, detecting ninhydrin for complete reaction, performing suction filtration, washing the DMF for 5 times with a proper amount, performing Fmoc-Gln (trt) -OH (3.664g, 6mmol), Fmoc-Gly-OH (1.784g, 6mmol), Fmoc-Glu (OtBu) -OH (2.553g, 6mmol) and Fmoc-Leu-OH (2.120g, 6mmol), Fmoc-Tyr (Boc) -OH (2.757g, 6mmol), Fmoc-Ser (tBu) -OH (2.301g, 6mmol), Fmoc-Ser (tBu) -OH (2.301g, 6mmol), Fmoc-Val-OH (2.036g,6mmol), Fmoc-Asp (OtBu) -OH (2.469g, 6mmol), Fmoc-Ser (tBu) -OH (2.301g, 6mmol), Fmoc-Thr (tBu) -OH (2.385g, 6mmol), Fmoc-Thr (tBu) -Phe-OH (3.162g, 6mmol), Fmoc-Gly-OH (1.784g, 6mmol), Fmoc-Glu (OtBu) -OH (2.553g, 6mmol), Boc-His Trt) -Aib-OH (3.496g, 6mmol), and finally the peptide resin is shrunk to obtain:

Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(Boc)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-2CTC Resin

a cracking step: the cleavage reagent used was TFA: DCM ═ 0.5:95 at room temperature, lysis was carried out for 2 hours, then 0.1M sodium hydroxide was added to adjust the pH to between 6 and 7, after DCM was concentrated, appropriate amount of water was added, solid precipitated, filtration was carried out to obtain solid, which was dried under reduced pressure to obtain compound I (fragment I):

Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(Boc)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-OH

EXAMPLE 3 side chain Synthesis of fragment II

Fmoc-Lys-OH (36.8g, 100mmol) and Boc-AEEA-OSu (37.8,105mmol) were weighed and dissolved in 500mL of THF, DIPEA (34.86ml,200 mmol) was added thereto, and the reaction was carried out at room temperature for 2 hours; TLC to monitor the reaction completion, concentrate, add 500ml HCl/EA saturated solution (Boc group removal, 10ml/g) to the oil solid for 2 hours; after the reaction is finished, concentrating to be dry. Repeating and coupling a second Boc-AEEA-OSu to obtain Fmoc-Lys (AEEA-AEEA) -OH; adding 100 ethyl acetate for dissolving, dropwise adding 300mL of petroleum ether, separating out a precipitate, and filtering to obtain a solid for later use;

weighing 46g of octadecanoic Acid tert-butyl monoester, adding the octadecanoic Acid tert-butyl monoester into 500ml of dry-treated HF for dissolving, then adding Boc anhydride and anhydrous pyridine, reacting for 0.5 hour at room temperature, then adding NH2-Glu (OtBu) -OH, stirring for reacting for 2 hours, adding diethyl ether, and separating out a solid to obtain gamma-Glu (OtBu) -octanedioic Acid;

dissolving the oily matter in the first step in DMF, adding a DMF solution of gamma-Glu (OtBu) -octanedioic Acid, stirring for dissolving, adding DCC, stirring at room temperature for reacting for 2 hours to obtain side chain monomer Fmoc-Lys (AEEA-AEEA-gamma-Glu-octanedioic Acid) -OH.

EXAMPLE 4 fragment II Synthesis

NH₂-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OH。

Weighing 10g of Fmoc-Gly-2CTCResin amino acid resin with the substitution degree of 0.45mmol/g, adding the Fmoc-Gly-2CTCResin amino acid resin into a reaction column, adding DMF for washing for 1 time, adding DCM for swelling for 30 minutes, draining, adding 20% piperidine/DMF solution for removing Fmoc twice (5 minutes +7 minutes), after removal, washing the DMF for 5 times, weighing Fmoc-Arg (Pbf) -OH (5.839g, 9mmol), HOBt (1.459g, 10.8mmol), adding the mixture into the reaction column, dissolving the DMF, adding DIC (1.83mL,11.7mmol), reacting for 2 hours at room temperature, detecting ninhydrin completely, performing suction filtration, washing for 5 times by proper amount of DMF, continuously repeating the above operations, and performing Fmoc-Gly-OH (2.676g, 9 mm) on the mixtureol), Fmoc-Arg (Pbf) -OH (5.839g, 9mmol), Fmoc-Vla-OH (3.055g, 9mmol), Fmoc-Leu-OH (3.181g, 9mmol), Fmoc-Trp (Boc) -OH (4.739g, 9mmol), Fmoc-Ala-OH (2.802g, 9mmol), Fmoc-Iie-OH (3.181g, 9mmol), Fmoc-Phe-OH (3.487g,9mmol), Fmoc-Glu (OtBu) -OH (3.830g, 9mmol), coupling was completed, after removal of Fmoc, DMF was washed 3 times, DCM was washed 3 times, Fmoc-Lys (AEEA-AEEA-gamma-Glu-Octadalediodic) -OH in DMF, HOAt (1.461g, 10.8mmol), DIC (1.83mL,11.7mmol), reaction time 3 hours, after removal of the piperidine ketone, clear detection for 20 min (7 min), washing 5 times each with DMF and DCM, and shrinking with methanol gave the peptide resin: NH (NH)₂-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-2CTC Resin。

A cracking step: the cracking reagent used was TFE: DCM 1:3 at room temperature, cleaved for 3 hours, concentrated to give a solid, added n-hexane, slurried, stirred and filtered. Drying under reduced pressure. Fragment II was obtained: NH (NH)₂-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-OH

Example 5 crude peptide Synthesis

The fragment I of example 1 was dissolved in DMF, added with HOSu, DIC, reacted at room temperature for 2 hours, followed by HPLC monitoring, and after completion of the reaction, filtered to obtain a filtrate.

Fragment II from example 4 was dissolved in DMF and dissolved by addition of 3 equivalents of DIPEA.

Condensation: and (3) dropwise adding the filtrate of the fragment I into the solution of the fragment II, stirring, carrying out condensation reaction, monitoring the reaction by HPLC, filtering after the reaction is finished, passing the filtrate through a preparative chromatograph, replacing DMF with an acetonitrile solvent, and collecting the fully-protected Somalide acetonitrile solution. Concentrating and drying to obtain a crude product of the Somalutide.

Cracking: 68g of total protected soxhlet peptide was dissolved in 420ml of frozen lysate (TFA: H2O: PhOH: EDT:. 5:5:5: 2.5), the reaction was stirred for 2.5 hours, and the lysate was precipitated with 4.2L of methyl tert-butyl ether to give the crude peptide. The chromatogram is shown in FIG. 3, and the crude peptide purity is 70.57%. .

Example 6 crude peptide Synthesis

Fragment I from example 2 was dissolved in DMF, followed by addition of HOSu, DIC, reaction at rt for 2 h, HPLC monitoring of the reaction, and after completion of the reaction, filtration of the filtrate was performed for future use.

Fragment II from example 4 was dissolved in DMF and dissolved by addition of 3 equivalents of DIPEA.

Condensation: and (3) dropwise adding the filtrate of the fragment I into the solution of the fragment II, stirring, carrying out condensation reaction, monitoring the reaction by HPLC, filtering after the reaction is finished, passing the filtrate through a preparative chromatograph, replacing DMF with an acetonitrile solvent, and collecting the fully-protected Somalide acetonitrile solution. Concentrating and drying to obtain a crude product of the Somalutide.

Cracking: 68g of total protected soxhlet peptide was dissolved in 420ml of frozen lysate (TFA: H2O: PhOH: EDT:. 5:5:5: 2.5), the reaction was stirred for 2.5 hours, and the lysate was precipitated with 4.2L of methyl tert-butyl ether to give the crude peptide. The chromatogram is shown in FIG. 4, and the purity of the crude peptide is 71.92%.

Example 7 crude peptide purification

The crude peptide of example 5 was purified by reverse phase high performance liquid chromatography to obtain the refined peptide, and the chromatogram was shown in FIG. 5 (99.88%).

Example 8 crude peptide purification

The crude peptide of example 6 was purified by reverse phase high performance liquid chromatography to obtain the refined peptide, and the chromatogram was shown in FIG. 6 (99.95%).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> Shenzhen Hanyu pharmaceutical stockings Limited

<120> synthetic method of Somalutide

<130> S21P001863

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> MOD_RES

<222> (2)..(2)

<223> xaa= Aib

<220>

<221> UNSURE

<222> (2)..(2)

<223> The 'Xaa' at location 2 stands for Gln, Arg, Pro, or Leu.

<400> 1

His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala

<210> 2

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

1 5 10