CN111057139A

CN111057139A - Novel process for preparing teriparatide

Info

Publication number: CN111057139A
Application number: CN201811206844.6A
Authority: CN
Inventors: 邹正才; 杨凯; 张孝清; 宋志春; 包金远
Original assignee: Nanjing Huawe Medicine Technology Group Co Ltd
Current assignee: Nanjing Huawe Medicine Technology Group Co Ltd
Priority date: 2018-10-17
Filing date: 2018-10-17
Publication date: 2020-04-24
Anticipated expiration: 2038-10-17
Also published as: CN111057139B

Abstract

The invention provides a method for synthesizing teriparatide. According to the method, the teriparatide is divided into a fragment [1-12] peptide resin and a fragment I and a fragment II according to a teriparatide main chain peptide sequence, the three short peptides can be synthesized respectively, then the fragment [1-12] peptide resin is connected with the fragment I and the fragment II one by one, the terminal Fmoc protecting group is removed to obtain the teriparatide resin, and finally, the teriparatide resin is subjected to cracking, purification and salification to finally obtain a target product. The structures of the segments and the segment resins are described in the specification. The method has the advantages of simple operation, higher purity of the obtained product, lower cost and contribution to industrial production.

Description

Novel process for preparing teriparatide

Technical Field

The invention relates to a polypeptide synthesis technology, in particular to a process for realizing teriparatide synthesis by utilizing a solid phase synthesis technology.

Background

Teriparatide forteo (terriparatide) is developed by the present of america and used for primary osteoporosis, hypogonadism osteoporosis and postmenopausal osteoporosis. The parathyroid hormone derivative can promote bone growth by increasing the activity and quantity of osteoblasts, while the current conventional osteoporosis medicines only act on osteoclasts to slow or block bone loss. Teriparatide from the original research was first approved in the united states for 12 months in 2002 and in china for 3 months in 2011. In a clinical study involving 1, 637 postmenopausal osteoporosis patients, the results showed that 96% of patients, after receiving this drug, exhibited a significant increase in bone (mineral) density BMD in both spine and buttocks, compared to those treated with calcium and vitamin D supplements alone. In addition, it was found that the drug was able to reduce the risk of vertebral fractures and other types of fractures by 65% and 53%, respectively. Another characteristic of the drug is that it has few side effects, usually only nausea, dizziness and leg cramps. Teriparatide reaches $ 15 billion worldwide in 2016. China is the country with the most number of osteoporosis worldwide, and data show that 9000 ten thousand osteoporosis patients in China currently, the number is expected to rise to 1.5 hundred million by 2025, and the market potential is huge.

The structural formula of teriparatide:

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

teriparatide [ hPTH (1-34) ] is a synthetic polypeptide hormone which is a 1-34 amino acid fragment of human parathyroid hormone PTH, which is the biologically active N-terminal region of the endogenous parathyroid hormone PTH containing 84 amino acids. Teriparatide has immunological and biological properties identical to those of endogenous parathyroid hormone PTH and bovine parathyroid hormone PTH (PTH), and is capable of stimulating bone formation and resorption, reducing the incidence of bone fracture in postmenopausal women, and increasing or decreasing bone density depending on the mode of administration. Continuous infusion results in a sustained increase in parathyroid hormone PTH concentration and therefore greater bone resorption than daily injections which cause only a transient increase in serum parathyroid hormone PTH. In addition, teriparatide does not inhibit platelet aggregation response of either the adenosine diphosphate-induced pathway or the collagen-induced pathway. The usage is intravenous injection or subcutaneous injection, particularly, the dosage of the old is within the range of 31-85 years old, the pharmacokinetics of the medicine is not obviously affected by age increase, and the dosage does not need to be adjusted.

The patents currently associated with teriparatide synthesis are as follows:

1) a stepwise solid phase synthesis method is adopted. CN107501408 and CN104530218 and other patents describe amino acid sequences, by connecting amino acids with resins one by one to obtain peptide resins, then performing acidolysis to obtain crude teriparatide, purifying the crude teriparatide, and performing transacetate extraction to obtain pure teriparatide acetate; CN104017064 couples Ser and Met at 17 and 18 positions to form dipeptide in sequence from C end to N end, and then docking into main chain; CN1103467595 couples Asn and Ser at positions 18 and 19 to form dipeptide in the order from C-terminal to N-terminal and then docks into the main chain. Generally, CN104017064 and CN1103467595 adopt a method of connecting in a cascade manner. Generally speaking, the technical method difference of the step-by-step solid phase synthesis method is mainly reflected in the difference of resin, condensation or activation reagent, cracking solvent and the like, and the advantages of the resin, the condensation or activation reagent, the cracking solvent and the like are not obvious.

2) Solid phase fragment synthesis. CN105384809, CN102731643 and the like divide an amino acid sequence into a plurality of fragments for synthesis, and then splice to obtain the final peptide resin, wherein the yield is 20-40%. The defects are more fragments, long steps and low yield, and improvement is needed.

In conclusion, the existing teriparatide synthesis method has the problems that the yield of the obtained peptide is basically 20-42%, the impurities are more, the yield is low, the racemization of amino acid is difficult, the yield is low and the like, and a technical scheme which is relatively suitable for industrial production is not seen yet. Therefore, the present inventors have studied a method for synthesizing teriparatide, and have arrived at a technical solution of the present invention.

Disclosure of Invention

The invention aims to solve the technical problems of more impurities, low purity and yield, high cost, complex operation steps, excessive waste liquid and inconvenience for industrial production in the conventional synthesis process, and provides a teriparatide synthesis method.

The technical problem to be solved by the invention is realized by the following technical scheme, and the method for synthesizing teriparatide comprises the following steps:

step one, according to the sequence of amino acids from the C end to the N end of a main chain, carrying out solid phase synthesis on a 1 st-12 th amino acid fragment [1-12] peptide resin, wherein a protecting group is coupled on the side chain of the peptide resin of the polypeptide fragment;

step two, according to the sequence of the amino acids from the C end to the N end of the main chain, carrying out solid phase synthesis on the peptide resin of the 13 th-22 th amino acid, and cutting the resin to obtain a segment I;

step three, according to the sequence of the amino acids from the C end to the N end of the main chain, carrying out solid phase synthesis on the peptide resin of the 23 rd to 34 th amino acids, and cutting the resin to obtain a second fragment;

fourthly, after removing the end Fmoc of the fragment [1-12] peptide resin by a solid phase method, carrying out coupling reaction with the fragment I to obtain a fragment [1-22] peptide resin;

fifthly, performing coupling reaction on the fragment [1-22] peptide resin and the fragment II after removing the terminal Fmoc by a solid phase method to obtain teriparatide peptide resin;

step six: carrying out a cracking reaction on the teriparatide resin under the action of a cracking agent to obtain a crude teriparatide product; step seven: and purifying, salifying and freeze-drying the crude teriparatide product to obtain the teriparatide acetate refined peptide.

In the method of the present invention, the resin is divided into a short-chain fragment resin and two short-chain fragments according to the peptide sequence of the main chain of teriparatide. Respectively synthesizing fragment peptide resin and the other two fragments, then connecting one by one, and cutting the resin to obtain a crude product of a final product. The amino acid sequence from the C end to the N end of the main chain of teriparatide is numbered, and the peptide sequence is shown as the following formula:

Ser³⁴-Val³³-Ser³²-Glu³¹-Ile³⁰-Gln²⁹-Leu²⁸-Met²⁷-His²⁶-Asn²⁵-Leu²⁴-Gly²³-Lys²²-His²¹-Leu²⁰-Asn¹⁹-Ser¹⁸-Met¹⁷-Glu¹⁶-Arg¹⁵-Val¹⁴-Glu¹³-Trp¹²-Leu¹¹-Arg¹⁰-Lys⁹-Lys⁸-Leu⁷-Gln⁶-Asp⁵-Val⁴-His³-Asn²-Phe¹-OH

further, the air conditioner is provided with a fan,

protecting groups are present in His, Asp, Gln, Lys, Arg, Trp, Glu, Ser and Asn of the fragment [1-12] peptide resin, the fragment [1-22] peptide resin and the teriparatide peptide resin;

protecting groups are arranged on His, Asp, Gln, Lys, Arg, Trp, Glu, Ser and Asn of the fragment I and the fragment II;

the protecting group is a protecting group which is commonly used in the field of amino acid synthesis and can protect amino groups and side chain functional groups on an amino acid main chain from interfering with synthesis, such as active hydrogen-containing nitrogen groups, hydroxyl groups or carboxyl groups and the like, and prevent the main chain amino groups or the side chain functional groups and the like from generating impurities in the process of preparing a target product.

Preferably, the side chain protecting groups of Asn and Gln are Trt;

the side chain protecting group of His is Adoc, Boc, Mmt, Mtt or Trt, further preferably Trt;

the side chain protecting groups of Lys and Trp are Boc or Trt, and further, Boc is preferred;

the side chain protecting group of Arg is Boc or Pbf, further preferably Pbf;

the side chain protecting group of Glu is preferably OtBu;

the side chain protecting group of Ser is TES, TMS, TBS, DHP, THP or tBu, and further, tBu is preferable.

A solid phase synthesis method of teriparatide, including but not limited to side chain protecting group and described with preferred protecting group, as a preferred scheme, the synthesis method comprises the following steps:

adding Fmoc-Phe-Wang Resin into a solid phase reactor, removing Fmoc reaction under the action of a deprotection agent, then carrying out coupling reaction with Fmoc-Asn (Trt) -OH under the action of a condensation agent, then repeating the deprotection reaction and the coupling reaction, and sequentially connecting the following amino acids of 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 and Fmoc-Trp (Boc) -OH according to a polypeptide sequence to obtain the polypeptide fragment [1-12] peptide Resin;

step two, coupling reaction is carried out on 2-CTC Resin and amino acid Fmoc-Glu (OtBu) -OH under the action of a condensing agent to obtain joint Resin, Fomc deprotection reaction is carried out on the joint Resin, and then Fmoc-Val-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, OtBu-OH and OtBu-OH are sequentially coupled according to a polypeptide sequence in a one-by-one solid phase coupling reaction mode,

Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH and Fmoc-Lys (Boc) -OH, repeating the coupling reaction and deprotection reaction operation until a fragment-peptide resin is synthesized, and finally cracking the resin under the action of a cracking agent to obtain a fragment I;

step three, adding the 2-CTC Resin into a solid phase reactor to perform coupling reaction with amino acid Fmoc-Gly-OH under the action of a condensing agent to obtain a joint Resin, performing Fomc deprotection reaction on the joint Resin, and then sequentially coupling the amino acid Fmoc-Leu-OH, the amino acid Fmoc-Gly-OH, the amino acid Fmoc-Leu-OH, the amino acid Fmoc-Gly-OH and the amino,

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 Fmoc-Ser (tBu) -OH, repeating the operations of coupling reaction and deprotection reaction until a fragment dipeptide resin is synthesized, and finally obtaining a fragment II after cracking the resin under the action of a cracking agent;

step four, carrying out deprotection reaction on the terminal Fmoc of the fragment [1-12] peptide resin by a solid phase method, and coupling the fragment I with the fragment I under the action of a condensing agent to obtain a fragment [1-22] peptide resin;

fifthly, carrying out deprotection reaction on the Fmoc at the tail end of the fragment [1-22] peptide resin by a solid phase method, and coupling the fragment II with the fragment II under the action of a condensing agent to obtain teriparatide peptide resin;

Further, the air conditioner is provided with a fan,

the structural formula of the fragment [1-12] peptide resin is as follows:

Fmoc-Trp¹²(Boc)-Leu¹¹-Arg¹⁰(pbf)-Lys⁹(Boc)-Lys⁸(Boc)-Leu⁷-Gln⁶(Trt)-Asp⁵(OtBu)- Val⁴-His³(Trt)-Asn²(Trt)-Phe¹-Wang resin;

the structural formula of the fragment is as follows:

Fmoc-Lys²²(Boc)-His²¹(Trt)-Leu²⁰-Asn¹⁹(Trt)-Ser¹⁸(tBu)-Met¹⁷-Glu¹⁶(OtBu)-Arg¹⁵(pb f)-Val¹⁴-Glu¹³(OtBu)-OH；

the structural formula of the fragment II is as follows:

Fmoc-Ser³⁴(tBu)-Val³³-Ser³²(tBu)-Glu³¹(OtBu)-Ile³⁰-Gln²⁹(Trt)-Leu²⁸-Met²⁷-His²⁶(Trt )-Asn²⁵(Trt)-Leu²⁴-Gly²³-OH；

the structural formula of the fragment [1-22] peptide resin is as follows:

Fmoc-Lys²²(Boc)-His²¹(Trt)-Leu²⁰-Asn¹⁹(Trt)-Ser¹⁸(tBu)-Met¹⁷-Glu¹⁶(OtBu)-Arg¹⁵(pb f)-Val¹⁴-Glu¹³(OtBu)-Trp¹²(Boc)-Leu¹¹-Arg¹⁰(pbf)-Lys⁹(Boc)-Lys⁸(Boc)-Leu⁷-Gln⁶(Tr t)-Asp⁵(OtBu)-Val⁴-His³(Trt)-Asn²(Trt)-Phe¹-Wang resin;

the structural formula of the teriparatide resin is as follows:

Ser³⁴(tBu)-Val³³-Ser³²(tBu)-Glu³¹(OtBu)-Ile³⁰-Gln²⁹(Trt)-Leu²⁸-Met²⁷-His²⁶(Trt)-Asn²⁵(Trt)-Leu²⁴-Gly²³-Lys²²(Boc)-His²¹(Trt)-Leu²⁰-Asn¹⁹(Trt)-Ser¹⁸(tBu)-Met¹⁷-Glu¹⁶(Ot Bu)-Arg¹⁵(pbf)-Val¹⁴-Glu¹³(OtBu)-Trp¹²(Boc)-Leu¹¹-Arg¹⁰(pbf)-Lys⁹(Boc)-Lys⁸(Boc)- Leu⁷-Gln⁶(Trt)-Asp⁵(OtBu)-Val⁴-His³(Trt)-Asn²(Trt)-Phe¹wang resin.

Further, the air conditioner is provided with a fan,

in any step from the first step to the fifth step, the coupling reaction is carried out under the action of a condensing agent, and the condensing agent is optionally and independently selected from one or more of DIC/HOBt, HBTU/HOBt/DIEA, PyBop/HOBt/DIEA, preferably DIC/HOBt; the reaction solvent used is preferably one or more of dichloromethane, DMF, NMP and DMSO, preferably DMF; the deprotection reaction is carried out under the action of a deprotection agent, wherein the deprotection agent is selected from any one of Pip/DMF, NaOH/MeOH and TEA/DMF, and is preferably a Pip/DMF solution with a volume ratio of 20%.

As a preferred embodiment of the present invention,

in any step of the second step or the third step, the cracking agent is selected from a trifluoroacetic acid/dichloromethane mixed solution or a trifluoroethanol/dichloromethane mixed solution.

Further, the dosage of the mixed solution is as follows: volume ratio TFA: DCM is 0.5-5: 95.0 to 99.5; a mixed solution of trifluoroethanol and dichloromethane in a volume ratio of TFE: DCM is 10-30: 70-90; further preferred is TFE: DCM ═ 20: 80 (volume ratio); the volume of the cracking agent is 5-20 times of the mass of the peptide resin, and preferably 8-12 times of the mass of the peptide resin.

In one embodiment, in the sixth step, the adopted cracking agent is a mixed solution of TFA, PhSMe, TIS and water; the volume ratio of the mixed solution is TFA: PhSMe: and (3) TIS: 70-97% of water: 10-1: 10-1: 10-1; or the adopted cracking agent is a mixed solution of TFA, EDT and water; the volume ratio of the mixed solution is TFA: EDT (electro-thermal transfer coating): water is 90-95: 1-5: 1-5, preferably 90: 5: 5;

further, in the sixth step, the volume of the cracking agent is 5-25 times of the mass of the peptide resin, and preferably 8-12 times of the mass of the peptide resin;

in one of the solutions, the first and second parts are,

in the solid phase synthesis method of teriparatide, solid phase carriers for solid phase synthesis are Wang resin with the degree of substitution of 0.2-1.0mmol/g, Fmoc-Phe-Wang resin with the degree of substitution of 0.2-0.7mmol/g, HMP resin with the degree of substitution of 0.2-1.0mmol/g, HMBA-AM resin with the degree of substitution of 0.2-1.0mmol/g or 2-CTC resin with the degree of substitution of 0.2-1.5 mmol/g; Fmoc-Phe-Wang resins with a degree of substitution of 0.3 to 0.5mmol/g or 2-CTC resins with a degree of substitution of 0.6 to 1.2mmol/g are preferred.

In one of the solutions, the first and second parts are,

the reaction solvent used in the invention is one or more of dichloromethane, DMF, NMP and DMSO.

In the invention, the mode of one-by-one solid phase coupling reaction is generally that a reaction substrate is sequentially connected with Fmoc protected amino acid, after each coupling reaction is finished, a deprotection agent is used for Fmoc protection group removing reaction, then solid phase coupling reaction is carried out with the next Fmoc protected amino acid, and the coupling reaction and deprotection reaction are sequentially repeated until a product with a target peptide sequence structure is synthesized.

The side chain protecting group of the present invention is a protecting group commonly used in the field of amino acid synthesis for protecting a functional group such as a hydrogen-containing nitrogen group, a hydroxyl group or a carboxyl group on an amino acid main chain and a side chain, and helps to prevent a part of the side chain from reacting with a chemical substance used in steps such as peptide synthesis and processing.

The condensing agent is a reagent capable of causing a coupling reaction, in particular a reagent capable of promoting the coupling of an amino group and a carboxyl group to form a peptide bond in the synthesis of polypeptide.

The activator refers to a reagent which can assist the condensing agent to better promote the coupling reaction in the polypeptide coupling reaction, such as: inhibiting the generation of racemic impurities in the coupling reaction, catalyzing and accelerating the reaction speed and the like.

In the method of the invention, the three short peptides can be synthesized firstly by dividing the resin into the basic segment [1-12] peptide resin and two segments according to the peptide sequence of the main chain of the teriparatide. And connecting the fragment [1-12] peptide resin with the fragment I and the fragment II one by one, and removing a terminal Fmoc protecting group to obtain the teriparatide peptide resin. Finally, the target product is obtained through cracking, purification and salification.

Description of the figures figure 1 is a flow diagram of teriparatide preparation

The invention has the following beneficial effects:

1) the invention adopts three fragment coupling methods to carry out solid phase synthesis of teriparatide, wherein the fragment [1-12] peptide resin, the fragment I and the fragment II are easy to synthesize and purify and have high purity;

2) compared with the step-by-step synthesis method, the method has the advantages that the step is shortened to 13 compared with the step-by-step synthesis method, different fragments can be synthesized simultaneously, and the teriparatide synthesis time is effectively shortened;

3) the fragment synthesis method adopted by the invention has short steps and mild reaction conditions, and can effectively reduce the problems of deletion of peptides, more difference of amino acid sequences of main components, isomerization, amino acid racemization and the like;

4) the product is easier to purify, the post-treatment operation is simple, and three wastes are reduced;

5) the method has stable process, the final purity of teriparatide can reach more than 99.5 percent, the total yield is 46 percent, and the industrial application is facilitated.

Detailed Description

For the sake of understanding, the present invention will be described in detail below by way of specific examples. It is specifically intended that the examples be given solely for the purpose of illustration and that all modifications that would be apparent to one skilled in the art from this disclosure are within the scope of the invention.

The abbreviations or English letters used in the description and claims of the present invention have the following meanings:

the first embodiment is as follows: synthesis of fragment [1-12] peptide resin

Fmoc-Phe-Wang resin (10.00g substitution 0.35mmol/g), 200mL of LPCM were added to a solid phase reaction flask, and the mixture was swollen and then drained. Mixing the filter cake with 300ml of 20% piperidine/DMF solution, stirring for 5 minutes and then pumping to dryness; then, 300mL of 20% piperidine/DMF solution was added, mixed and stirred for 15 minutes, and then, the mixture was drained. And washing the filter cake with a proper amount of DMF, draining, and placing in a solid-phase reaction bottle for later use. Fmoc-Asn (Trt) -OH (6.45g), HOBT (1.46g) and 30ml DMF were added to another dry reaction flask in sequence, stirred, DIC (1.36g) was added over 30min in ice bath, and stirred to obtain an activated solution. And adding the new solution into the solid-phase reaction bottle, and stirring for 2h under the protection of nitrogen until the coupling reaction is detected to be complete by ninhydrin. Pumping, washing the filter cake with DMF, and pumping. The deprotection and coupling reaction steps are repeated, 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 and Fmoc-Trp (Boc) -OH are connected in sequence. After the coupling reaction is finished, pumping, washing a filter cake by DMF, alternately washing by MeOH and DCM, and pumping. Drying in vacuo to give the fragment [1-12] peptide resin, which was used directly in the next step.

0.10g of peptide resin was put into 1mL of a lysate (TFA: EDT: water, 90: 5: 5, volume ratio), and stirred at room temperature for 3 hours. Filtration, washing of the resin with a small amount of TFA, combining the filtrates, mixing with 15mL of glacial ethyl ether for sedimentation, centrifugation, washing of the white precipitate with ethyl ether and draining. Vacuum drying to obtain solid with purity of 95% by detection.

EXAMPLE two Synthesis of fragment one

2-CTC Resin (25.00g, the degree of substitution is 1.12mmol/g) and 400ml of EDCM are sequentially added into a solid phase reaction bottle, and filtration is carried out after swelling to obtain a filter cake. Fmoc-Glu (OtBu) -OH (8.51g), 100mL DCM were added sequentially, with stirring under nitrogen, and DIEA (ca. 9.53g) was added dropwise. After dropping, the reaction was stirred for 2 h. MeOH (25mL) was added and stirring was continued for 30 min. And (4) pumping, alternately washing a filter cake by MeOH and DCM, pumping, and drying in vacuum at room temperature to obtain the joint resin. The detection shows that the degree of substitution is 0.73 mmol/g;

and adding the resin and 200ml of LPCM into a solid-phase reaction bottle in sequence, swelling and then pumping to dry. Adding 100mL of 20% piperidine/DMF solution, mixing and stirring for 5 minutes, and pumping to dry; 100mL of 20% piperidine/DMF solution was added, mixed and stirred for 15 minutes, and then the mixture was drained. The filter cake is washed with an appropriate amount of DMF and then drained. In another dry reaction flask were placed Fmoc-Val-OH (18.32g), HOBT (8.75g), and 100mL DMF in this order, followed by stirring, and DIC (8.18g) was added under nitrogen protection while cooling on ice to obtain an activated solution. And adding the new solution into the solid-phase reaction bottle under the protection of nitrogen, and stirring for 2 hours until the ninhydrin detection shows that the coupling reaction is complete. The mixture was drained and the filter cake was washed with DMF. Repeating the steps of deprotection and coupling reaction, and connecting Fmoc-Arg (pbf) -OH,

Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH, Fmoc-Lys (Boc) -OH. And after the coupling reaction is finished, pumping out, washing a filter cake by DMF, alternately washing by MeOH and DCM, pumping out, and drying in vacuum at room temperature to obtain the fragment-peptide resin.

The peptide resin and 500mL of 20% TFE/DCM were added in sequence to a reaction flask, nitrogen was bubbled, stirred and reacted for 2h, then suction filtration was carried out, the filter cake was washed with an appropriate amount of DCM, the filtrate was collected, and the solvent was removed by concentration under reduced pressure at 30 ℃. Adding 200ml of LCCM into the residue, dissolving, adding ethyl acetate, crystallizing, centrifuging, washing with ethyl acetate, draining, and drying at room temperature to obtain fragment I41.23 g, yield 91%, and purity 89%.

Example three: synthesis of fragment two

2-CTC Resin (25.00g, the degree of substitution is 1.12mmol/g) and 400ml of EDCM are sequentially added into a solid phase reaction bottle, and a filter cake is obtained by pumping after swelling. 100ml of LCCM and Fmoc-Gly-OH (5.95g) were added in this order, stirred under nitrogen, and DIEA (9.53g) was added dropwise. After dropping, the reaction was stirred for 2h, MeOH (25mL) was added, and the reaction was stirred with nitrogen bubbling for 30 min. It was drained and the filter cake was washed alternately with DCM and MeOH. And (5) pumping, and drying in vacuum at room temperature to obtain the joint resin. The degree of substitution was determined to be 0.74 mmol/g.

The mass was swollen with 200mL of DCM and drained. Mixing the filter cake with 100mL of 20% piperidine/DMF solution, stirring for 5 minutes, and pumping to dry; 100mL of 20% piperidine/DMF solution was added, mixed and stirred for 15 minutes, and then the mixture was drained. The filter cake is washed with an appropriate amount of DMF and then drained. 400ml of DMF was added to the reaction flask and stirred, and Fmoc-Leu-OH (19.08g), HOBT (8.75g) and DIC (8.18g) were sequentially added to the flask in ice bath for 30 min. Stirring the mixture for reaction for 2h under the protection of nitrogen until the reaction is detected to be complete. The solution was drained and the peptide resin was washed with DMF. And repeating the steps of deprotection and coupling reaction, and sequentially connecting 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 Fmoc-Ser (tBu) -OH. After the coupling reaction is finished, pumping to dry, washing a filter cake by a proper amount of DMF, alternately washing by a proper amount of DCM and MeOH, and pumping to dry. Vacuum drying at room temperature to obtain the fragment dipeptide resin.

Adding 500mL of 20% TFE/DCM into the peptide resin, stirring and reacting for 2h under nitrogen, carrying out suction filtration, washing a filter cake by using an appropriate amount of DCM, collecting a filtrate, carrying out reduced pressure concentration at room temperature to remove a solvent, dissolving a residue by using 200mL of CCM, adding glacial ethyl ether for crystallization, centrifuging, washing by using ethyl ether, carrying out suction drying, and drying at room temperature to obtain a second fragment 40.96g, wherein the yield is 95%, and the purity is 90%.

Example four: synthesis of fragment [1-22] peptide resin

The peptide resin (13.65g) obtained in example one, 200ml of LPCM was swollen with the above material in a solid phase reaction flask and then drained. Mixing the filter cake with 100mL of 20% piperidine/DMF solution, stirring for 5 minutes, and pumping to dry; 100mL of 20% piperidine/DMF solution was added, mixed and stirred for 15 minutes, and then the mixture was drained. The filter cake is washed by a proper amount of DMF and is pumped to be used. In another dry reaction flask, fragment one (9.65g), HOBT (2.96g), DMF (50mL) and DIC (3.00g) were added in this order, mixed and activated to give a solution, which was then added to the above solid phase reaction flask. The reaction was carried out under nitrogen bubbling until the reaction was complete as detected by ninhydrin. 100mL of 30% acetic anhydride/pyridine/DCM solution was added, stirred for 1h, and pumped dry. The filter cake was washed with appropriate amount of DMF and was taken up directly in the next step.

Example five: synthesis of teriparatide resin

The peptide resin obtained in example four was swollen with 200ml of LPCM and then was dried by suction. Mixing the filter cake with 100mL of 20% piperidine/DMF solution, stirring for 5 minutes, and pumping to dry; 100mL of 20% piperidine/DMF solution was added, stirred for 15 minutes and pumped to dryness. Washing with DMF, and draining. To another dry reaction flask were added successively fragment two (13.26g), HOBT (2.96g), DMF (50mL) and DIC (3.00g), and the mixture was stirred to activate a solution, which was then added to the above solid-phase reaction flask. And (5) reacting until the ninhydrin detection reaction is complete. 100mL of 30% acetic anhydride/pyridine/DCM solution was added, stirred for 1h, and pumped dry. Washed with DMF and drained. Mixing the filter cake with 100mL of 20% piperidine/DMF solution, stirring for 5 minutes, and pumping to dry; 100mL of 20% piperidine/DMF solution was added, mixed and stirred for 15 minutes, and then the mixture was drained. The filter cake was washed with DMF, then washed with DCM and MeOH alternately, dried and dried in vacuo to obtain 25.00g of peptide resin, which was used directly in the next step.

Example six: synthesis of crude teriparatide

The crude resin from example five was mixed with 150mL of a cleavage agent (TFA: water: EDT, 90: 5: 5, v/v) and cleaved at 25 ℃ for 3 hours to the end of the reaction. Filtering, adding a proper amount of isopropyl ether into the filtrate, settling, centrifuging, washing, separating, and vacuum-drying at room temperature to obtain 12.25g of crude teriparatide with HPLC purity of 75% and total yield of 85%.

Example seven: preparation of teriparatide acetate

Adding three drops of ammonia water and 1.00g of crude teriparatide into water in sequence, shaking for dissolution, and purifying by a Hanbang preparative HPLC system (the wavelength is 210nm, a chromatographic column is a C8 reverse phase column, a 20M ammonium acetate solution, and pH3.0/acetonitrile is a mobile phase) to obtain a teriparatide refined peptide solution with the purity of more than 99.5 percent. Transferring the refined peptide solution into a preparative HPLC system (the chromatographic column is a C18 reversed phase column, and 0.2% acetic acid/acetonitrile is a mobile phase), collecting the target component, concentrating under reduced pressure, and lyophilizing to obtain teriparatide acetate pure product 0.55g, with purity of 99.7%, purification yield of 55%, and total yield of 46%; mass spectrum (M + H)⁺＝1373.5,Calculated：4117.5。

Claims

1. A method for synthesizing teriparatide, which comprises the following steps:

step one, according to the sequence of amino acids from the C end to the N end of a main chain, carrying out solid phase synthesis on a fragment [1-12] peptide resin of 1-12 amino acids, wherein a protecting group is coupled on the side chain of the fragment [1-12] peptide resin;

step six: carrying out a cracking reaction on the teriparatide resin under the action of a cracking agent to obtain a crude teriparatide product;

step seven: and purifying, salifying and freeze-drying the crude teriparatide product to obtain the teriparatide acetate refined peptide.

2. The method of claim 1, wherein the first and second fragments have protecting groups for His, Asp, Gln, Lys, Arg, Trp, Glu, Ser, Asn.

3. The method of claim 2, wherein the side chain protecting groups of Asn and Gln are

Trt; the side chain protecting group of His is Adoc, Boc, Mmt, Mtt or Trt;

the side chain protecting groups of Lys and Trp are Boc or Trt;

the side chain protecting group of Arg is Boc or Pbf;

the side chain protecting group of Glu is preferably OtBu;

the side chain protecting group of Ser is TES, TMS, TBS, DHP, THP or tBu.

4. A method for synthesizing teriparatide, which is characterized by comprising the following steps:

step two, performing coupling reaction on 2-CTC Resin and amino acid Fmoc-Glu (OtBu) -OH under the action of a condensing agent to obtain joint Resin, performing Fomc deprotection reaction on the joint Resin, then sequentially coupling Fmoc-Val-OH, Fmoc-Arg (pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH and Fmoc-Lys (Boc) -OH according to a polypeptide sequence in a one-by-one solid phase coupling reaction mode, repeating the coupling reaction and the deprotection reaction until a fragment-peptide Resin is synthesized, and finally cracking the Resin under the action of a cracking agent to obtain a fragment I;

step three, adding the 2-CTC Resin into a solid phase reactor to perform coupling reaction with amino acid Fmoc-Gly-OH under the action of a condensing agent to obtain a linker Resin, performing Fomc deprotection reaction on the linker Resin, then sequentially coupling amino acids 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 Fmoc-Ser (tBu) -OH according to a polypeptide sequence by adopting a one-by-one solid phase coupling reaction mode, repeating the coupling reaction and deprotection reaction operation until a dipeptide resin fragment is synthesized, and finally obtaining a second fragment after cracking the resin under the action of a cracking agent;

5. The method according to any one of claims 3 or 4, wherein the fragment [1-12] peptide resin has the structural formula:

Fmoc-Trp¹²(Boc)-Leu¹¹-Arg¹⁰(pbf)-Lys⁹(Boc)-Lys⁸(Boc)-Leu⁷-Gln⁶(Trt)-Asp⁵(OtBu)-Val⁴-His³(Trt)-Asn²(Trt)-Phe¹-Wang resin;

the structural formula of the fragment is as follows:

Fmoc-Lys²²(Boc)-His²¹(Trt)-Leu²⁰-Asn¹⁹(Trt)-Ser¹⁸(tBu)-Met¹⁷-Glu¹⁶(OtBu)-Arg¹⁵(pbf)-Val¹⁴-Glu¹³(OtBu)-OH；

the structural formula of the fragment II is as follows:

Fmoc-Ser³⁴(tBu)-Val³³-Ser³²(tBu)-Glu³¹(OtBu)-Ile³⁰-Gln²⁹(Trt)-Leu²⁸-Met²⁷-His²⁶(Trt)-Asn²⁵(Trt)-Leu²⁴-Gly²³-OH；

the structural formula of the fragment [1-22] peptide resin is as follows:

Fmoc-Lys²²(Boc)-His²¹(Trt)-Leu²⁰-Asn¹⁹(Trt)-Ser¹⁸(tBu)-Met¹⁷-Glu¹⁶(OtBu)-Arg¹⁵(pbf)-Val¹⁴-Glu¹³(OtBu)-Trp¹²(Boc)-Leu¹¹-Arg¹⁰(pbf)-Lys⁹(Boc)-Lys⁸(Boc)-Leu⁷-Gln⁶(Trt)-Asp⁵(OtBu)-Val⁴-His³(Trt)-Asn²(Trt)-Phe¹-Wang resin;

the structural formula of the teriparatide resin is as follows:

Ser³⁴(tBu)-Val³³-Ser³²(tBu)-Glu³¹(OtBu)-Ile³⁰-Gln²⁹(Trt)-Leu²⁸-Met²⁷-His²⁶(Trt)-Asn²⁵(Trt)-Leu²⁴-Gly²³-Lys²²(Boc)-His²¹(Trt)-Leu²⁰-Asn¹⁹(Trt)-Ser¹⁸(tBu)-Met¹⁷-Glu¹⁶(OtBu)-Arg¹⁵(pbf)-Val¹⁴-Glu¹³(OtBu)-Trp¹²(Boc)-Leu¹¹-Arg¹⁰(pbf)-Lys⁹(Boc)-Lys⁸(Boc)-Leu⁷-Gln⁶(Trt)-Asp⁵(OtBu)-Val⁴-His³(Trt)-Asn²(Trt)-Phe¹wang resin.

6. The process according to any one of claims 4 or 5, wherein in any one of steps one to five, the coupling reaction is carried out under the action of a condensing agent, which is optionally independently selected from one or more of DIC/HOBt, HBTU/HOBt/DIEA, PyBop/HOBt/DIEA.

7. The method according to claim 4 or 5, wherein the deprotection reaction is performed by the action of a deprotection agent selected from any one of Pip/DMF, NaOH/MeOH, and TEA/DMF.

8. The method according to any one of claims 4 or 5, wherein in any of the second or third steps, the cleavage agent is selected from a trifluoroacetic acid/dichloromethane mixed solution or a trifluoroethanol/dichloromethane mixed solution.

9. The method of claim 8, wherein the volume of the cleavage agent is 5 to 20 times the mass of the peptide resin.

10. The method according to claim 4, wherein in the sixth step, the lysis reagent is mixed with trifluoroacetic acid. The volume ratio of the trifluoroacetic acid mixed solution is TFA: PhSMe: and (3) TIS: 70-97% of water: 10-1: 10-1: 10-1; or the volume ratio is TFA: EDT (electro-thermal transfer coating): water is 90-95: 1-5: 1-5; the volume of the cracking solvent is 5-25 times of the mass of the peptide resin.