阅读说明：本技术 一种制备特立帕肽的方法 (Method for preparing teriparatide ) 是由 汪伟 宓鹏程 陶安进 袁建成 于 2018-06-26 设计创作，主要内容包括：本发明属于药物化学技术领域,公开了一种制备特立帕肽的方法。本发明所述制备特立帕肽的方法采用片段缩合制备特立帕肽。首先分别合成特立帕肽第1-16位肽序列(片段A)和第17-34位肽序列(片段B),然后,将两个片段偶联得到特立帕肽粗肽,纯化获得特立帕肽。本发明中的片段侧链没有保护基,在水中有较好的溶解度,不存在偶联困难的问题,操作简单,生产效率高。得到的特立帕肽产品纯度高,易于纯化。实验表明,本发明得到的特立帕肽的粗肽纯度可得到80％,总收率能达到45％。经过简单的纯化精肽纯度可达到99.92％,单个最大杂质0.05％。与现有技术相比,本发明具有产品质量高、成本低、适合工业化生产等特点。(The invention belongs to the technical field of medicinal chemistry, and discloses a method for preparing teriparatide. The method for preparing teriparatide adopts fragment condensation to prepare teriparatide. Firstly, respectively synthesizing the peptide sequences (segment A) at the 1 st to 16 th positions and the peptide sequences (segment B) at the 17 th to 34 th positions of teriparatide, then coupling the two segments to obtain crude teriparatide peptide, and purifying to obtain the teriparatide. The side chain of the fragment has no protecting group, has better solubility in water, does not have the problem of difficult coupling, and has simple operation and high production efficiency. The obtained teriparatide product has high purity and is easy to purify. Experiments show that the purity of the crude peptide of the teriparatide obtained by the invention can be 80%, and the total yield can reach 45%. The purity of the refined peptide can reach 99.92% through simple purification, and the single maximum impurity is 0.05%. Compared with the prior art, the invention has the characteristics of high product quality, low cost, suitability for industrial production and the like.)

1. A method of preparing teriparatide, comprising:

step 1: coupling 3-Fmoc-4-diaminobenzoic acid with a solid phase carrier, sequentially coupling Fmoc-Asn (Trt) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Glu-OH and PG-Ser (tBu) -OH according to a peptide sequence from C terminal to N terminal, forming benzimidazolone by p-nitro phenyl chloroformate, finally obtaining a fragment A PG-Ser-Val-Ser-Ill-Ser-Val by the cleavage of salicylaldehyde and TFA -Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-SAL;

step 2: coupling Fmoc-Phe-OH with a solid-phase carrier, sequentially coupling Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH and Fmoc-Ser tBu) -OH from C end to N end according to the peptide sequence, and cleaving TFA to obtain fragment B-Glu-Arg-Val-Glu-Leu-Lys-OH -Leu-Gln-Asp-Val-His-Asn-Phe-OH;

and step 3: coupling the segment A and the segment B, and then removing a protecting group of Ser at the 1 position in the segment A to obtain crude teriparatide peptide;

and 4, step 4: purifying the crude teriparatide peptide to obtain teriparatide;

wherein the sequence of the step 1 and the step 2 is not divided into sequence.

2. The method of claim 1, wherein the solid phase carrier in step 1 is Rink Amide Resin or 2-Cl-CTC Resin.

3. The method of claim 1, wherein the coupling agent of step 1 is HOBt/DIPCDI, HOBt/PyBop/DIPEA, HATU/HOAt/DIPEA, HOAt/PyAop/DIPEA, or HBTU/HOBt/DIPEA.

4. The method of claim 1, wherein PG in PG-Ser (tBu) -OH in step 1 is Msz protecting group, Teoc protecting group, Fmoc protecting group.

5. The method of claim 1, wherein the cleaving agent used in the cleavage in step 1 is a mixed solution of TFA and water.

6. The method of claim 1, wherein the solid support of step 2 is Wang Resin.

7. The method of claim 1, wherein the coupling reagent in step 2 is HOBt/DIPCDI, HOBt/DMAP/DIPCDI, HOBt/PyBop/DIPEA, HATU/HOAt/DIPEA, HOAt/PyAop/DIPEA or HBTU/HOBt/DIPEA.

8. The method of claim 1, wherein the cleaving agent used in the step 2 is a mixed solution of TFA and TIS.

9. The method as claimed in claim 1, wherein the coupling in step 3 is carried out by dissolving in pyridine/acetic acid buffer solution for 2-4 hours.

10. The method according to claim 1, wherein the step 3 of deprotecting Ser at position 1 in segment a is specifically performed by:

when PG of PG-Ser (tBu) -OH in the segment A is an Msz protecting group, TFA/ammonium iodide/dimethyl sulfide is added to remove the protecting group Msz after the coupling of the segment A and the segment B is finished, and ether precipitation is carried out;

when PG of PG-Ser (tBu) -OH in the segment A is a Teoc protecting group, tetrabutylammonium fluoride is added to remove the Teoc protecting group after the coupling of the segment A and the segment B is finished;

and when PG of PG-Ser (tBu) -OH in the segment A is the Fmoc protecting group, adding diethylamine to remove the protecting group Fmoc after the coupling of the segment A and the segment B is finished.

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a method for preparing teriparatide.

Background

Teriparatide (Teriparatide) is a fragment from 1 to 34 in human parathyroid hormone, has the same biological activity as human parathyroid hormone, is developed by Eli Lilly company in the united states for primary osteoporosis, hypogonadal osteoporosis and osteoporosis in menopausal women, and has a wide market prospect. The structural formula is as follows:

the peptide sequence is:

H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH。

patent US6590081 uses genetic recombination to obtain teriparatide. However, the gene recombination method has the problems of complex process, high cost, serious three wastes and the like.

Patent CN201510005427 adopts Wang resin or 2-Cl-CTC resin to synthesize teriparatide by coupling amino acids from C end to N end one by one, belonging to conventional solid phase synthesis method. However, the method has incomplete reaction in the coupling to the later stage, so that the final product is difficult to purify and has low purity.

Patent CN201310403743 is synthesized by coupling one by one, and unlike patent CN201510005427, the free hydroxyl group at Ser 17 and carboxyl group at Asn 16 are subjected to ester condensation, and then teriparatide is obtained by O → N acyl transfer. Although the method can reduce the coupling difficulty of subsequent sites by changing the spatial configuration of the target peptide, the method still has the problems of more solid-phase coupling steps and difficult purification.

In patent CN201410262511, pseudo proline dipeptide Fmoc-Asn (Trt) -Ser (psi)^Me,MePro) -OH replaces two amino acids at the original 16-17 positions to be coupled one by one, and finally the teriparatide is obtained through cleavage. The method adopts the mode of pseudo proline dipeptide to feed materials, avoids the generation of oxidation impurities, but cannot avoid various deletion peptides generated by overlong peptide chains, and meanwhile, the pseudo proline dipeptide is expensive and is not easy to obtain.

In patent CN201511024053, multiple dipeptide or tripeptide fragments are used to replace single amino acid for coupling, and finally cleavage is performed to obtain teriparatide. The method needs a liquid phase synthesis method to obtain 11 short peptide fragments, and has complex operation and low production efficiency.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preparing teriparatide, which is simple in operation, high in production efficiency, suitable for large-scale production of teriparatide, and easy to purify, and the prepared teriparatide has high purity.

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

a method of preparing teriparatide comprising:

step 1: coupling 3-Fmoc-4-diaminobenzoic acid with a solid phase carrier, sequentially coupling Fmoc-Asn (Trt) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-O H, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val and PG-Ser (tBu) -OH from the C end to the N end according to the peptide sequence, forming benzimidazolone by p-nitrophenyl chloroformate ring, finally obtaining fragment A-PG-Val-Ser-Val-Glu-OH and PG-Gl-Val-OH by the cleavage of salicylaldehyde and TFA Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-SAL;

step 2: coupling Fmoc-Phe-OH with a solid-phase carrier, sequentially coupling Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH and Fmoc-Ser tBu) -OH from C end to N end according to the peptide sequence, and cleaving TFA to obtain fragment B-Glu-Arg-Val-Glu-Leu-Lys-OH -Leu-Gln-Asp-Val-His-Asn-Phe-OH;

and step 3: coupling the segment A and the segment B, and then removing a protecting group of Ser at the 1 position in the segment A to obtain crude teriparatide peptide;

and 4, step 4: purifying the crude teriparatide peptide to obtain teriparatide;

wherein the sequence of the step 1 and the step 2 is not divided into sequence.

Preferably, the solid phase carrier in the step 1 is Rink Amide Resin or 2-Cl-CTC Resin.

Preferably, the coupling agent in step 1 is HOBt/DIPCDI, HOBt/PyBop/DIPEA, HATU/HOAt/DIPEA, HOAt/PyAop/DIPEA or HBTU/HOBt/DIPEA.

Preferably, PG of PG-Ser (tBu) -OH in step 1 is an Msz protecting group, a Teoc protecting group or an Fmoc protecting group.

Preferably, the cleavage agent for cleavage in step 1 is a mixed solution of TFA and water.

Preferably, the solid phase carrier in step 2 is Wang Resin.

Preferably, the coupling agent for coupling in step 2 is HOBt/DIPCDI, HOBt/DMAP/DIPCDI, HOBt/PyBop/DIPEA, HATU/HOAt/DIPEA, HOAt/PyAop/DIPEA or HBTU/HOBt/DIPEA.

Preferably, the cleavage agent for cleavage in step 2 is a mixed solution of TFA and TIS.

Preferably, the specific operation of the coupling in step 3 is dissolving in pyridine/acetic acid buffer solution for 2-4 hours.

Preferably, the specific operation of removing the protecting group of Ser at position 1 in segment a in step 3 is:

when PG of PG-Ser (tBu) -OH in the segment A is an Msz protecting group, TFA/ammonium iodide/dimethyl sulfide is added to remove the protecting group Msz after the coupling of the segment A and the segment B is finished, and ether precipitation is carried out;

when PG of PG-Ser (tBu) -OH in the segment A is a Teoc protecting group, tetrabutylammonium fluoride is added to remove the Teoc protecting group after the coupling of the segment A and the segment B is finished;

and when PG of PG-Ser (tBu) -OH in the segment A is the Fmoc protecting group, adding diethylamine to remove the protecting group Fmoc after the coupling of the segment A and the segment B is finished.

The method for preparing teriparatide adopts fragment condensation to prepare teriparatide. Firstly, respectively synthesizing the peptide sequences (segment A) at the 1 st to 16 th positions and the peptide sequences (segment B) at the 17 th to 34 th positions of teriparatide, then coupling the two segments to obtain crude teriparatide peptide, and purifying to obtain the teriparatide. The side chain of the fragment has no protecting group, has better solubility in water, does not have the problem of difficult coupling, and has simple operation and high production efficiency. The obtained teriparatide product has high purity and is easy to purify. Experiments show that the purity of the crude peptide of the teriparatide obtained by the invention can be 80%, and the total yield can reach 45%. The purity of the refined peptide can reach 99.92% through simple purification, and the single maximum impurity is 0.05%. Compared with the prior art, the invention has the characteristics of high product quality, low cost, suitability for industrial production and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a synthetic route to fragment A;

FIG. 2 is a chromatogram of crude teriparatide obtained in example 5;

FIG. 3 is a chromatogram of crude teriparatide peptide obtained in example 6;

FIG. 4 is a chromatogram of crude teriparatide peptide from example 7;

FIG. 5 is a chromatogram of teriparatide depsipeptide obtained in example 8.

Detailed Description

The embodiment of the invention discloses a method for preparing teriparatide. 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 of the present 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 of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

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

a method of preparing teriparatide comprising:

step 1: coupling 3-Fmoc-4-diaminobenzoic acid with a solid phase carrier, sequentially coupling Fmoc-Asn (Trt) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Glu-OH and PG-Ser (tBu) -OH according to a peptide sequence from C terminal to N terminal, forming benzimidazolone by p-nitro phenyl chloroformate, finally obtaining a fragment A PG-Ser-Val-Ser-Ill-Ser-Val by the cleavage of salicylaldehyde and TFA -Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-SAL;

step 2: coupling Fmoc-Phe-OH with a solid-phase carrier, sequentially coupling Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH and Fmoc-Ser tBu) -OH from C end to N end according to the peptide sequence, and cleaving TFA to obtain fragment B-Glu-Arg-Val-Glu-Leu-Lys-OH -Leu-Gln-Asp-Val-His-Asn-Phe-OH;

and step 3: coupling the segment A and the segment B, and then removing a protecting group of Ser at the 1 position in the segment A to obtain crude teriparatide peptide;

and 4, step 4: purifying the teriparatide to obtain teriparatide;

wherein the sequence of the step 1 and the step 2 is not divided into sequence.

The method for preparing teriparatide adopts fragment condensation to prepare teriparatide. Firstly, respectively synthesizing the peptide sequences (segment A) at the 1 st to 16 th positions and the peptide sequences (segment B) at the 17 th to 34 th positions of teriparatide, then coupling the two segments to obtain crude teriparatide peptide, and purifying to obtain the teriparatide. The side chain of the fragment has no protecting group, has better solubility in water, does not have the problem of difficult coupling, and has simple operation and high production efficiency. The obtained teriparatide product has high purity and is easy to purify.

The preparation method comprises the following steps of 1, sequentially coupling and synthesizing amino acids at positions 1-16 in a peptide sequence of teriparatide from a C end to an N end by adopting a solid-phase synthesis method, then forming benzimidazolone by a closed loop, and finally cracking to obtain a peptide fragment A (PG-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-SAL). The specific route is shown in fig. 1.

Wherein, the solid phase carrier in the step 1 is Rink Amide Resin or 2-Cl-CTC Resin.

Further, the initial substitution degree of the solid phase carrier in the step 1 is preferably 0.4mmol/g to 1.0 mmol/g. In some embodiments, the initial degree of substitution of the solid support described in step 1 is 0.5mmol/g, and in some embodiments the initial degree of substitution of the solid support described in step 1 is 0.6 mmol/g.

Coupling agents are often required during coupling of the amino acids of the Fmoc protecting group to the solid support to activate the amino acids. Preferably, the coupling agent for coupling in step 1 is HOBt/DIPCDI, HOBt/PyBop/DIPEA, HATU/HOAt/DIPEA, HOAt/PyAop/DIPEA or HBTU/HOBt/DIPEA. . In some embodiments, the coupling agent of step 1 is HOBt/DIPCDI.

Specifically, the 3-Fmoc-4-diaminobenzoic acid and the HOBt are dissolved by an organic solvent, DIPCDI is added under ice bath to activate the mixture, and then the mixture is added into a solid phase reaction column to perform coupling reaction with a solid phase carrier dissolved by the organic solvent in advance.

Preferably, the organic solvent for dissolving the 3-Fmoc-4-diaminobenzoic acid and the solid phase carrier is DMF.

Preferably, the coupling reaction is carried out at room temperature for 2 hours.

After the coupling reaction in the step 1 is finished, the reaction solution needs to be purified. The purification method is specifically washing with DMF.

Furthermore, the method also comprises a step of removing Fmoc after the coupling reaction is finished. In some embodiments, the Fmoc-removing reagent is a 20% piperidine solution (piperidine: DMF ═ 1: 4).

The preparation method comprises the step 1 of coupling Fmoc-Asn (Trt) -OH, Fmoc-Leu-OH, Fmoc-His (T rt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH and PG-Ser (tBu) -OH one by one through a polypeptide solid phase synthesis method after 3-Fmoc-4-diaminobenzoic acid is coupled with a solid phase carrier.

Wherein, the coupling agent used in the one-by-one coupling mode in the step 1 is preferably HOBt/DIPCDI, HOBt/PyBop/DIPEA, HATU/HOAt/DIPEA, HOAt/PyAop/DIPEA or HBTU/HOBt/DIPEA.

Further, the coupling-by-coupling mode in the step 1 further comprises a step of removing Fmoc before each coupling step. In some embodiments, the Fmoc-removing reagent is a 20% piperidine solution (piperidine: DMF ═ 1: 4).

Further, PG of PG-Ser (tBu) -OH in the one-by-one coupling in the step 1 is Msz protecting group, Teoc protecting group and Fmoc protecting group. In the one-by-one coupling, the serine at the 1 position adopts Msz-Ser (tBu) -OH, Teoc-Ser (tBu) -OH or Fmoc-Ser (tBu) -OH as raw materials.

In the method, step 1, amino acids are sequentially coupled, and then p-nitrophenyl chloroformate is subjected to ring closure to form benzimidazolone resin. In some embodiments, the ring closing operation is specifically performed by dissolving phenyl p-nitrochloroformate in dichloromethane, adding the solution into a solid-phase reaction column, reacting at room temperature for 1 hour, adding DIPEA, and reacting for 30 minutes.

In the method of the invention, in step 1, the generated resin by ring closing reaction firstly reacts with salicylaldehyde. In some embodiments, the salicylaldehyde reaction is specifically performed by dissolving sodium carbonate and salicylaldehyde in a mixed solution of DCM and THF, adding to the above-produced peptide resin, reacting overnight at room temperature, filtering, and concentrating the filtrate to dryness under reduced pressure. Wherein the volume ratio of DCM to THF in the mixed solution of DCM and THF is 1: 3.

The cleavage agent for the cleavage in step 1 of the method is a mixed solution of TFA and water. In some embodiments, the volume ratio of TFA to water in the TFA in water mixture is 95: 5.

The preparation method comprises the step 2 of adopting a solid-phase synthesis method to sequentially couple and synthesize the 17 th-34 th amino acids in a peptide sequence of the teriparatide from a C end to an N end, and cracking to obtain a peptide fragment B (Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH).

Wherein, the solid phase carrier in the step 2 is Wang Resin.

Further, the initial substitution degree of the solid phase carrier in the step 2 is preferably 0.4mmol/g to 1.0 mmol/g. In some embodiments, the initial degree of substitution of the solid support in step 1 is 0.8 mmol/g.

The coupling agent in step 2 of the preparation method is HOBt/DIPCDI, HOBt/DMAP/DIPCDI, HOBt/PyBop/DIPEA, HATU/HOAt/DIPEA, HOAt/PyAop/DIPEA or HBTU/HOBt/DIPEA.

In some embodiments, the coupling reagent of step 2 is HOBt/DMAP/DIPCDI.

Specifically, Fmoc-Phe-OH, HOBt and DMAP are dissolved by an organic solvent, DIPCDI is added under ice bath to activate the mixture, and then the mixture is added into a solid phase reaction column to perform coupling reaction with a solid phase carrier dissolved by the organic solvent in advance.

Preferably, the organic solvent for dissolving Fmoc-Phe-OH and the solid phase carrier is DMF.

Preferably, the coupling reaction is carried out at room temperature for 2 hours.

And (3) sealing the reaction liquid after the coupling reaction in the step 2 is finished. The sealing method specifically comprises the step of adding a mixed solution of pyridine and acetic anhydride to seal the resin for 6 hours.

After the blocking reaction in the step 2 is finished, the reaction solution needs to be purified. The purification method is specifically washing with DMF.

Step 2 of the preparation method of the present invention coupling Fmoc-Asn (Trt) -OH, Fmoc-His (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-OH by the polypeptide solid phase synthesis method one by one according to the method of step 1, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH and Fmoc-Ser (tBu) -OH.

The cleavage agent for cleavage in step 2 of the method is a mixed solution of TFA and TIS. In some embodiments, the volume ratio of TFA to TIS in the mixed TFA and TIS solution is 95: 5.

The preparation method comprises the step 3 of coupling the segment A and the segment B, and then removing the protecting group of Ser at the 1-position in the segment A to obtain the crude teriparatide peptide.

Wherein, the specific operation of the coupling in the step 3 is dissolving in pyridine/acetic acid buffer solution, and reacting for 2-4 hours.

The molar ratio of pyridine to acetic acid in the pyridine/acetic acid buffer solution is 1: 1. The temperature of the coupling reaction was room temperature.

And 3, coupling the segment A and the segment B, and then removing a protecting group of Ser at the 1-position in the segment A to obtain the crude teriparatide peptide.

The protecting group of Ser at position 1 is different, and the method for removing the protecting group is different.

In some embodiments, the protecting group for Ser at position 1 is an Msz protecting group, i.e., PG of PG-Ser (tBu) -OH in fragment A is a Msz protecting group, TFA/ammonium iodide/dimethyl sulfide is added to remove the protecting group Msz after coupling of fragment A and fragment B, and ether precipitation is performed. Wherein the TFA/ammonium iodide/dimethyl sulfide volume ratio is preferably 90:5: 5.

In some embodiments, the protecting group for Ser at position 1 is a Teoc protecting group, i.e., PG of PG-Ser (tBu) -OH in segment A is a Teoc protecting group, and tetrabutylammonium fluoride is added to remove the Teoc protecting group after segment A and segment B are coupled;

in some embodiments, the protecting group for Ser at position 1 is an Fmoc protecting group, i.e., PG of PG-Ser (tBu) -OH in fragment A is an Fmoc protecting group, and upon coupling of fragment A and fragment B, diethylamine is added to remove the protecting group Fmoc.

In the step 4 of the preparation method, the crude teriparatide peptide is purified to obtain the teriparatide, wherein the purification is preferably reverse phase high performance liquid chromatography purification.

Reversed-phase high performance liquid chromatography, english name reversed phase high performance liquid chromatography, RP-HPLC, is a liquid chromatography system consisting of a non-polar stationary phase and a polar mobile phase. It is exactly the opposite of a liquid chromatography system consisting of a polar stationary phase and a weakly polar mobile phase (normal phase chromatography). RP-HPLC is the most dominant separation mode of liquid chromatography today and is used for the separation and purification of almost all organic materials that are soluble in polar or weakly polar solvents.

Preferably, the reversed-phase high performance liquid chromatography is specifically as follows: and (3) taking reverse-phase octadecylsilane or octaalkylsilane bonded silica gel as a stationary phase, loading the obtained crude peptide solution of the teriparatide, purifying by using a 0.1% TFA/acetonitrile mobile phase, and collecting the target peak fraction.

Further, the teriparatide is obtained by freeze-drying after purification by reversed phase high performance liquid chromatography.

The purity detection method of teriparatide comprises the following steps:

octadecylsilane chemically bonded silica was used as a filler (C184.6X 250mm X5 μm 300A); taking 50mmol/L ammonium sulfate solution (pH is adjusted to 2.5 by dilute sulfuric acid) as a mobile phase A, and taking acetonitrile as a mobile phase B; gradient elution was performed using the following conditions. The flow rate is 1.0 ml/min; the column temperature was 25 ℃; the detection wavelength was 214 nm.

For a further understanding of the present invention, reference will now be made in detail to the following examples.

The meanings of abbreviations used in the specification and claims are listed in the following table: