阅读说明：本技术 固液相结合制备特立帕肽的方法 (Method for preparing teriparatide by solid-liquid combination ) 是由 王浩 朱明磊 周建华 于 2019-10-18 设计创作，主要内容包括：本发提供了本发明公开了固液相结合制备特立帕肽的方法,本发明所述方法包括如下步骤：以2-Cl-CTC Resin为固相载体合成特立帕肽1-15个氨基酸的全保护肽作为第一片段；以PS-Wang Resin为固相载体合成特立帕肽16-34个氨基酸的全保护肽作为第二片段；在液相里将第一片段和第二片段偶联,得到特立帕肽全保护肽,经裂解、纯化、冻干得特立帕肽成品。该方法可以有效的缩短50％的合成周期、降低缺失肽、消旋肽的生成,提高了总收率、降低物料成本,适合于工业化生产。(The invention provides a method for preparing teriparatide by solid-liquid phase combination, which comprises the following steps: synthesizing a whole protective peptide of 1-15 amino acids of teriparatide as a first segment by taking 2-Cl-CTC Resin as a solid phase carrier; synthesizing a total protective peptide of 16-34 amino acids of teriparatide as a second segment by taking PS-Wang Resin as a solid phase carrier; and coupling the first fragment and the second fragment in a liquid phase to obtain teriparatide full-protection peptide, and cracking, purifying and freeze-drying to obtain a teriparatide finished product. The method can effectively shorten the synthesis period by 50 percent, reduce the generation of deletion peptide and racemic peptide, improve the total yield, reduce the material cost and is suitable for industrial production.)

1. The method for preparing teriparatide by solid-liquid combination is characterized by comprising the following steps:

(1) solid-phase synthesis of a whole protective peptide of 1-15 amino acids of teriparatide as a first fragment;

(2) synthesizing total protection of 16-34 amino acids of teriparatide as a second segment by taking PS-Wang Resin as a solid phase carrier;

(3) coupling the first segment and the second segment in a liquid phase to synthesize teriparatide complete protective peptide;

(4) the finished product of teriparatide is obtained by cracking, purifying and freeze-drying.

2. The method for preparing teriparatide by solid-liquid phase combination according to claim 1, wherein: the Resin used in the step (1) is 2-Cl-CTC Resin, and the substitution degree is 0.1-1.2 mmol/g.

3. The method for preparing teriparatide by solid-liquid phase combination according to claim 1, wherein: the cracking reagent of the total protection segment in the step (1) is 15-30% V/V TFE/DCM or 0.5-3% V/V TFA/DCM.

4. The method for preparing teriparatide by solid-liquid phase combination according to claim 1, wherein: Fmoc-Val-Ser [ Psi (Me, Me) Pro ] -OH was used in step (1) to synthesize the first fragment fully protected.

5. The method for preparing teriparatide by solid-liquid phase combination according to claim 1, wherein: the structure of the solid phase carrier PS-Wang Resin used in the step (2) is as follows:

。

6. the description of the structure of PS-Wang Resin according to claim 5, wherein: r₁、R₂、R₃、R₄May be one or more of C1-C3 alkyl, C1-C3 alkoxy, hydrogen, chlorine and other halogen atoms, and may be the same or different.

7. The method for preparing teriparatide by solid-liquid phase combination according to claim 1, wherein: the cleavage reagent of the total protection fragment in the step (2) is 1-4% V/V TFA/DCM.

8. The method for preparing teriparatide by solid-liquid phase combination according to claim 1, wherein: the cleavage reagent used in step (4) was TFA/TIS/m-cresol, using TFA: 80-95; and (3) TIS: 3-10; m-cresol: 2-10.

9. The method for preparing teriparatide by solid-liquid phase combination according to claim 8, wherein: the cleavage reagent ratio used was TFA/TIS/m-cresol 90/5/5.

10. The method for preparing teriparatide by solid-liquid phase combination according to claim 1, which comprises the following specific steps:

(1) preparation of Fmoc-Leu-CTC Resin:

adding 2-Cl-CTC resin into a solid phase reactor, and adding DMF to swell the resin; draining the solvent, adding DMF solution of Fmoc-Leu-OH and DIEA, stirring for reaction, draining the solvent, adding DIEA/methanol/DCM solution, and sealing; washing DCM and methanol alternately, and drying in vacuum to obtain Fmoc-Leu-CTC Resin;

(2) preparation of the first fragment:

adding Fmoc-Leu-CTC Resin into a reaction column, adding DMF (dimethyl formamide) for swelling, draining a solvent, and adding piperidine/DMF solution for reaction; pumping out the solvent, and adding DMF for washing; positive by indene detection; dissolving Fmoc-His (trt) -OH, HOBt and DIC in DMF, and activating in ice bath; adding the activating solution into a reaction column for reaction, and draining the solvent after the indene detection result is negative; adding DMF for washing; repeating the steps, sequentially adding 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 and Fmoc-Val-Ser [ Psi (Me, Me) Pro ] -OH, BOC-Ser tBu) -OH according to the amino acid sequence, and carrying out coupling reaction; after coupling, washing DCM and methanol alternately; vacuum drying to obtain teriparatide first fragment peptide resin, adding the peptide resin into TFE/DCM, and reacting; collecting the filtrate, and carrying out vacuum spin-drying on the filtrate; obtaining a first fragment of teriparatide;

(3) Fmoc-Phe-PS-Wang Resin preparation:

adding 2-Cl-CTC resin into a solid phase reactor, and adding DMF to swell the resin; and (3) draining the solvent, adding a DMF (dimethyl formamide) solution of p-hydroxybenzyl alcohol, stirring uniformly, adding DIEA, reacting at 30 ℃ for 2 hours, adding MeOH into the reaction liquid to seal Cl which is not completely reacted, and washing the resin with DMF after the reaction is finished. Adding DMF solution of Fmoc-Phe-OH and DIEA, stirring for reaction, draining the solvent, adding DIEA/methanol/DCM solution, and sealing; washing DCM and methanol alternately, and drying in vacuum to obtain Fmoc-Phe-PS-Wang Resin;

(4) preparation of the second fragment:

adding Fmoc-Phe-PS-Wang Resin into a reaction column, adding DMF (dimethyl formamide) for swelling, draining a solvent, and adding a piperidine/DMF solution for reaction; pumping out the solvent, and adding DMF for washing; positive by indene detection; dissolving Fmoc-Asn (trt) -OH, HOBt and DIC in DMF, and activating in ice bath; adding the activating solution into a reaction column for reaction, and draining the solvent after the indene detection result is negative; adding DMF for washing; repeating the above steps, and sequentially adding Fmoc-His (trt) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -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, Fmoc-Ser (tBu) -OH and Fmoc-Asn (trt) -OH according to the amino acid sequence to perform coupling reaction; after coupling, washing DCM and methanol alternately; vacuum drying to obtain teriparatide second fragment peptide resin, adding the peptide resin into 3% TFA/DCM, and reacting; collecting the filtrate, and carrying out vacuum spin-drying on the filtrate; obtaining a second fragment of teriparatide;

(5) preparing teriparatide full-protection peptide:

dissolving the first fragment, the second fragment and PyBoP in a mixed solution of DMF/DCM, adjusting pH to about 8 with DIEA, reacting for 3h, performing rotary evaporation to obtain oil, dissolving in ethyl acetate, adding 5% NaHCO₃And alternately washing the organic phase with 5% citric acid aqueous solution, washing the organic phase with pure water, and drying by rotary evaporation to obtain the teriparatide full-protection peptide.

(6) Teriparatide preparation

Preparing a lysate TFA/TIS/m-cresol (90/5/5), adding peptide resin for reaction, performing suction filtration after the reaction is completed, taking a filtrate, adding the filtrate into isopropyl ether for sedimentation, performing centrifugal drying to obtain a crude teriparatide product, and purifying to obtain the finished teriparatide.

Technical Field

The invention relates to the field of solid-phase synthesis of polypeptide drugs, in particular to a method for preparing teriparatide by solid-liquid phase combination.

Background

Chinese name: teriparatide

English name: teraramide:

the peptide sequence structure is as follows:

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

structural formula (xvi):

cas number: 52232-67-4

Molecular weight: 4117.77

The molecular formula is as follows: c₁₈₁H₂₉₁N₅₅O₅₁S₂

Teriparatide is an N-terminal region 1-34 amino acid fragment with bioactivity of endogenous parathyroid hormone, is developed by li corporation, is firstly marketed in the United states in 12 months in 2002, is used for treating primary osteoporosis, hypogonadism osteoporosis and postmenopausal osteoporosis, has the immunological and biological properties identical to endogenous parathyroid hormone PTH, can increase the number of osteoblasts, enhance the activity of osteoblasts and prevent the apoptosis of osteoblasts, has small toxic and side effects, rarely causes accumulative poisoning, and has wide market prospect.

At present, methods for preparing teriparatide have been widely reported at home and abroad. In addition to the original tendency of the factories to obtain teriparatide by gene expression, there are many patents on gene expression, such as CZ20100374A3, which describes a method of expressing teriparatide in Escherichia coli by gene recombination technology, followed by isolation and purification. US2017129935a1 describes a method for purifying teriparatide based on ion exchange chromatography, which is also based on teriparatide produced by genetic engineering techniques. However, the gene expression method has obvious defects of high technical threshold, complex process, serious three wastes and the like.

Another common preparation method of teriparatide is a solid-phase chemical synthesis method, wherein CN104017064A describes a method for feeding a peptide chain at 16-17 position by adopting a pseudo proline dipeptide mode, which avoids the generation of special process impurities, but cannot avoid various missing peptides caused by overlong peptide chain; CN104530218A describes a method for gradually synthesizing teriparatide from C end to N end of a peptide chain by adopting a traditional solid phase synthesis method, which is simple and convenient to operate, but cannot solve the problem of difficult late coupling of long peptides, and the final target peptide has low purity and difficult purification; CN105384809A describes a method of synthesizing dipeptide or tripeptide in liquid phase, and then synthesizing the dipeptide or tripeptide in solid phase, which reduces the generation of missing peptide to some extent, but increases a large amount of liquid phase dipeptide synthesis work, and the process is complicated; CN104910269 describes a method for synthesizing peptide sequences by coupling one by one using HMP Linker as a solid phase carrier, which is not effective in avoiding deletion and racemic peptides generated by coupling one by one.

Disclosure of Invention

In order to overcome the defects in the synthesis of teriparatide, the invention provides a novel method for preparing teriparatide by fragment method solid-liquid combination.

According to one aspect of the technical scheme of the invention, the method for preparing teriparatide by solid-liquid phase combination is provided, and comprises the following steps:

(1) solid-phase synthesis of a whole protective peptide of 1-15 amino acids of teriparatide as a first fragment;

(2) synthesizing total protection of 16-34 amino acids of teriparatide as a second segment by taking PS-Wang Resin as a solid phase carrier;

(3) coupling the first segment and the second segment in a liquid phase to synthesize teriparatide complete protective peptide;

(4) the finished product of teriparatide is obtained by cracking, purifying and freeze-drying.

According to one aspect of the technical scheme, the invention provides a method for preparing teriparatide by solid-liquid phase combination, wherein the Resin used in the step (1) is 2-Cl-CTC Resin, and the substitution degree is 0.1-1.2 mmol/g.

According to one aspect of the technical scheme of the invention, the condensing agent used in the step (1) and/or the step (2) is a + B or a + C + D, wherein a is any one of HoBt, HoAt, Cl-HoBt or HOOBt, B is any one of DIC, DCC, edc.hcl, C is any one of HATU, HBTU, HCTU, TATU, TBTU, PyBOP or PyAOP, and D is any one of DIEA, NMM, TMP or TEA; when an A + B condensation system is used, the molar mass ratio of the resin to the amino acid to the A, B is 1: 1.5-3: 1.8-3.6, and when A + C + D is used, the molar mass ratio of the resin to the amino acid to the A, C, D is 1: 1.5-3: 1.8-3.6: 1.35-2.7: 4.5-9; the reaction solvent is one or a combination of DMF, DCM, DMSO, DMA and NMP; the Fmoc removing reagent is piperidine/DMF solution with 10-30% V/V or DBU/DMF solution with 1-5% V/V, and the removing is carried out twice, 5-20 min each time.

According to one aspect of the technical scheme, the invention provides a method for preparing teriparatide by solid-liquid phase combination, wherein the cracking reagent of the total protection fragment in the step (1) is 15-30% V/V TFE/DCM or 0.5-3% V/V TFA/DCM.

According to one aspect of the technical scheme, the invention provides a method for preparing teriparatide by solid-liquid phase combination, wherein Fmoc-Val-Ser [ Psi (Me, Me) Pro is used in the step (1)]-OH instead of Fmoc-Val²-OH、Fmoc-Ser³(tBu) -OH to synthesize the first fragment full protection.

According to one aspect of the technical scheme, the invention provides a method for preparing teriparatide by solid-liquid phase combination, and a condensation system adopted in Fmoc-Val-Ser [ Psi (Me, Me) Pro ] -OH coupling is any one of A + C + D.

According to one aspect of the technical scheme of the invention, the method for synthesizing teriparatide is provided, wherein the solid phase carrier PS-Wang Resin used in the step (2) has a structure as follows:

PS-Wang Resin

according to one aspect of the technical scheme, the invention provides a method for preparing teriparatide by solid-liquid combination, wherein R in the step (2)₁、R₂、R₃、R₄Can be one or more of alkyl containing 1-3 carbon atoms, alkoxy containing 1-3 carbon atoms, hydrogen, chlorine and other halogen atoms, and can be the same or different; r₁、R₂、R₃、R₄One or more of CH3 and CH H, Cl are preferably selected;R₁、R₂、R₃、R₄h is more preferred.

According to one aspect of the technical scheme of the invention, a method for preparing teriparatide by solid-liquid phase combination is provided, wherein n in the step (2) can be 0, 1, 2, 3 or 4; n is 0, 1 and 2 preferably; 0 is selected more preferentially.

According to one aspect of the technical scheme of the invention, the solid-liquid combined method for preparing teriparatide is provided, and the cleavage reagent of the full-protection fragment in the step (2) is 1% -4% V/V TFA/DCM, and the preferable ratio of the cleavage reagent is 3% V/V TFA/DCM.

According to one aspect of the technical scheme of the invention, a method for preparing teriparatide by solid-liquid phase combination is provided, wherein in the step (3), a condensing agent for coupling the first fragment and the second fragment in a liquid phase is one or more of PyBop, PyAop, HCTU, HBTU, TBTU and TCTU; the solvent is one or more of DMF, DMA, NMP and DCM; the base used is any of DIEA, NMM, TMP or TEA.

According to one aspect of the technical scheme of the invention, the method for preparing teriparatide by solid-liquid phase combination is provided, wherein the cleavage reagent used in the step (4) is TFA/TIS/m-cresol, and TFA: 80-95; and (3) TIS: 3-10; m-cresol: 2-10.

According to one aspect of the present invention, there is provided a method for preparing teriparatide by solid-liquid phase combination, wherein the cleavage reagent used in the step (4) is TFA/TIS/m-cresol 90/5/5.

According to one aspect of the technical scheme of the invention, a method for preparing teriparatide by solid-liquid phase combination is provided, wherein in the synthetic method of teriparatide of the invention, a further preferable technical scheme is that the steps are as follows:

(1) preparation of Fmoc-Leu-CTC Resin:

adding 2-Cl-CTC resin into a solid phase reactor, and adding DMF to swell the resin; draining the solvent, adding DMF solution of Fmoc-Leu-OH and DIEA, stirring for reaction, draining the solvent, adding DIEA/methanol/DCM solution, and sealing; washing DCM and methanol alternately, and drying in vacuum to obtain Fmoc-Leu-CTC Resin;

(2) preparation of the first fragment:

adding Fmoc-Leu-CTC Resin into a reaction column, adding DMF (dimethyl formamide) for swelling, draining a solvent, and adding piperidine/DMF solution for reaction; pumping out the solvent, and adding DMF for washing; positive by indene detection; dissolving Fmoc-His (trt) -OH, HOBt and DIC in DMF, and activating in ice bath; adding the activating solution into a reaction column for reaction, and draining the solvent after the indene detection result is negative; adding DMF for washing; repeating the steps, and sequentially adding 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-Val-Ser [ Psi (Me, Me) Pro ] -OH and BOC-Ser tBu-OH according to the amino acid sequence to carry out coupling reaction; after coupling, washing DCM and methanol alternately; vacuum drying to obtain teriparatide first fragment peptide resin, adding the peptide resin into TFE/DCM, and reacting; collecting the filtrate, and carrying out vacuum spin-drying on the filtrate; obtaining a first fragment of teriparatide;

(3) Fmoc-Phe-PS-Wang Resin preparation:

adding 2-Cl-CTC resin into a solid phase reactor, and adding DMF to swell the resin; and (3) draining the solvent, adding a DMF (dimethyl formamide) solution of p-hydroxybenzyl alcohol, stirring uniformly, adding DIEA, reacting at 30 ℃ for 2 hours, adding MeOH into the reaction liquid to seal Cl which is not completely reacted, and washing the resin with DMF after the reaction is finished. Adding DMF solution of Fmoc-Phe-OH and DIEA, stirring for reaction, draining the solvent, adding DIEA/methanol/DCM solution, and sealing; washing DCM and methanol alternately, and drying in vacuum to obtain Fmoc-Phe-PS-Wang Resin;

(4) preparation of the second fragment:

adding Fmoc-Phe-PS-Wang Resin into a reaction column, adding DMF (dimethyl formamide) for swelling, draining a solvent, and adding a piperidine/DMF solution for reaction; pumping out the solvent, and adding DMF for washing; positive by indene detection; dissolving Fmoc-Asn (trt) -OH, HOBt and DIC in DMF, and activating in ice bath; adding the activating solution into a reaction column for reaction, and draining the solvent after the indene detection result is negative; adding DMF for washing; repeating the above steps, and sequentially adding Fmoc-His (trt) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -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, Fmoc-Ser (tBu) -OH and Fmoc-Asn (trt) -OH according to the amino acid sequence to perform coupling reaction; after coupling, washing DCM and methanol alternately; vacuum drying to obtain teriparatide second fragment peptide resin, adding the peptide resin into 3% TFA/DCM, and reacting; collecting the filtrate, and carrying out vacuum spin-drying on the filtrate; obtaining a second fragment of teriparatide;

(5) preparing teriparatide full-protection peptide:

dissolving the first fragment, the second fragment and PyBoP in a mixed solution of DMF/DCM, adjusting pH to about 8 with DIEA, reacting for 3h, performing rotary evaporation to obtain oil, dissolving in ethyl acetate, adding 5% NaHCO₃And alternately washing the organic phase with 5% citric acid aqueous solution, washing the organic phase with pure water, and drying by rotary evaporation to obtain the teriparatide full-protection peptide.

(6) Teriparatide preparation

Preparing a lysate TFA/TIS/m-cresol (90/5/5), adding peptide resin for reaction, performing suction filtration after the reaction is completed, taking a filtrate, adding the filtrate into isopropyl ether for sedimentation, performing centrifugal drying to obtain a crude teriparatide product, and purifying to obtain the finished teriparatide.

Drawings

FIG. 1: HPLC profile of the finished teriparatide product in example 5.

Detailed Description

The following examples are intended to further illustrate some, but not all, preferred embodiments of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention. The invention will be further described with reference to the accompanying drawings.

The meanings of the abbreviations used in the present invention are listed in the following table:

example 1:

preparation of teriparatide first fragment full protection peptide, Scheme 1:

1) swelling resin: 60g of 2-Cl-CTC Resin with a substitution degree of 1.0mmol/g are weighed, 600ml of DCM swelling Resin is added for 0.5h, the solvent is drained, 300ml of DMF is added for washing the Resin twice, and the solvent is drained.

2) Preparation of Fmoc-Leu-CTC Resin: weighing 42.4g of Fmoc-Leu-OH according to the molar mass ratio of the resin, Fmoc-Leu-OH and DIEA of 1: 2: 6, dissolving in 300ml of DMF, adding 63ml of DIEA, uniformly mixing, adding the solution into a reaction column for reaction for 2 hours, and draining the solvent; adding 300ml DMF to wash the resin for three times, and pumping out the washing solvent; preparing 800ml of sealing liquid according to the volume ratio of DCM, methanol and DIEA of 17: 2: 1, sealing the resin twice for 10min each time; adding 400ml DMF to wash the resin for three times, and pumping off the washing solvent; the solution is washed three times with methanol for 10min each time, the solvent is pumped off, and after vacuum drying, 80.6g of Fmoc-Leu-CTC Resin is obtained, and the measured substitution degree is 0.65 mmol/g.

3) Teriparatide first fragment peptide resin preparation: a) swelling amino acid Resin, weighing 76.9g of Fmoc-Leu-CTC Resin, adding 700ml of DCM swelling Resin for 0.5h, draining the solvent, adding 500ml of DMF, washing the Resin twice, and draining the solvent; b) removing Fmoc protecting groups, adding 500ml of 20% piperidine/DMF solution into the resin, deprotecting twice, wherein the first deprotection time is 5min, the second deprotection time is 15min, after the deprotection is finished, adding 500ml of DMF to wash the resin, draining to dissolve, repeating for 5 times, and detecting positive indene; c) according to the molar mass ratio of resin, Fmoc-His (trt) -OH, HOBt and DIC of 1: 2: 2.4, weighing 62.0g of Fmoc-His (trt) -OH and 16.2g of HOB, dissolving in 500ml of DMF, adding 19ml of DIC, activating in ice-water bath for 5min, adding the solution into a reaction column at 30 ℃ for reaction for 2h, detecting negative by indene, draining the solvent, washing the resin for 3 times by 500ml of DMF, and draining the solvent; d) and (3) extending a peptide chain, repeating the steps, and sequentially adding 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-Val-Ser [ Psi (Me, Me) Pro ] -OH and BOC-Ser (tBu) -OH according to the amino acid sequence to perform coupling reaction to obtain 206.3g of teriparatide first-segment peptide resin.

4) Preparation of teriparatide first fragment fully protected peptide: preparing 1600 ml of 20% TFE/DCM solution, adding teriparatide first fragment peptide resin, reacting for 2 hours, collecting filtrate, and carrying out vacuum spin-drying on the filtrate; 149.9g of the first fragment of teriparatide is obtained.

Example 2:

Fmoc-Phe-PS-Wang Resin, Scheme 2:

1) swelling resin: 120g of 2-Cl-CTC Resin with a substitution degree of 1.0mmol/g is weighed, 1200 ml of DCM swelling Resin is added for 0.5h, the solvent is drained, 900ml of DMF is added for washing the Resin twice, and the solvent is drained.

2) Preparation of Fmoc-Phe-PS-Wang Resin: 1) weighing 44.7g of p-hydroxybenzyl alcohol and 900ml of DMF according to the molar mass ratio of the resin to the p-hydroxybenzyl alcohol to the DIEA of 1: 3: 12, dissolving the p-hydroxybenzyl alcohol in 251ml of DMF, adding 251ml of DIEA, uniformly mixing, adding the solution into a reaction column for reaction for 3 hours, adding 100ml of MeOH into reaction liquid to seal Cl which is not completely reacted, reacting for 20 minutes, and draining the solvent; adding 900ml DMF to wash the resin for three times, and pumping out the washing solvent; 2) weighing 93.0g of Fmoc-Ala-OH and 38.9g of HoBt according to the molar mass ratio of resin, Fmoc-Phe-OH, HoBt, DIC and DMAP of 1: 2: 2.4: 0.2, dissolving in 900ml of DMF, adding 44.6ml of DIC for activation for 5min, adding the activated solution into the resin for reaction at 30 ℃ for 10min, weighing 2.9g of DMAP for dissolving in 100ml of DMF, slowly dropwise adding into a reaction column for continuous reaction at 30 ℃ for 1.5h, and draining the solvent; adding 900ml DMF to wash the resin for three times, and pumping out the washing solvent; adding confining liquid according to the molar ratio of the resin, the acetic anhydride and the pyridine of 1: 20: 5, and reacting for 5 hours; adding 900ml DMF to wash the resin for three times, and pumping out the washing solvent; methanol was washed three times each for 10min, and after draining the solvent and drying in vacuo, 148.2g of Fmoc-Phe-PS-Wang Resin was obtained, which was found to have a degree of substitution of 0.38 mmol/g.

Example 3

Preparation of teriparatide second fragment full protection peptide:

1) preparation of teriparatide second fragment peptide resin: a) swelling amino acid Resin, weighing 132.0g of Fmoc-Phe-PS-Wang Resin, adding 1500ml of DCM swelling Resin for 0.5h, draining the solvent, adding 1000 ml of DMF, washing the Resin twice, and draining the solvent; b) removing Fmoc protecting groups, adding 1000 ml of 20% piperidine/DMF solution into the resin, deprotecting twice, wherein the first deprotection time is 5min, the second deprotection time is 15min, after the deprotection is finished, adding 1000 ml of DMF to wash the resin, draining and dissolving, repeating for 5 times, and detecting positive indene; c) according to the molar mass ratio of resin, Fmoc-Asn (trt) -OH, HOBt and DIC of 1: 2: 2.4, 59.7g of Fmoc-Asn (trt) -OH and 16.2g of HOB are weighed and dissolved in 1000 ml of DMF, 19ml of DIC is added, after ice-water bath activation for 5min, the solution is added into a reaction column for reaction at 30 ℃ for 2h, negative indene detection is realized, solvent is drained, resin is washed for 3 times by 1000 ml of DMF, and the solvent is drained; d) the peptide chain was extended by repeating the above steps, and Fmoc-His (trt) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -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, Fmoc-Ser (tBu) -OH, and Fmoc-Asn (trt) -OH were sequentially added according to the amino acid sequence to carry out coupling reaction to obtain 321.5g of the isolated peptide resin.

4) Preparation of teriparatide second fragment full protection peptide: preparing 2500 ml of 20% TFE/DCM solution, adding teriparatide first fragment peptide resin, reacting for 2 hours, collecting filtrate, and carrying out vacuum spin-drying on the filtrate; obtaining 208.4g of a second fragment of teriparatide.

Example 4

Preparing teriparatide full-protection peptide:

press the first and second piecesThe molar mass ratio of PyBoP is 1: 1.01, 149.9g of the first fragment, 208.4 of the second fragment and 26.2g of PyBoP are weighed and dissolved in 5000ml of 10% DMF/DCM mixed solution, the pH value is adjusted to about 8 by using DIEA, the reaction is carried out for 3h at 40 ℃, after the reaction is finished, the mixture is steamed to oil, 3000ml of ethyl acetate is added for dissolution, and 1500ml of 5% NaHCO is used₃And alternately washing the organic phase with 5% citric acid aqueous solution for five times, washing the organic phase with 1500ml of pure water for five times, and drying the organic phase by rotary evaporation to obtain 446.2g of teriparatide full-protection peptide.

Example 5

Teriparatide preparation:

preparing 3000ml of lysate according to lysate TFA/TIS/m-cresol (90/5/5), slowly adding teriparatide full-protection peptide after uniformly stirring, reacting for 3 hours at 30 ℃, adding the reaction solution into 28000 ml of isopropyl ether after dissolving, centrifuging and drying to obtain a teriparatide crude product, purifying by using a C18 preparation column, using a mobile phase 0.1% TFA aqueous solution at the flow rate of 50-100 ml/min, detecting the wavelength of 230nm, collecting the required effluent by HPLC tracking, and then freeze-drying by using a conventional method to obtain the teriparatide refined peptide, wherein the mass of the teriparatide refined peptide is 82.5g, the purity of the teriparatide refined peptide is 99.59%, the maximum single impurity content of 0.22%, the total impurity content of 0.41%, and the total yield of 40.0%; ESI-MS: [ M +4H ]]⁴⁺＝1030.5，[M+3H]³⁺The HPLC results are shown in fig. 1, 1373.9.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.