CN112679603B - Method for preparing teriparatide by solid-liquid phase combination - Google Patents

Abstract

The invention provides a method for preparing teriparatide by combining solid and liquid phases, which comprises the following steps: 2-Cl-CTC Resin is used as a solid phase carrier to synthesize full-protection peptide of 1-15 amino acids of teriparatide as a first fragment; synthesizing full-protection peptide of 16-34 amino acids of teriparatide by taking PS-Wang Resin as a solid phase carrier as a second fragment; coupling the first fragment and the second fragment in a liquid phase to obtain the teriparatide full-protection peptide, and obtaining the teriparatide finished product through cracking, purifying and freeze-drying. The method can effectively shorten the synthesis period by 50%, reduce the generation of missing peptide and racemized peptide, improve the total yield, reduce the material cost and is suitable for industrial production.

Description

Method for preparing teriparatide by solid-liquid phase combination

Technical Field

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

Background

Chinese name: teriparatide

English name: teiratides:

peptide sequence structure:

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:

cas number: 52232-67-4

Molecular weight: 4117.77

The molecular formula: c (C) ₁₈₁ H ₂₉₁ N ₅₅ O ₅₁ S ₂

Teriparatide is an N-terminal region 1-34 amino acid fragment with biological activity of endogenous parathyroid hormone, is developed by Gift corporation, is first marketed in the United states in 12 months of 2002, is used for treating primary osteoporosis and hypogonadal osteoporosis, has the same immunological and biological properties as endogenous parathyroid hormone PTH and postmenopausal osteoporosis, can increase the number of osteoblasts, enhance the activity and prevent the apoptosis of the osteoblasts, has little toxic and side effects, seldom causes accumulation poisoning, and has a very broad market prospect.

At present, a preparation method of teriparatide is widely reported at home and abroad. Besides the fact that the original mill's house uses gene expression to obtain teriparatide, there are various patents on gene expression, for example, CZ20100374A3 describes a method of expressing in colibacillus by using gene recombination technology, and then obtaining the finished product by separation and purification technology. A method for purifying teriparatide based on ion exchange chromatography is described in US2017129935A1, which is also based on teriparatide produced by genetic engineering techniques. However, the gene expression method has the obvious defects of high technical threshold, complex process, serious three wastes and the like.

Another more common preparation method of teriparatide is a solid-phase chemical synthesis method, and CN104017064a describes a method for feeding a pseudo-proline dipeptide at the 16-17 position of a peptide chain, which avoids the production of special process impurities, but cannot avoid various missing peptides caused by overlong peptide chain; CN104530218A describes a method for gradually synthesizing teriparatide from the C-terminal to the N-terminal 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 coupling of long peptides in the later period, and the final target peptide has low purity and difficult purification; CN105384809a describes a method of first synthesizing a dipeptide or tripeptide by liquid phase and then synthesizing in solid phase, which reduces the generation of missing peptide to some extent but increases a lot of liquid phase dipeptide synthesis work, which is complicated in procedure; CN104910269 describes a method for completing peptide sequence synthesis by coupling one by one using HMP Linker as solid phase carrier, which cannot effectively avoid deletion and racemization of peptides caused by coupling one by one.

Disclosure of Invention

In order to solve the defects existing 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, a method for preparing teriparatide by solid-liquid phase combination is provided, and the method comprises the following steps:

(1) Solid phase synthesis of a full-protection peptide of 1-15 amino acids of teriparatide as a first fragment;

(2) Synthesizing full protection of 16-34 amino acids of teriparatide by taking PS-Wang Resin as a solid phase carrier as a second fragment;

(3) Coupling the first fragment and the second fragment in a liquid phase to synthesize the teriparatide full-protection peptide;

(4) And obtaining a teriparatide finished product through cracking, purifying and freeze-drying.

According to one aspect of the technical scheme of the invention, a method for preparing teriparatide by solid-liquid phase combination is provided, 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 present invention, there is provided a method for preparing teriparatide by solid-liquid phase combination, wherein 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 HOBt, 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 the A+B condensation system is used, the molar mass ratio of the resin to the amino acid to the A, B is 1:1.5-3:1.8-3.6:1.8-3.6, and when the A+C+D condensation system 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 a plurality of DMF, DCM, DMSO, DMA, NMP; the Fmoc-removing reagent is piperidine/DMF solution with the concentration of 10-30% V/V or DBU/DMF solution with the concentration of 1-5% V/V, and the Fmoc-removing reagent is removed twice for 5-20 min each time.

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 of the full-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 present invention, there is provided a method for preparing teriparatide by solid-liquid phase binding, wherein Fmoc-Val-Ser [ Psi (Me, me) is used in the step (1)Pro]-OH instead of Fmoc-Val ² -OH、Fmoc-Ser ³ (tBu) -OH to synthesize the first fragment full protection.

According to one aspect of the technical scheme of the invention, a method for preparing teriparatide by solid-liquid phase combination is provided, 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, a synthetic method of teriparatide is provided, wherein in the step (2), a solid-phase carrier PS-Wang Resin structure is adopted as follows:

PS-Wang Resin

according to one aspect of the present invention, there is provided a method for preparing teriparatide by solid-liquid phase combination, wherein R in the step (2) ₁ 、R ₂ 、R ₃ 、R ₄ Can 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; r is R ₁ 、R ₂ 、R ₃ 、R ₄ One or more of CH3 and H, cl are preferably selected; r is R ₁ 、R ₂ 、R ₃ 、R ₄ More preferred is selection H.

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

According to one aspect of the present invention there is provided a method for preparing teriparatide by solid-liquid phase conjugation, wherein the cleavage reagent of the fully protected fragment in step (2) is 1% to 4%V/V TFA/DCM, preferably the cleavage reagent is in a ratio of 3%V/V TFA/DCM.

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

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 TFA/TIS/m-cresol used in the step (4) is respectively TFA: 80-95 percent; TIS:3 to 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 preferably TFA/TIS/m-cresol=90/5/5.

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

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

adding 2-Cl-CTC resin into a solid phase reactor, and adding DMF swelling 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 with DCM and methanol alternately, and vacuum drying to obtain Fmoc-Leu-CTC Resin;

(2) Preparation of the first fragment:

Fmoc-Leu-CTC Resin is taken and added into a reaction column, DMF is added for swelling, solvent is pumped out, and piperidine/DMF solution is added for reaction; draining the solvent, and adding DMF for washing; indene detection is positive; fmoc-His (trt) -OH, HOBt, DIC is taken and dissolved in DMF and activated in ice bath; adding the activating solution into a reaction column for reaction, and pumping out 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 for coupling reaction; after the coupling is finished, DCM and methanol are alternately washed; vacuum drying to obtain teriparatide first segment peptide resin, adding the peptide resin into TFE/DCM, and reacting; collecting filtrate, and performing 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 swelling resin; the solvent was drained, a DMF solution of parahydroxyben-nol was added, after stirring well, DIEA was added, the reaction was carried out for 2h at 30℃and MeOH was added to the reaction to block the incompletely reacted Cl, after which the resin was washed with DMF. Adding a DMF solution of Fmoc-Phe-OH and DIEA, stirring for reaction, pumping the solvent, adding a DIEA/methanol/DCM solution, and sealing; the DCM and methanol are alternately washed, and Fmoc-Phe-PS-Wang Resin is obtained after vacuum drying;

(4) Preparation of the second fragment:

Fmoc-Phe-PS-Wang Resin is taken and added into a reaction column, DMF is added for swelling, solvent is pumped out, and piperidine/DMF solution is added for reaction; draining the solvent, and adding DMF for washing; indene detection is positive; fmoc-Asn (trt) -OH, HOBt, DIC is taken and dissolved in DMF and activated in ice bath; adding the activating solution into a reaction column for reaction, and pumping out the solvent after the indene detection result is negative; adding DMF for washing; repeating the 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 for coupling reaction; after the coupling is finished, DCM and methanol are alternately washed; vacuum drying to obtain teriparatide second fragment peptide resin, adding the peptide resin into 3% TFA/DCM, and reacting; collecting filtrate, and performing vacuum spin drying on the filtrate; obtaining a second fragment of teriparatide;

(5) Preparation of teriparatide full-protection peptide:

dissolving the first fragment, the second fragment and PyBoP in a mixed solution of DMF/DCM, adjusting the pH to about 8 with DIEA for 3h, finishing the post-rotary evaporation to oily state, dissolving in ethyl acetate, adding 5% NaHCO ₃ And (3) alternately washing the organic phase with 5% citric acid aqueous solution, washing the organic phase with pure water, and spin-evaporating and drying to obtain the teriparatide full-protection peptide.

(6) Preparation of teriparatide

Preparing a lysate TFA/TIS/m-cresol=90/5/5, adding peptide resin for reaction, filtering after completion, taking filtrate, adding the filtrate into isopropyl ether for sedimentation, centrifugally drying to obtain a crude teriparatide product, and purifying to obtain the final product teriparatide.

Drawings

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

Detailed Description

The following examples are provided to further illustrate some, but not all, of the preferred embodiments of the present invention. Other embodiments of the invention, which are based on the invention, will be apparent to those skilled in the art without undue burden, and are within the scope of the 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 of the resin: 60g of 2-Cl-CTC Resin with a substitution of 1.0mmol/g was weighed, 600ml of DCM was added to swell the Resin for 0.5h, the solvent was drained off, 300ml of DMF was added to wash the Resin twice, and the solvent was drained off.

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

3) Preparation of teriparatide first fragment peptide resin: a) Swelling amino acid Resin, weighing 76.9g of Fmoc-Leu-CTC Resin, adding 700ml of DCM to swell the Resin for 0.5h, draining the solvent, adding 500ml of DMF to wash the Resin twice, and draining the solvent; b) Removing Fmoc protecting group, 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, adding 500ml DMF after deprotection is finished to wash the resin, pumping to dissolve, repeating for 5 times, and detecting indene positive; c) According to the molar mass ratio of the resin to Fmoc-His (trt) -OH, HOBt, DIC being 1:2:2.4:2.4, weighing 62.0g Fmoc-His (trt) -OH and 16.2g HOB, dissolving in 500ml DMF, adding 19ml DIC, activating in an ice water bath for 5min, adding the solution into a reaction column to react for 2h at 30 ℃, performing indene detection negative, pumping out the solvent, washing the resin for 3 times by 500ml DMF, and pumping out the solvent; d) And (3) extending the 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 for coupling reaction to obtain 206.3g of teriparatide first-stage peptide resin.

4) Preparation of teriparatide first-fragment full-protection peptide: preparing 1 600ml of 20% TFE/DCM solution, adding teriparatide first-stage peptide resin, reacting for 2h, collecting filtrate, and carrying out vacuum spin drying on the filtrate; 149.9g of teriparatide first fragment.

Example 2:

Fmoc-Phe-PS-Wang Resin preparation, scheme 2:

1) Swelling of the resin: 120g of 2-Cl-CTC Resin with a substitution of 1.0mmol/g was weighed, 1.200 ml of DCM was added to swell the Resin for 0.5h, the solvent was drained off, 900ml of DMF was added to wash the Resin twice, and the solvent was drained off.

2) Preparation of Fmoc-Phe-PS-Wang Resin: 1) According to the molar mass ratio of resin, parahydroxyben-nol and DIEA of 1:3:12, weighing 44.7g of parahydroxyben-nol to be dissolved in 900ml of DMF, adding 251ml of DIEA, uniformly mixing, adding the solution into a reaction column to react for 3h, adding 100ml of MeOH into the reaction liquid to seal the Cl which is not completely reacted, reacting for 20min, and pumping out the solvent; adding 900ml DMF to wash the resin three times, and pumping down the washing solvent; 2) According to the molar mass ratio of the resin to Fmoc-Phe-OH, hoBt, DIC, DMAP being 1:2:2.4:2.4:0.2, weighing 93.0g Fmoc-Ala-OH and 38.9g HoBt to dissolve in 900ml DMF, adding 44.6ml DIC to activate for 5min, adding the activated liquid into the resin to react for 10min at 30 ℃, weighing 2.9g DMAP to dissolve in 100ml DMF, slowly dripping into a reaction column to react for 1.5h at 30 ℃, and draining the solvent; adding 900ml DMF to wash the resin three times, and pumping down the washing solvent; adding a sealing liquid according to the mole ratio of the resin to the acetic anhydride to the pyridine of 1:20:5, and reacting for 5 hours; adding 900ml DMF to wash the resin three times, and pumping down the washing solvent; after washing with methanol three times each for 10min, the solvent was drained and dried under vacuum, 148.2g of Fmoc-Phe-PS-Wang Resin was obtained, and the degree of substitution was determined to be 0.38mmol/g.

Example 3

Preparation of teriparatide second fragment full-protection peptide:

1) Preparation of teriparatide second fragment peptide resin: a) Swelling the amino acid Resin, weighing 132.0g of Fmoc-Phe-PS-Wang Resin, adding 1.500 ml of DCM to swell the Resin for 0.5h, draining the solvent, adding 1.000 ml of DMF to wash the Resin twice, and draining the solvent; b) Removing Fmoc protecting group, adding 1 000ml of 20% piperidine/DMF solution into the resin, deprotecting twice, wherein the first deprotection time is 5min, the second deprotection time is 15min, adding 1 000ml DMF after deprotection is finished, washing the resin, pumping to dissolve, repeating for 5 times, and performing indene detection positive; c) According to the molar mass ratio of the resin to Fmoc-Asn (trt) -OH, HOBt, DIC being 1:2:2.4:2.4, 59.7g of Fmoc-Asn (trt) -OH and 16.2g of HOB are weighed and dissolved in 1 000ml of DMF, 19ml of DIC is added, after ice water bath activation is carried out for 5min, the solution is added into a reaction column for reaction for 2h at 30 ℃, indene detection is negative, the solvent is pumped out, 1 000ml of DMF is used for washing the resin for 3 times, and the solvent is pumped out; d) The above steps are repeated, 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, fmoc-Asn (trt) -OH are sequentially added according to the amino acid sequence, and the coupling reaction is carried out to obtain 321.5g of teriparatide first-stage peptide resin.

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

Example 4

Preparation of teriparatide full-protection peptide:

according to the mole mass ratio of the first fragment, the second fragment and the PyBoP being 1:1:1.01, 149.9g of the first fragment, 208.4g of the second fragment and 26.2g of the PyBoP are weighed and dissolved in 5000ml of 10% DMF/DCM mixed solution, the pH is regulated to about 8 by using DIEA, the mixture is reacted for 3 hours at 40 ℃, the mixture is distilled to oily state after completion, 3000ml of ethyl acetate is added for dissolution, 1500ml of 5% NaHCO is used ₃ The organic phase is alternately washed with 5% citric acid aqueous solution for five times, washed with 1500ml pure water for five times, and the organic phase is spin-evaporated and dried to obtain 446.2g of teriparatide full-protection peptide.

Example 5

Preparation of teriparatide:

preparing 3,000 ml of lysate according to the ratio of TFA/TIS/m-cresol=90/5/5, stirring uniformly, slowly adding teriparatide full-protection peptide, dissolving, reacting at 30 deg.C for 3h, adding the reaction solution into 28,000 ml of isopropyl ether, settling, centrifuging and drying to obtain teriparatidePurifying the crude peptide by a C18 preparation column, wherein the mobile phase is 0.1% TFA aqueous solution, the flow rate is 50-100 ml/min, the detection wavelength is 230nm, tracking and collecting the required effluent by using HPLC, and then freeze-drying by adopting a conventional method to obtain teriparatide refined peptide with the mass of 82.5g, the purity of 99.59%, the maximum single impurity of 0.22%, the total impurity of 0.41% and the total yield of 40.0%; ESI-MS: [ M+4H ]] ⁴⁺ ＝1030.5，[M+3H] ³⁺ =1373.9, and the hplc results are shown in fig. 1.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (1)

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

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

adding 2-Cl-CTC resin into a solid phase reactor, and adding DMF swelling 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 with DCM and methanol alternately, and vacuum drying to obtain Fmoc-Leu-CTC Resin;

(2) Preparation of the first fragment:

Fmoc-Leu-CTC Resin is taken and added into a reaction column, DMF is added for swelling, solvent is pumped out, and piperidine/DMF solution is added for reaction; draining the solvent, and adding DMF for washing; indene detection is positive; fmoc-His (trt) -OH, HOBt, DIC is taken and dissolved in DMF and activated in ice bath; adding the activating solution into a reaction column for reaction, and pumping out 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, BOC-Ser (tBu) -OH and performing coupling reaction according to the amino acid sequence; after the coupling is finished, DCM and methanol are alternately washed; vacuum drying to obtain teriparatide first segment peptide resin, adding the peptide resin into TFE/DCM, and reacting; collecting filtrate, and performing 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 swelling resin; extracting solvent, adding DMF solution of p-hydroxybenzyl alcohol, stirring uniformly, adding DIEA, reacting at 30 ℃ for 2h, adding MeOH into the reaction solution to seal the incompletely reacted Cl, and washing the resin with DMF after completion; adding a DMF solution of Fmoc-Phe-OH and DIEA, stirring for reaction, pumping the solvent, adding a DIEA/methanol/DCM solution, and sealing; the DCM and methanol are alternately washed, and Fmoc-Phe-PS-Wang Resin is obtained after vacuum drying;

(4) Preparation of the second fragment:

Fmoc-Phe-PS-Wang Resin is taken and added into a reaction column, DMF is added for swelling, solvent is pumped out, and piperidine/DMF solution is added for reaction; draining the solvent, and adding DMF for washing; indene detection is positive; fmoc-Asn (trt) -OH, HOBt, DIC is taken and dissolved in DMF and activated in ice bath; adding the activating solution into a reaction column for reaction, and pumping out the solvent after the indene detection result is negative; adding DMF for washing; repeating the 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 for coupling reaction; after the coupling is finished, DCM and methanol are alternately washed; vacuum drying to obtain teriparatide second fragment peptide resin, adding the peptide resin into 3% TFA/DCM, and reacting; collecting filtrate, and performing vacuum spin drying on the filtrate; obtaining a second fragment of teriparatide;

(5) Preparation of teriparatide full-protection peptide:

dissolving the first fragment, the second fragment and PyBoP in a mixed solution of DMF/DCM, and adjusting the pH to about 8 by using DIEA to react for 3h to finishSpin-evaporating to obtain oil, dissolving in ethyl acetate, adding 5% NaHCO ₃ Alternately washing the organic phase with 5% citric acid aqueous solution, washing the organic phase with pure water, and spin-evaporating and drying to obtain teriparatide full-protection peptide;

(6) Preparation of teriparatide

Preparing a lysate TFA/TIS/m-cresol=90/5/5, adding peptide resin for reaction, filtering after completion, taking filtrate, adding the filtrate into isopropyl ether for sedimentation, centrifugally drying to obtain a crude teriparatide product, and purifying to obtain the final product teriparatide.