1. A method for preparing Somaloutide, which comprises preparing Somaloutide by solid phase synthesis, wherein Ala²⁴-Ala²⁵The coupling of (2) was carried out using Fmoc-Ala- (Dmb) Ala-OH as starting material.

2. The method of claim 1, wherein the solid phase synthesis comprises coupling each amino acid sequentially in order from C-terminus to N-terminus of the sequence of the somaglutide on a resin to give a somaglutide backbone-resin,

wherein Lys²⁶The coupling of (a) was performed using Fmoc-Lys (Ivdde) -OH as the starting material, His⁷The coupling of (2) was performed using Boc-His (Trt) -OH as starting material.

3. The method of claim 2, further comprising removing Lys from the somaglutide backbone²⁶The side chain protecting group Ivdde of (1), then at Lys²⁶And coupling the side chain of the Somalou peptide to obtain the Somalou peptide-resin.

4. A preparation method of the Somalutide is characterized by comprising the following steps:

step 1: coupling Gly and solid phase carrier resin to obtain Fmoc-Gly-resin;

step 2: sequentially coupling the rest amino acids according to the sequence of the somaglutide from the C end to the N end by using the Fmoc-Gly-resin obtained in the step 1 to obtain the somaglutide main chain-resin,

wherein, Ala²⁴-Ala²⁵The Fmoc-Ala- (Dmb) Ala-OH is used as a raw material for coupling;

and step 3: desorption of Lys in the backbone of Somatocride²⁶Side chain protecting groups of (3), then at Lys²⁶Coupling the side chain of the Somaloutide to obtain Somaloutide-resin; and

and 4, step 4: and (4) cracking the Somalou peptide-resin obtained in the step (3), and purifying to obtain the Somalou peptide.

5. The process of claim 4, wherein the resin in step 1 is Wang resin or 2-CTC resin.

6. The process of claim 5, wherein the degree of substitution of the Wang resin or 2-CTC resin coupled to Gly is 0.15-0.30 mmol/g.

7. The method of any one of claims 4-6, wherein Lys is present in step 2²⁶The coupling of (a) was performed using Fmoc-Lys (Ivdde) -OH as the starting material, His⁷The coupling of (2) was performed using Boc-His (Trt) -OH as starting material.

8. The method of any one of claims 1-7, wherein an Arg is coupled³⁶The condensing agent used in the method is HATU/DIEA or DIC/HOBt.

9. The method of claim 8, wherein Arg-removing is coupled³⁶The condensation reagent used in the other amino acids is one or two of DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and the solvent is one or two of DMF, DCM and NMP.

10. The method of any one of claims 1-9, wherein Lys is coupled²⁶、Ala²⁴-Ala²⁵、His⁷And Gln²³The condensation reagent used in any one or more of DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and the solvent is one or a mixture of DMF, DCM and NMP.

11. The method of claim 10, wherein Lys is present in step 2²⁶、Ala²⁴-Ala²⁵、His⁷And Gln²³The coupling of any one or more of the following is performed: coupling with condensing agent DIC/HOBt for 20-60min in DMF solvent, vacuum filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 20-60min in volume ratio of 3:1-5:1DCM is the DMF of (1).

12. The method of any one of claims 3 and 7-11, wherein Lys²⁶The side chain protecting group Ivdde is removed by using 2 percent hydrazine hydrate/DMF for 3 to 6 times, and each time lasts for 5 to 30 min.

13. The process of any one of claims 1-12, comprising Lys in the deprotection of side chain protecting groups²⁶Fmoc-NH-PEG coupled in sequence₂-CH₂COOH、Fmoc-NH-PEG₂-CH₂COOH, Fmoc-Glu (OH) -OtBu and mono-tert-butyl octadecanedioate.

14. The method of claim 13, wherein Fmoc-NH-PEG is coupled₂-CH₂COOH、Fmoc-NH-PEG₂-CH₂The condensation reagent used for COOH and Fmoc-Glu (OH) -OtBu is independently one of HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and the solvent is DMF; the condensation reagent used for coupling the octadecanedioic acid mono-tert-butyl ester is DIC, and the solvent is DMF: DCM with the volume ratio of 1:1-2: 1.

15. The method of any one of claims 1-14, wherein the reagent used to cleave the synthesized somaglutide from the solid phase synthesis support is a mixture of TFA, thioanisole, phenol, EDT, and water.

16. The method of claim 15, wherein the volume ratio of TFA, thioanisole, phenol, EDT and water is 85-90: 2.5-7.5: 1-4: 2.5-7.5.

17. A preparation method of a Somali peptide salt is characterized by comprising the following steps:

step 1: producing a somaglutide according to the method of any one of claims 1-16; and

step 2: and (3) carrying out salt conversion on the Somalutide prepared in the step (1) to obtain the Somalutide salt.

18. The method of claim 17, wherein the salt of somaglutide is a TFA salt of somaglutide.

19. A compound of the formula R-Ala- (Dmb) Ala-OH, wherein R is an amino protecting group, for use in solid phase synthesis of a polypeptide comprising two adjacent alanine residues.

20. The compound of claim 19, wherein R is Fmoc or Boc, preferably Fmoc.

21. A compound according to claim 19 or 20, wherein the polypeptide is a somaglutide.

22. Use of a compound according to any one of claims 19 to 21 for the solid phase synthesis of somaglutide.

Background

Diabetes mellitus is a group of lifelong metabolic diseases characterized by chronic hyperglycemia caused by multiple causes. According to the latest edition of the international diabetes union (IDF) data, the number of adult diabetics reaches 4.25 hundred million worldwide, with type II diabetes accounting for 90%. According to the statistics of the world health organization, the diabetes complication is more than 100, and is the most known disease at present. More than half of the deaths due to diabetes are caused by cardiovascular and cerebrovascular diseases, and 10% of the deaths are caused by nephropathy. Clinical data show that about 10 years after the onset of diabetes, 30-40% of patients will develop at least one complication, and once the complication occurs, the drug treatment is difficult to reverse, so that the early prevention of the diabetic complication is emphasized.

The commonly used hypoglycemic drugs at present mainly comprise insulin and analogues thereof, GLP-1 receptor agonist, DDP-4 enzyme inhibitor, metformin and other various types. The Somatode approved by the US FDA and marketed on 6.12.2017 is obtained, and has great advantages in the fields of blood sugar reduction and weight loss, and the Somatode is also a hypoglycemic agent which follows the empagliflozin and the liraglutide, and the third Somatode shows cardiovascular benefits. The somaglutide is GLP-1 analogue, and has 94% homology with human GLP-1. The modification of the somaglutide at position 8 to alpha-aminoisobutyric acid serves to increase the stabilization against degradation by the DPP-4 enzyme. In addition, through structural modification, the position of the 26 th lysine of the peptide chain is connected with the 18-carbon fatty diacid side chain, compared with the liraglutide of C16, the affinity of the increased carbon chain to albumin is enhanced by 5-6 times, and the peptide is combined with albumin, so that the phenomenon that the peptide is rapidly cleared by the kidney, the metabolic degradation is prevented, and the half-life period in vivo is prolonged. In terms of safety, the thaumatin is similar to liraglutide and dolaglutide, and adverse reactions can disappear with time. On 20/3/2019, noh and noded announced two new drug marketing applications (NDA) for oral somaglutide were filed by the FDA. Somaglutide will be the business leg and growth driver for norand noded in the coming years, with analysts predicting its peak sales is expected to exceed $ 100 billion. The structural formula of the somaglutide is as follows:

commonly used side chain protecting groups of 26-bit K in the prior art comprise Mtt and Alloc, the acidic condition is difficult to avoid influencing other side chain protecting groups such as Trt and Boc when Mtt is removed, and complete removal is difficult to ensure in the amplification process; multiple acid treatments also result in cleavage of the polypeptide from the 2-CTC resin; meanwhile, one of the problems of the removal of the Alloc protecting group is that the sample is possibly dyed and metal remains, so that the sample has poor properties; in addition, in the Alloc removal process, if the capture agent of Alloc is not in place, the MS is made 40 larger and the subsequent amino acids cannot be coupled. Both methods result in poor yields and purity.

The existence of hydrophobic amino acid in the sequence of the somaglutide enables hydrogen bonds between peptide chains to be more stable, severe beta folding is generated, acting force between the peptide chains is enhanced, resin polycondensation is caused, and the reaction activity and efficiency of coupling are reduced. At present, there are patents which mention the use of Ser-Thr pseudoprolyl dipeptide, Ser-Ser coupled in the form of a dipeptide fragment to increase the efficiency of coupling. In addition, it can also be used in (Gly)¹⁰-Thr¹¹)、(Phe¹²-Thr¹³)、(Thr¹³-Ser¹⁴)、(Val¹⁶-Ser¹⁷) And (Ser)¹⁷-Ser¹⁸) Coupling one of the five positions with an O-isoacylated dipeptide followed by O → N transfer reaction gives the crude peptide Somaloutide, but the racemization problem is more likely to occur in this process.

Therefore, there is still a need in the art to develop a simple and efficient preparation method of somaglutide.

Disclosure of Invention

The invention aims to provide a simple and efficient preparation method of the somaglutide, and solves the problems of complex preparation process, low efficiency or high purification difficulty of the somaglutide at present. The purpose of the invention can be realized by the following technical scheme.

The invention provides a preparation method of Somalutide, which is characterized in that the Somalutide is prepared by solid phase synthesis, wherein Ala²⁴-Ala²⁵The coupling of (2) was carried out using Fmoc-Ala- (Dmb) Ala-OH as starting material.

In some embodiments of the invention, the solid phase synthesis method is to couple each amino acid sequentially in the order of the sequence of the somaglutide from the C-terminus to the N-terminus to obtain the somaglutide backbone-resin. In some embodiments of the invention, Lys²⁶The coupling of (a) was performed using Fmoc-Lys (Ivdde) -OH as the starting material, His⁷The coupling of (2) was performed using Boc-His (Trt) -OH as starting material.

In some embodiments of the invention, the preparation method further comprises removing Lys from the somaglutide backbone²⁶The side chain protecting group Ivdde of (1), then at Lys²⁶And coupling the side chain of the Somalou peptide to obtain the Somalou peptide-resin.

The invention also provides a preparation method of the Somalutide, which comprises the following steps:

step 1: coupling Gly and solid phase carrier resin to obtain Fmoc-Gly-resin;

step 2: sequentially coupling the rest amino acids according to the sequence of the somaglutide from the C end to the N end by using the Fmoc-Gly-resin obtained in the step 1 to obtain the somaglutide main chain-resin,

wherein Ala²⁴-Ala²⁵The Fmoc-Ala- (Dmb) Ala-OH is used as a raw material for coupling;

and step 3: desorption of Lys in the backbone of Somatocride²⁶Side chain protecting groups of (3), then at Lys²⁶Coupling the side chain of the Somaloutide to obtain Somaloutide-resin; and

and 4, step 4: and (4) cracking the Somalou peptide-resin obtained in the step (3), and purifying to obtain the Somalou peptide.

In some embodiments of the invention, the resin in step 1 is a Wang resin or a 2-CTC resin. In some embodiments of the invention, the degree of substitution of the Wang resin or 2-CTC resin coupled to Gly is in the range of 0.15 to 0.30 mmol/g. In one embodiment, the resin coupled to Gly in step 1 is 2-CTC resin with degree of substitution of 0.20 mmol/g. In another embodiment, the resin that couples Gly in step 1 is Wang resin with degree of substitution of 0.25 mmol/g.

In some embodiments of the invention, Lys²⁶The coupling of (a) was performed using Fmoc-Lys (Ivdde) -OH as the starting material, His⁷The coupling of (2) was performed using Boc-His (Trt) -OH as starting material. In some embodiments, the feed equivalents of amino acid starting material are from 2 to 4 equivalents and the reaction concentration is from 0.10 to 0.40 mmol/mL. In one embodiment, the feed equivalent of amino acid starting material is 3 equivalents and the reaction concentration is 0.15mmol/mL or 0.30 mmol/mL. In one embodiment, the feed equivalent of amino acid starting material is 3 equivalents and the reaction concentration is 0.15 mmol/mL. In one embodiment, the feed equivalent of amino acid starting material is 3 equivalents and the reaction concentration is 0.30 mmol/mL.

In some embodiments of the invention, Arg is coupled³⁶HATU/DIEA or DIC/HOBt was used as a condensing agent. In one embodiment, the Arg is coupled³⁶DIC/HOBt was used as a condensing agent. In another embodiment, an Arg is coupled³⁶HATU/DIEA was used as the condensing agent.

In some embodiments of the invention, coupling to remove Arg³⁶When other amino acids are used, the condensation reagent may be one or two of DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and the solvent may be one or two of DMF, DCM and NMP. Preferably, DIC/HOBt is used as the condensation reagent and DMF is used as the solvent.

In some embodiments of the invention, the Wang resin may be coupled to the first amino acid by adding DMAP to catalyze the reaction, preferably for a period of time ranging from 1.5h to 2h, wherein the amount of DMAP added may be routinely determined by one skilled in the art, for example 1/10 moles of amino acid starting material.

In some embodiments of the invention, Lys²⁶、Ala²⁴-Ala²⁵、His⁷And Gln²³The coupling of any one or more of the following is performed: coupling with condensing agent DIC/HOBt for 20-60min in DMF, suction filtering, washing resin with DMF, and coupling with condensing agent HATU/HOBT/DIEA20-60min, wherein the solvent is DMF and DCM with the volume ratio of 3:1-5: 1. In a preferred embodiment, Lys²⁶、Ala²⁴-Ala²⁵、His⁷And Gln²³The coupling of any one or more of the following is performed: coupling with condensing agent DIC/HOBt for 40min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 40min again in DMF/DCM in 4:1 volume ratio.

In some embodiments of the invention, Lys is coupled²⁶When Fmoc-Lys (Ivdde) -OH is used as a raw material, two condensation reagents are DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and a solvent is one or a mixture of two of DMF, DCM and NMP; specifically, coupling of Fmoc-Lys (Ivdde) -OH was performed as follows: coupling with condensing agent DIC/HOBt for 20-60min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 20-60min in DMF/DCM in 3:1-5:1 volume ratio. Preferably, the coupling of Fmoc-Lys (Ivdde) -OH is performed as follows: coupling with condensing agent DIC/HOBt for 40min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 40min again in DMF/DCM in 4:1 volume ratio.

In some embodiments of the invention, the His is conjugated⁷When in use, Boc-His (Trt) -OH is used as a raw material, two condensation reagents are DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and a solvent is one or a mixture of two of DMF, DCM and NMP; specifically, coupling of Boc-His (Trt) -OH was performed as follows: coupling with condensing agent DIC/HOBt for 20-60min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 20-60min in DMF/DCM in 3:1-5:1 volume ratio. Preferably, the coupling of Boc-His (Trt) -OH is performed as follows: coupling with condensing agent DIC/HOBt for 40min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 40min again in DMF/DCM in 4:1 volume ratio.

In some embodiments of the invention, the coupling isAla²⁴-Ala²⁵The Fmoc-Ala- (Dmb) Ala-OH is used as a raw material, so that the aggregation of polypeptide and the generation of deletion peptide can be effectively avoided, and the purity of a sample is improved. In some embodiments of the invention, Fmoc-Ala- (Dmb) Ala-OH is coupled using two condensation reagents DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, the solvent being one or a mixture of two of DMF, DCM and NMP; specifically, coupling of Fmoc-Ala- (Dmb) Ala-OH was performed as follows: coupling with condensing agent DIC/HOBt for 20-60min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 20-60min in DMF/DCM in 3:1-5:1 volume ratio. Preferably, the coupling of Fmoc-Ala- (Dmb) Ala-OH is carried out as follows: coupling with condensing agent DIC/HOBt for 40min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 40min again in DMF/DCM in 4:1 volume ratio.

In some embodiments of the invention, the conjugated Gln²³The condensation reagent is two of DIC/HOBt, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and the solvent is one or a mixture of two of DMF, DCM and NMP; in particular, Gln²³The coupling of (a) is carried out as follows: coupling with condensing agent DIC/HOBt for 20-60min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 20-60min in DMF/DCM in 3:1-5:1 volume ratio. Preferably, Gln in step 2²³The coupling of (a) is carried out as follows: coupling with condensing agent DIC/HOBt for 40min in DMF, suction filtering, washing resin with DMF, coupling with condensing agent HATU/HOBT/DIEA for 40min again in DMF/DCM in 4:1 volume ratio.

In some embodiments of the invention, Lys²⁶The side chain protecting group Ivdde is removed by using 2 percent hydrazine hydrate/DMF for 3 to 6 times, each time lasts for 5 to 30min, and no side reaction occurs basically. In some embodiments of the invention, Lys²⁶The side chain protecting group Ivdde is removed by using 2 percent hydrazine hydrate/DMF for 4 times, each time is 10min, and basically no side reaction occurs; preferably, a detectable amount of the resin is taken to be cleaved, usuallyThe reaction was followed by MS detection of amu + 206.26.

In some embodiments of the invention, Lys is left after removal of side chain protecting groups²⁶Fmoc-NH-PEG coupled in sequence₂-CH₂COOH、Fmoc-NH-PEG₂-CH₂COOH, Fmoc-Glu (OH) -OtBu and mono-tert-butyl octadecanedioate, in which Fmoc-NH-PEG was coupled₂-CH₂COOH、Fmoc-NH-PEG₂-CH₂The condensation reagent used for COOH and Fmoc-Glu (OH) -OtBu is independently one of HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA, and the solvent is DMF; the condensation reagent used for coupling the octadecanedioic acid mono-tert-butyl ester is DIC, and the solvent is DMF: DCM with the volume ratio of 1:1-2: 1. Preferably, the condensation reagent HATU/HOBT/DIEA used in the coupling of Fmoc-NH-PEG2-CH2COOH, Fmoc-NH-PEG2-CH2COOH and Fmoc-Glu (OH) -OtBu, wherein the solvent is DMF; the condensation reagent used for coupling the mono-tert-butyl octadecanedioate is DIC, and the solvent is DMF: DCM in a volume ratio of 1: 1. In some embodiments of the invention, Fmoc-NH-PEG₂-CH₂The charging equivalent of raw materials of COOH, Fmoc-Glu (OH) -OtBu and mono-tert-butyl octadecanedioate is 3 equivalents, and the reaction concentrations are all 0.3 mmol/mL.

In some embodiments of the invention, the polypeptide-conjugated resin is dried by suction with DCM or methanol prior to cleavage.

In some embodiments of the invention, the cleavage reagent used for cleaving the synthesized somniferin from the solid phase synthesis support is a mixture of TFA, thioanisole, phenol, EDT and water, preferably the volume ratio of TFA, thioanisole, phenol, EDT and water is 85-90: 2.5-7.5: 1-4: 2.5-7.5, and more preferably the volume ratio of TFA, thioanisole, phenol, EDT and water is 87.5:5:2.5: 5.

During the solid phase synthesis of Somalutide, 2 hydrophobic Ala (i.e. Ala) were in series²⁴-Ala²⁵) After Gln²³Possibly lost. Thus, in some preferred embodiments of the invention, in Gln²³After coupling, a detectable amount of the resin can be removed from Fmoc and cleaved, and mass spectrometric detection ensures Gln²³Then the next step is carried out.

Compared with the somaglutide, the somaglutide salt is more stable, is not easy to decompose, oxidize, denature and the like under natural conditions, and is convenient to store and use. Therefore, the invention also provides a preparation method of the salt of the somalvidin, which comprises the following steps:

step 1: preparing the Somalutide according to the method of the invention; and

step 2: and (3) carrying out salt conversion on the Somalutide prepared in the step (1) to obtain the Somalutide salt.

In some embodiments of the present invention, the solvent for dissolving the somaglutide prepared in step 1 in step 2 is water and acetonitrile, preferably in a volume ratio of about 3: 1.

In some embodiments of the invention, the salt of the somagluteptide is lyophilized after the transformation of the somagluteptide to the salt of somagluteptide. In some preferred embodiments of the invention, the salt of somaglutide is the TFA salt of somaglutide.

The invention also provides a compound having the structural formula R-Ala- (Dmb) Ala-OH, wherein R is an amino protecting group, for use in solid phase synthesis of polypeptides comprising two adjacent alanine residues.

In some embodiments of the invention, R is Fmoc or Boc, preferably Fmoc. In a specific embodiment, the compound is Fmoc-Ala- (Dmb) Ala-OH.

In some embodiments of the invention, the polypeptide is a somaglutide.

The use of the compound R-Ala- (Dmb) Ala-OH of the invention for the solid phase synthesis of Somatoglutide, in particular Ala for coupling Somatoglutide²⁴-Ala²⁵。

The invention provides a preparation method of a compound Fmoc-Ala- (Dmb) Ala-OH, which comprises the following steps:

step 1: dissolving alanine, 2, 4-dimethoxybenzaldehyde and cyano sodium borohydride in methanol and water to react to obtain (S) -2- (2, 4-dimethoxyphenylenediamine) propionic acid;

step 2: Fmoc-Ala-OH was reacted with oxalyl chloride in DCM to afford Fmoc-Ala-Cl;

and step 3: and (3) reacting the Fmoc-Ala-Cl prepared in the step (1), triethylamine and (S) -2- (2, 4-dimethoxyphenylenediamine) propionic acid prepared in the step (2) in DCM to obtain Fmoc-Ala- (Dmb) Ala-OH.

The term "solid phase synthesis" as used herein refers to a synthetic method in which reactants are attached to an insoluble solid phase support. The solid phase synthesis of polypeptide is to connect one amino acid to the insoluble solid phase carrier and to connect other amino acids successively to the amino acid connected to the solid phase carrier. In solid phase synthesis of polypeptides, resins are generally used as solid phase carriers. The solid phase synthesis of polypeptide usually includes linking the first amino acid C terminal of target peptide to solid phase carrier via covalent bond, taking the amino acid N terminal as synthesis starting point, removing amino protecting group and excessive activated second amino acid to react, lengthening peptide chain, repeating operation to reach ideal synthetic peptide chain length, finally cracking the peptide chain from resin, separating and purifying to obtain target polypeptide. As is well known in the art, resins for solid phase synthesis of polypeptides include, but are not limited to, polystyrene-divinyl benzene cross-linked resins, polyacrylamide resins, polyethylene-glycol resins and derivatives thereof, which may be directly linked with (first) amino acids after introduction of reactive groups. These resins and resin derivatives can be classified into chloromethyl resins, carboxyl resins, amino resins, or hydrazide resins depending on the introduced reactive group. Resins currently used in the art include, but are not limited to, PAM resins, MBHA resins, Wang resins, 2-CTC resins, Rink-Amide resins, and the like. In some embodiments of the invention, the resin used is Wang resin or 2-CTC resin, the meaning and structural formula of which are given in Table 1 below.

The term "condensation reagent" as used herein refers to a reagent used for amino acid coupling in solid phase synthesis of polypeptides. In solid phase synthesis of polypeptides, different coupling methods and/or different condensing reagents may be chosen depending on different resins, different amino acid sequences and/or different protecting groups. Condensation reagents include, but are not limited to, carbodiimide type and onium salt type. The carbodiimide type mainly includes DCC, DIC, edc.hcl, and the like. The carbodiimide type condensation reagent may be used alone or in combination with other condensation reagents. When a carbodiimide type condensation reagent is used in combination with HOBt, HOAt, etc., the side reaction can be controlled to a low range. The onium salt type condensation reagent mainly includes HBTU, HATU, PyBOP and the like. The onium salt type condensing agent may be added with an organic base such as DIEA for activating amino acids during use. The selection of suitable condensing reagents based on the resin, amino acid sequence and protecting group is within the ordinary skill in the art.

In the present invention, when a plurality of condensing agents are used, these condensing agents may be used in combination. In the present invention, the condensing agent may be DIC, DIC/HOBt, HATU/DIEA, HATU/HOBT/DIEA, HBTU/HOBT/DIEA or PyBOP/HOBT/DIEA. Wherein "DIC/HOBt" represents a combination of DIC and HOBt, "HATU/DIEA" represents a combination of HATU and DIEA, "HATU/HOBT/DIEA" represents a combination of HATU, HOBT and DIEA, "HBTU/HOBT/DIEA" represents a combination of HBTU, HOBT and DIEA, and "PyBOP/HOBT/DIEA" represents a combination of PyBOP, HOBT and DIEA. It will be understood by those skilled in the art that the use of two or more condensing agents in combination means that the two or more condensing agents are added in one coupling reaction. It is within the knowledge of the skilled person how to use two or more condensing reagents in combination, and the skilled person knows how to add said two or more condensing reagents in one coupling reaction, e.g. in amounts, timing and order of addition, etc. In the present invention, various combinations of condensation reagents may also be used in the coupling with a certain starting material (which may be coupling of one amino acid, coupling of a short peptide of two or more amino acids, or coupling of part or all of a side chain). For example, in the present invention, the condensing agent to be used may be two or more of DIC, DIC/HOBt, HATU/DIEA, HATU/HOBT/DIEA, HBTU/HOBT/DIEA and PyBOP/HOBT/DIEA combinations. When two or more combinations of condensing agents are used, the coupling reactions may be performed multiple times sequentially, each using one of the combinations of condensing agents, as known to those skilled in the art.

In the present invention, the reagents and reaction conditions used for the coupling reaction of each amino acid may be the same as or different from each other. For example, the reagents and reaction conditions used for the coupling reaction of all amino acids may be the same as each other, or the reagents and reaction conditions used for the coupling reaction of a part of amino acids may be the same as each other. The reagents and reaction conditions used for the coupling reaction of each amino acid may be independently selected.

In the present invention, when referring to a specific amino acid or side chain in the sequence of the conjugated thaumatin, it means the same as the starting material for the coupling of the specific amino acid or side chain, it will be understood by those skilled in the art that it refers to the starting material for the synthesis of the amino acid or side chain attached to the resin or to the amino acid that has been attached to the resin. The starting material usually contains the amino acid or side chain, and further contains a protecting group or the like. For example, in the present invention, when Fmoc-Lys (Ivdde) -OH is used as a starting material for coupling Lys²⁶When is "coupled Lys²⁶"can have the same meaning as" coupled Fmoc-Lys (Ivdde) -OH ".

In the present invention, the side chain of the somagluteptide refers to the bond Lys²⁶An attached side chain moiety. In the invention, the structural formula of the side chain of the Somalutide is shown asWhen the Somalutide is synthesized by a solid-phase synthesis method, Lys may be eliminated in the backbone²⁶After side chain protecting groups of (5), in Lys²⁶The side chain of the somulutide is coupled. At Lys²⁶When the side chain of the soxhlet peptide is coupled, the side chain can be divided into a plurality of parts, the parts are coupled step by step, or the whole side chain can be synthesized firstly and then coupled to Lys²⁶The above.

In the present invention, when an amino acid in the somaglutide amino acid sequence is described using an amino acid having a numerical symbol in the upper right corner, the numerical symbol in the upper right corner indicates the position number of the amino acid from the N-terminus to the C-terminus in the somaglutide amino acid sequence. For example, "Arg³⁶"denotes the 36 th Arg from N-terminus to C-terminus of the thaumatin amino acid sequence; similarly, "Lys²⁶”、“Ala²⁴-Ala²⁵”、“Gln²³"and“His⁷"denotes Lys, Ala-Ala at positions 24 to 25, Gln at position 23, and His at position 7, respectively, in the amino acid sequence of Somalutide from N-terminus to C-terminus. Other similar expressions are synonymous.

As is known to those skilled in the art, protection of the coupled amino acid starting material is often required during solid phase synthesis of polypeptides. In solid phase synthesis of polypeptides, if the polypeptide contains two adjacent alanines, it is prone to form a secondary structure of β -sheet, which leads to aggregation of the peptide. The present inventors have found that when two adjacent alanines are synthesized, R-Ala- (Dmb) Ala-OH, which can be any suitable protecting group such as Fmoc or Boc, can be used as a starting material instead of two R-Ala-OH, and that the formation of hydrogen bonds between peptide chains is disrupted by introducing Dmb (i.e., 2, 4-dimethoxybenzyl) on the N of Ala, so that the synthesized peptides do not easily form a secondary structure of β -sheet, and the aggregation of peptides is greatly reduced, thereby effectively increasing the condensation efficiency. Therefore, in the present invention, Fmoc-Ala- (Dmb) Ala-OH is used instead of Fmoc-Ala-OH in the solid phase synthesis of two amino acids from the N-to C-terminal positions 24-25 of the sequence of Somalouin.

In the present invention, for Lys²⁶The Ivdde can be removed by 2% hydrazine in DMF and is relatively stable in TFA and piperidine solution, protected by Ivdde. In the present invention, for His⁷Protection is performed using Trt. Both Trt and Dmb can be removed together by a cleavage reagent in the final step of cracking the somaglutide-resin without adding other reagents additionally, so that other impurities cannot be additionally introduced in the synthesis process.

In the process of solid-phase synthesis of the somalutide in the present invention, other amino acids may be selected from suitable protecting groups according to circumstances, which protecting groups are used for different amino acids is well known to those skilled in the art, and amino acids with protecting groups may be obtained commercially or synthesized by themselves. For example, protection of amino acids may include alpha-amino protection and side chain protection. As known to those skilled in the art, the α -amino group can be protected by Boc, Fmoc, etc.; the side chain hydroxyl of serine and threonine can be protected by benzyl, tert-butyl and the like; the side chain carboxyl groups of aspartic acid and glutamic acid can be protected by tert-butyl and the like; the lysine side chain can be protected by t-butyloxycarbonyl group, 1- (1-adamantyl) -1-methylethoxycarbonyl (Adpoc), Alloc, etc.; tryptophan may be protected by t-butyloxycarbonyl or the like.

In the present invention, after all the amino acids of the backbone and side chains of the somagluteptide are attached, the somagluteptide needs to be cleaved from the solid support (e.g., resin), and this process is performed using a cleavage reagent. In solid phase polypeptide synthesis, different cleavage methods and/or different cleavage reagents may be selected depending on the different solid phase supports (e.g., resins), different amino acid sequences, and/or different protecting groups. For example, the cleavage under acidic conditions may be selected, and for PAM and MBHA resins, HF cleavage may be used, and reagents such as p-cresol, p-mercaptophenol, and/or anisole may be added during the cleavage. For Wang resin, Rink-Amide resin, 2-CTC resin and Trt resin, TFA can be used for cracking, and ethanedithiol, thioanisole, water, triisopropylsilane and/or phenol and the like can be added in the cracking process. The additive agents can be used as a carbonium ion capture agent for capturing carbonium ions generated in the cracking reaction process and reducing side reactions caused by the attack of the carbonium ions on partial amino acid (such as Trp, Tyr and the like) side chains. In addition, 2-CTC resin can also be cut by weak acid AcOH/TFE/DCM or 0.5% TFA, and polypeptide with fully protected side chain active group can be obtained. The selection of suitable cleavage reagents based on resin, amino acid sequence and protecting groups is within the routine skill in the art. In the invention, the cleavage reagent used can be a mixture of TFA, thioanisole, phenol, EDT and water, preferably, the volume ratio of TFA, thioanisole, phenol, EDT and water is 85-90: 2.5-7.5: 1-4: 2.5-7.5, and more preferably, the volume ratio of TFA, thioanisole, phenol, EDT and water is 87.5:5:2.5: 5.

In the present invention, "salt of somagluteptide" includes, but is not limited to, salts of somagluteptide with acids formed from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with organic acids; such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or salts formed when the acidic proton present on the somaglutide is replaced by a metal ion, for example an alkali metal ion or an alkaline earth metal ion; or a coordination compound of somaglutide with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like. In some preferred embodiments of the invention, the salt of somaglutide is the TFA salt of somaglutide.

In the present invention, "salt transfer" refers to a process of producing a desired salt from a synthesized polypeptide by an ion exchange reaction, which can be performed by an ion exchange method and an HPLC method using a column. In some embodiments of the invention, the thaumatin is subjected to salt transfer using an HPLC method using a C18 reverse phase column.

In the present invention, the "detection amount" means an amount sufficient to achieve the desired detection purpose, which can be determined by a person skilled in the art by conventional technical means according to a specific detection method.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The meanings of some common abbreviations in the present invention and their structural formulas are shown in the following table 1.

TABLE 1

The invention synthesizes the Somatobrutide by a solid-phase synthesis method, which comprises gradually coupling the Somatobrutide on a solid-phase carrier on a main chainThen Lys is removed²⁶The side chain protecting group of (a) is then coupled with the amino acid on the side chain of the somaglutide. The method can reduce the synthesis of difficult peptide sequences in solid phase synthesis, improve the synthesis efficiency and reduce the purification difficulty. Specifically, the method of the present invention comprises: (1) coupling Gly with Wang resin or 2-CTC resin to obtain Fmoc-Gly-resin; (2) using Fmoc-Gly-resin, obtaining the Somaloutide backbone-resin, wherein, if 2-CTC resin is selected, Arg is coupled³⁶HATU/DIEA is selected as condensation reagent to avoid Gly³⁷The double condensation phenomenon reduces the probability of the generation of the inserted peptide and the difficulty of purification; in the coupling of Ala²⁴-Ala²⁵The Fmoc-Ala- (Dmb) Ala-OH is selected as a raw material, so that the aggregation of polypeptide and the generation of deletion peptide can be effectively avoided, and the purity of a sample is improved; in coupling His⁷Boc-His (Trt) -OH is selected as a raw material, so that the influence on Fmoc when Ivdde is removed by hydrazine hydrate can be avoided, and racemization of histidine can be effectively avoided; (3) desorption of Lys in the backbone of Somatocride²⁶Side chain protecting groups of (3), then at Lys²⁶Coupling the side chain of the Somaloutide to obtain Somaloutide-resin; and (4) the resin peptide of the Somalutide is obtained by cracking and purifying. The measures of the invention can effectively avoid the generation of inserted peptides and deleted peptides, and improve the coupling efficiency and the purity of crude peptides. In addition, the method can remove excessive amino acid and condensation reagent only by suction filtration and washing, avoids the complicated step of separating and purifying through a silica gel column in the liquid phase reaction, and greatly improves the efficiency.

Drawings

FIG. 1: schematic solid phase synthesis of somaglutide.

FIG. 2: example 14 HPLC profile of the final product.

FIG. 3: and when Fmoc-Ala- (Dmb) Ala-OH is not used as a raw material, a mass spectrum of the molecular weight detection is obtained by coupling 15 amino acids.

FIG. 4: and when Fmoc-Ala- (Dmb) Ala-OH is not used as a raw material, a mass spectrum of the molecular weight detection is obtained by coupling 19 amino acids.

FIG. 5: when Fmoc-Ala- (Dmb) Ala-OH is used as a raw material, a mass spectrum of the molecular weight detection is obtained by coupling 16 amino acids.

FIG. 6: and when Fmoc-Ala- (Dmb) Ala-OH is used as a raw material, a molecular weight detection mass spectrum is formed by coupling 22 amino acids.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the invention. Any modification, equivalent replacement or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The amino acid raw materials, the condensation reagent, the resin and the like used in the invention are all sold in the market; resins were purchased from west anlan and amino acid feedstocks from gill biochemistry.

The amino acid starting materials used in the present invention are shown in table 2 below.

TABLE 2

The present invention will be described in detail with reference to the following examples.

Example 1 Synthesis of Fmoc-Ala- (Dmb) Ala-OH

Alanine (20g, 225mmol),2, 4-dimethoxybenzaldehyde (44.82g, 270mmol), sodium cyanoborohydride (28g, 450mmol) were added to a 2L three-necked flask and dissolved in methanol (800mL) and water (400mL), the reaction was stirred at room temperature for 48 hours, after completion of the reaction, the reaction solution was evaporated by rotary evaporator at 45 ℃ to remove methanol, the aqueous phase was washed 3 times with DCM, the aqueous phase was lyophilized and then dissolved in methanol and applied to the column and eluted with DCM/MeOH ═ 10:1 to give (S) -2- (2, 4-dimethoxyphenylenediamine) propionic acid (27g) in 50% yield.

Adding Fmoc-Ala-OH (5.0g, 16.1mmol) into a 250mL three-necked flask, adding 150mL dry DCM for dissolution, enabling the reaction liquid to be turbid, dropwise adding 2-3 drops of DMF, stirring for 5 minutes, cooling to 0 ℃ under ice bath, dropwise adding oxalyl chloride (6.13g, 48.2mmol), stirring for 2 hours at normal temperature after dropwise adding, and after the reaction is finished, performing rotary evaporation and rotary drying on the reaction liquid at 45 ℃ by using a rotary evaporator to obtain a white solid compound Fmoc-Ala-Cl (5.0g), wherein the yield is 94.3%.

Adding Fmoc-Ala-Cl (6.45g, 27.4mmol) and triethylamine (9.2g, 91.2mmol) into a 500mL three-necked flask, cooling to 0 ℃ in ice bath, dissolving (S) -2- (2, 4-dimethoxyphenylenediamine) propionic acid (10.0g, 30.4mmol) in 100mL DCM, slowly dropping into the reaction system at a temperature below 5 ℃, naturally heating to react for 3 hours after dropping, adjusting pH to acidity with 1mol/L hydrochloric acid after reaction is completed, stirring for 30 minutes, then layering, extracting the aqueous phase with DCM for 2 times, combining the organic phases, washing with saturated saline, drying, filtering and evaporating to dryness. The pure product is obtained by column chromatography separation, and the yield is as follows: 24.3 percent.

EXAMPLE 2 Synthesis of Fmoc-Gly-CTC resin having a degree of substitution of 0.20mmol/g

Weighing 1g of 2-CTC resin with the substitution degree of 0.98mmol/g, adding the resin into a cup-shaped reaction column, swelling the resin with DCM for 30 minutes, and filtering to remove DCM; weighing 0.3mmol Fmoc-Gly-OH, adding 10mL of DCM and 0.15mL of DIEA into a reaction cup, and carrying out nitrogen bubbling reaction for 90 min; adding 0.2mL of methanol and 0.2mL of DIEA to perform end capping reaction for 30min, washing with industrial DMF for 4 times, washing with DCM for 1 time, and then pumping out methanol to obtain Fmoc-Gly-CTC resin; a small amount of the resin was used and the degree of substitution was measured by UV spectrophotometer to be 0.16mmol/g (three-time average).

EXAMPLE 3 Synthesis of Fmoc-Gly-Wang resin having degree of substitution of 0.25mmol/g

Weighing 1g of Wang resin with the substitution degree of 0.79mmol/g, adding the Wang resin into a cup-shaped reaction column, swelling the resin with DCM for 30min, and filtering to remove DCM; weighing 0.35mmol of Fmoc-Gly-OH, adding HOBT/DIC/DMAP (the equivalent ratio of the amino acid to the amino acid is 1/1/0.1) into a reaction cup, wherein the solvent is DCM/DMF (1: 1), and carrying out nitrogen bubbling reaction for 120 min; washing with industrial DMF for 5 times, adding 0.2mL acetic anhydride/pyridine (volume ratio of 1/1), keeping the solvent as DCM, and carrying out end capping reaction for 60 min; industrial DMF was washed 4 times, DCM was washed 1 time and then methanol was used for suction drying to obtain Fmoc-Gly-Wang resin. A small amount of the resin was used and the degree of substitution was measured by UV spectrophotometer to be 0.24mmol/g (three-time average).

Example 42-CTC resin Arg³⁶Of (2) coupling

The Fmoc-Gly-CTC resin with the degree of substitution of 0.20mmol/g prepared in example 2 was placed in a cup-shaped reaction column, the resin was swollen with DCM for 30min, and DCM was removed by suction filtration; adding the mixture in a volume ratio of 1:4, removing the Fmoc protecting group by the Pip/DMF, wherein the removing time is 25min, adding industrial DMF for washing for 5 times, DCM for washing for 1 time, washing with the Chinese medicament DMF for 1 time, detecting the color development of the resin by ninhydrin, and simultaneously ensuring that piperidine is washed cleanly without residues. 0.31g of Fmoc-Arg (Pbf) -OH (0.48mmol), 0.19g of HATU (0.5mmol) was weighed out, dissolved in 5mL of DMF, and 0.16mL of DIEA (1mmol) was added thereto, reacted for 2min, poured into a cup-shaped reaction column, and subjected to nitrogen bubbling for 90 min.

In all coupling reactions, the process needing pre-activation is carried out in an ice-water bath, and racemization and other byproducts are avoided in the activation process. Ninhydrin is used for detecting the resin in the coupling reaction, and if the resin has no color, the coupling is complete; if the resin is developed, the coupling is not complete, and the reaction time needs to be prolonged until the coupling is complete; under the condition, the prolonged reaction time cannot exceed 3h, if the resin still develops color after 3h, the reaction solution needs to be removed by suction filtration, and after the resin is washed for 1 time by adding the traditional Chinese medicine DMF, the material is fed again for coupling until the resin has no color by ninhydrin detection.

All reagents for removing Fmoc protecting groups are Pip/DMF with the volume ratio of 1:4, the removal time is 25min, then industrial DMF is added for washing for 5 times, DCM is used for washing for 1 time, the Chinese medicine DMF is used for rinsing for 1 time, and ninhydrin detection resin develops color. In order to ensure that the piperidine is washed cleanly and has no residue, the filtrate after the last washing needs to be detected by chloranil when necessary, the color of the filtrate does not become dark purple, the washing is clean, the resin has no residual piperidine, or the washing times need to be added.

The stirring mode except for the cracking was nitrogen bubbling.

Example 5 Wang resin Arg³⁶Of (2) coupling

The Fmoc-Gly-Wang resin with the degree of substitution of 0.24mmol/g, prepared in example 3, was placed in a cup-shaped reaction column, the resin was swollen with DCM for 30min, and DCM was removed by suction filtration; adding Pip/DMF with the volume ratio of 1:4 to remove Fmoc, removing for 5min, then performing suction filtration, adding Pip/DMF with the volume ratio of 1:4 again to perform reaction for 5min, performing suction filtration, adding industrial DMF to wash for 5 times, washing for 1 time with DCM, washing for 1 time with Chinese medicine DMF, and detecting the color development of the resin by ninhydrin detection. 0.37g of Fmoc-Arg (Pbf) -OH (0.57mmol), 65mg of HOBT (0.48mmol) was weighed out and dissolved in 4mL of DMF, and after pre-activation in an ice-water bath, 0.075mL of DIC (0.45mmol) was added and poured onto a cup-shaped reaction column and subjected to nitrogen bubbling for 70 min.

In all coupling reactions, the process needing pre-activation is carried out in an ice-water bath, and racemization and other byproducts are avoided in the activation process. Ninhydrin is used for detecting the resin in the coupling reaction, and if the resin has no color, the coupling is complete; if the resin is developed, the coupling is not complete, and the reaction time needs to be prolonged until the coupling is complete; under the condition, the prolonged reaction time cannot exceed 3h, if the resin still develops color after 3h, the reaction solution needs to be removed by suction filtration, and after the resin is washed for 1 time by adding the traditional Chinese medicine DMF, the material is fed again for coupling until the resin has no color by ninhydrin detection.

All reagents for removing Fmoc protecting groups are Pip/DMF with the volume ratio of 1:4, the removal time is 25min, then industrial DMF is added for washing for 5 times, DCM is used for washing for 1 time, the Chinese medicine DMF is used for rinsing for 1 time, and ninhydrin detection resin develops color. In order to ensure that the piperidine is washed cleanly and has no residue, the filtrate after the last washing needs to be detected by chloranil when necessary, the color of the filtrate does not become dark purple, the washing is clean, the resin has no residual piperidine, or the washing times need to be added.

The stirring mode except for the cracking was nitrogen bubbling.

Example 62 preparation of CTC resin Somatocride backbone resin peptide

Taking the already coupled Arg in example 4³⁶Adding Pip/DMF with the volume ratio of 1:4 to remove the Fmoc protecting group, then adding industrial DMF to wash for 5 times, DCM to wash for 1 time, Chinese medicine DMF to wash for 1 time, and detecting the color development of the resin by ninhydrin. Then coupling the two in sequence according to the amino acid sequence of the Somalou peptide, wherein the charging equivalent of the amino acid raw material is 3 equivalents, the reaction concentration is 0.15mmol/mL, and the condensation reagentCan be DIC/HOBt, and the solvent is DMF.

The coupling method of Fmoc-Lys (Ivdde) -OH, Boc-His (Trt) -OH and Fmoc-Ala- (Dmb) Ala-OH comprises the steps of feeding amino acid raw materials with the equivalent weight of 3 equivalents, reacting with the concentration of 0.3mmol/mL, DIC/HOBt as a condensation reagent, coupling and suction-filtering DMF with a solvent for 40min, washing the resin with DMF for 1 time, then coupling again with HATU/HOBT/DIEA for 40min, and using DMF: DCM with the volume ratio of 4:1 as the solvent.

In addition, Gln after 2 consecutive hydrophobic Ala²³The coupling method is characterized in that the feeding equivalent of amino acid raw materials is 3 equivalents, the reaction concentration is 0.3mmol/mL, DIC/HOBt is a condensation reagent, DMF is used as a solvent, the resin is coupled for 40min and then is subjected to suction filtration, HATU/HOBT/DIEA is used for coupling for 40min again after DMF is used for washing the resin for 1 time, and the solvent is DMF: DCM with the volume ratio of 4: 1. Taking a small amount of resin to remove Fmoc and then cracking to ensure Gln²³The next step can be performed.

Example 7 preparation of Wang resin Somatolutide backbone resin peptide

Taking the already coupled Arg in example 5³⁶Adding Pip/DMF with the volume ratio of 1:4 to remove the Fmoc protecting group, then adding industrial DMF to wash for 5 times, DCM to wash for 1 time, Chinese medicine DMF to wash for 1 time, and detecting the color development of the resin by ninhydrin. And then coupling the two in sequence according to the amino acid sequence of the Somalouptides, wherein the feeding equivalent of the amino acid raw material is 3 equivalents, the reaction concentration is 0.15mmol/mL, the condensation reagent can be DIC/HOBt, and the solvent is DMF.

The coupling method of Fmoc-Lys (Ivdde) -OH, Boc-His (Trt) -OH and Fmoc-Ala- (Dmb) Ala-OH comprises the steps of feeding amino acid raw materials with the equivalent weight of 3 equivalents, reacting with the concentration of 0.3mmol/mL, DIC/HOBt as a condensation reagent, coupling and suction-filtering DMF with a solvent for 40min, washing the resin with DMF for 1 time, then coupling again with HATU/HOBT/DIEA for 40min, and using DMF: DCM with the volume ratio of 4:1 as the solvent.

In addition, Gln after 2 consecutive hydrophobic Ala²³The coupling method is characterized in that the feeding equivalent of amino acid raw materials is 3 equivalents, the reaction concentration is 0.3mmol/mL, DIC/HOBt is a condensation reagent, DMF is used as a solvent for coupling for 40min, then suction filtration is carried out, HATU/HOBT/DIEA is used for coupling for 40min again after DMF is used for washing resin for 1 time, and the solvent is HATU/HOBT/DIEAThe ratio by volume of DMF to DCM was 4: 1. Taking a detected amount of resin, removing Fmoc, cracking, and ensuring Gln²³The next step can be performed.

Example 82 CTC resin Somatode backbone resin peptide removal of Ivdde

Taking the backbone resin peptide of the Somaluotide obtained in the embodiment 6, adding 2% hydrazine hydrate/DMF solution with the volume 2-3 times of the volume of the resin, carrying out nitrogen bubbling reaction for 10min, and then carrying out suction filtration to remove the reaction liquid; adding 2% hydrazine hydrate/DMF solution with the volume 2-3 times of that of the resin again, and repeating the operation for 4 times; a measured amount of the resin was cleaved and MS detected without amu +206.26 for the next step.

Example 9 Wang resin Somalutide backbone resin peptide removal of Ivdde

Taking the backbone resin peptide of the Somaluotide obtained in the embodiment 7, adding a DMF solution of 2% hydrazine hydrate, the volume of which is 2-3 times of the volume of the resin, carrying out nitrogen bubbling reaction for 10min, and then carrying out suction filtration to remove the reaction liquid; adding 2% hydrazine hydrate solution DMF solution 2-3 times of the resin volume again, and repeating the operation for 4 times; a small amount of the resin was cleaved and MS detected without amu +206.26 for the next step.

Example 102 CTC resin Somatode backbone resin peptide modified side chains

Sequentially coupling the Somalou peptide backbone resin peptide from which Ivdde is removed in example 8 with Fmoc-NH-PEG according to the sequence of the side chain fragments of the Somalou peptide₂-CH₂COOH、Fmoc-NH-PEG₂-CH₂COOH, Fmoc-Glu (OH) -OtBu and mono-tert-butyl octadecanedioate.

Wherein Fmoc-NH-PEG₂-CH₂COOH and Fmoc-Glu (OH) -OtBu feeding equivalent weight are 3 equivalent, reaction concentration is 0.3mmol/mL, condensation reagent is HATU/HOBT/DIEA, and solvent is DMF. The reaction was carried out at room temperature for 90 min.

The condensation reagent of octadecanedioic acid mono-tert-butyl ester is DIC and preactivated for 5min, the charge equivalent is 3 equivalents, the reaction concentration is 0.3mmol/mL, the solvent is DMF: DCM with the volume ratio of 1:1, the reaction is carried out at room temperature for 70min, after washing with DMF for 5 times, washing with DCM for 2 times, and then draining the resin with methanol or dichloromethane.

Example 11 Wang resin Somalutide backbone resin peptide modified side chain

Coupling the Somalou peptide backbone resin peptide from which Ivdde had been removed in example 9 with Fmoc-NH-PEG in sequence according to the sequence of the side chain groups of the Somalou peptide₂-CH₂COOH、Fmoc-NH-PEG₂-CH₂COOH, Fmoc-Glu (OH) -OtBu and mono-tert-butyl octadecanedioate.

Wherein Fmoc-NH-PEG₂-CH₂COOH and Fmoc-Glu (OH) -OtBu feeding equivalent weight are 3 equivalent, reaction concentration is 0.3mmol/mL, condensation reagent is HATU/HOBT/DIEA, and solvent is DMF. The reaction was carried out at room temperature for 90 min.

The condensation reagent of octadecanedioic acid mono-tert-butyl ester is DIC and is preactivated for 5min, the feeding equivalent is 3 equivalents, the reaction concentration is 0.3mmol/mL, the solvent is DMF: DCM with the volume ratio of 1:1, the reaction is carried out for 70min at room temperature, after washing with DMF for 5 times, washing with DCM for 2 times, and then draining the resin with methanol or dichloromethane.

Example 122-cleavage of CTC somaglutide resin peptide

The cleavage reagent was TFA, thioanisole, phenol, EDT and water in a ratio of 87.5:5:2.5:5: 2.5. Placing in a 2-8 deg.C display cabinet for use, and placing diethyl ether in a 2-8 deg.C display cabinet for use. Pouring the tree cracking reagent into a large beaker, placing the beaker into an ice water bath, adding the drained resin while stirring, reacting for 30min in the ice water bath, and then gradually raising the temperature to room temperature to continue reacting for 2.5 h. And (4) detecting by MS that the protecting group is not completely cut off, and supplementing a capture agent corresponding to the protecting group if the MS shows that the protecting group is not cut off. After the cracking is finished, filtering to obtain filtrate, adding 20 times of the volume of the filtrate into the filtrate, and centrifuging and precipitating; adding water and acetonitrile into the precipitate to dissolve, filtering and freeze-drying the precipitate to prepare the product. After lyophilization, 0.49g of crude peptide was obtained in 74.4% yield and 57.8% purity.

Example 13 cleavage of Wang Somatode resin peptide

The ratio of the cracking reagent to TFA, thioanisole, phenol, EDT and water is 85: 2.5: 2.5:5: 2.5. placing in a 2-8 deg.C display cabinet for use, and placing diethyl ether in a 2-8 deg.C display cabinet for use. Pouring the tree cracking reagent into a large beaker, placing the beaker into an ice water bath, adding the drained resin while stirring, reacting for 30min in the ice water bath, and then gradually raising the temperature to room temperature to continue reacting for 2.5 h. And (4) detecting by MS that the protecting group is not completely cut off, and supplementing a capture agent corresponding to the protecting group if the MS shows that the protecting group is not cut off. After the cracking is finished, filtering to obtain filtrate, adding 20 times of the volume of the filtrate into the filtrate, and centrifuging and precipitating; adding water and acetonitrile into the precipitate to dissolve, filtering and freeze-drying the precipitate to prepare the product. After freeze-drying, 0.76g of crude peptide is obtained, the yield is 77.0 percent, and the purity of the crude peptide is 62.0 percent.

EXAMPLE 14 preparation of Somalutide TFA salt

Crude peptide 0.49g of crude peptide of Somalutide was weighed, a sample was dissolved by adding water and acetonitrile (about 3:1) in a total volume of about 50mL, and prepared by LC20-AP (Shimadzu corporation, model 20-AP) using a 50X 250mm C18 reverse phase column, wavelength 220nm and 254nm, column temperature 40 ℃ and mobile phase of 1 ‰ TFA/water and 1 ‰ TFA/acetonitrile, and the target fraction was collected and lyophilized to obtain a product peptide with a purity of greater than 98.5%. 0.16g of a solid was obtained in a purification yield of 32% and a total yield of 23.8%.

Example 15 preparation of the Somalutide TFA salt

Weighing 0.5g of crude peptide of the somaltulin, adding formic acid water/acetonitrile with the total volume of about 30mL to dissolve a sample, preparing the sample by using LC20-AP, wherein a chromatographic column is a 50 x 250mm C18 reverse phase column, the wavelengths are 220nm and 254nm, the column temperature is 40 ℃, a mobile phase is 1 thousandth TFA/water and 1 thousandth TFA/acetonitrile, collecting a target component, and freeze-drying to obtain the product peptide with the purity of more than 98.5%. 0.12g of solid was obtained in 24% yield and 18.5% overall yield.

Example 16 comparative experiment

To verify the synthesis of Ala Using Fmoc-Ala- (Dmb) Ala-OH starting Material²⁴-Ala²⁵Whether Gln can be avoided during the synthesis of Somalutide²³The loss of (d) was demonstrated using comparative experiments. Taking the already coupled Arg in example 4³⁶The Fmoc protecting group of the resin is removed by adding 20 percent of Pip/DMF, and then the resin is washed 5 times by adding industrial DMF, 1 time by DCM and 1 time by national medicine DMF, and the color development of the resin is detected by ninhydrin detection. Then coupling according to the amino acid sequence from C end to N end of the Somaloutide, Ala²⁴-Ala²⁵Fmoc-Ala- (Dmb) Ala-OH is not used as a raw material, but is used as a raw material, wherein ammoniaThe equivalent of the raw material of the amino acid is 3 equivalents, the reaction concentration is 0.2mmol/mL, the coupling reagent is DIC/HOBt, and the solvent is DMF. The Fmoc-Lys (Ivdde) -OH coupling method comprises the steps of feeding 3 equivalents of amino acid raw materials, performing suction filtration after coupling 40min by using DIC/HOBt as a coupling reagent and DMF as a solvent, washing resin by using DMF for 1 time, then performing coupling again for 40min by using HATU/HOBT/DIEA, wherein the volume ratio of the solvent to the DMF is 4: 1: DCM. Gln²³The coupling method comprises the steps of feeding 3 equivalent amino acid raw materials, reacting 0.3mmol/mL, DIC/HOBt as a coupling reagent, coupling DMF as a solvent for 40min, then carrying out suction filtration, washing resin with DMF for 1 time, then selecting HATU/HOBT/DIEA, and coupling again for 40min, wherein the solvent is DMF with the volume ratio of 4: 1: DCM. Taking the intermediate resin peptide for mass spectrum detection, and calculating the molecular weight according to mass spectrum data by the following formula: m-bivalent mass spectrum value 2-2 or M-trivalent mass spectrum value 3-3. When the peptide chain length is 15 amino acids, the target molecular weight is 1907.26, and the detection molecular weights are 1908 and 1779.8, the latter being the molecular weight of the lost Q, as shown in FIG. 3; when the peptide chain length was 19 amino acids coupled, the target molecular weight was 2370.73, and the detected molecular weights were 2370.6 and 2242.2, the latter being the molecular weight of the lost Q, see fig. 4.

Similarly, when the intermediate resin peptide of example 6 was subjected to mass spectrometric detection and the peptide chain length was 16 amino acids, the target molecular weight was 1964.3, the detected molecular weight was 1965, and no loss of Q was found at 1836.3 (see FIG. 5); when the peptide chain length was 22 amino acids, the target molecular weight was 2644.01, the detected molecular weight was 2643.8, and the molecular weight of lost Q was 2515.8, as shown in fig. 6. As can be seen, the raw material of Fmoc-Ala- (Dmb) Ala-OH can effectively avoid Gln²³Probably due to the fact that the Dmb protecting group prevents the formation of hydrogen bonds which could lead to polypeptide aggregation, resulting in Gln²³Can be fully condensed; meanwhile, the Dmb protecting group replaces H on a nitrogen atom capable of forming a hydrogen bond, so that the hydrogen bond cannot be formed and the aggregation of the polypeptide can be avoided.

While the foregoing is directed to the preferred embodiment of the present invention, and it is not intended that the invention be limited thereto, it will be appreciated by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention.