CN114685614B

CN114685614B - Solid-phase synthesis method of atosiban

Info

Publication number: CN114685614B
Application number: CN202011609835.9A
Authority: CN
Inventors: 吴峰; 胡鹏; 刘自成; 李培祥; 钟祥龙; 王宏欣
Original assignee: Hubei Jianxiang Biopharmaceutical Co ltd
Current assignee: Hubei Jianxiang Biopharmaceutical Co ltd
Filing date: 2020-12-30
Publication date: 2024-07-12
Anticipated expiration: 2040-12-30

Abstract

The invention belongs to the field of solid-phase synthesis of polypeptides, and discloses a solid-phase synthesis method of atosiban, which comprises the following steps: synthesizing Fmoc-Gly-Sieber Resin by taking Sieber Resin as starting Resin; according to a solid phase synthesis method, sequentially coupling corresponding protected amino acids or fragments to prepare atosiban linear peptide resin, wherein Fmoc-Orn (Dde) -OH is adopted as the 8 th amino acid; and removing Dde protection of Fmoc-Orn (Dde) -OH by using hydrazine hydrate, and then directly carrying out solid-phase oxidation and cracking to obtain atosiban. The method solves the problem that ditBu impurities and dimer impurities are easy to generate after atosiban resin is cracked; the problem that the stability of the peptide is affected when the high-concentration acid is used for separating the peptide from the resin is avoided, and the method is simple and convenient to operate, easy to control the quality, small in environmental pollution and suitable for large-scale production.

Description

Solid-phase synthesis method of atosiban

Technical Field

The invention relates to the field of solid-phase synthesis of polypeptides, in particular to a solid-phase synthesis method of atosiban.

Background

Atosiban injection (Atosiban Acetate Injection) was developed by the company Hui Ling (Ferring AB), and was first marketed in Austria at 3/23/2000 under the trade name: Atosiban (Atosiban) can inhibit the binding of oxytocin and oxytocin receptors, thereby directly inhibiting the action of oxytocin on the uterus and inhibiting uterine contractions; can also inhibit hydrolysis of phosphatidylinositol, block generation of second messenger and activity of Ca ⁺, thereby indirectly inhibiting uterine response to oxytocin, and inhibit uterine contraction.

Atosiban is a nonapeptide, the peptide chain contains 3 unnatural amino acids D-Tyr (Et), mpa and Orn and a pair of disulfide bonds forming a ring between Mpa and Cys, and the molecular structural formula is: cyclo [ Mpa-D-Tyr (Et) -Ile-Thr-Asn-Cys ] -Pro-Orn-Gly-NH ₂

The Chinese patent with the publication number of CN101357937B adopts solid phase gradual coupling to obtain Mpa (SX) -D-Tyr (OEt) -Ile-Thr (tBu) -Asn (Trt) -Cys (Trt) -Pro-Orn (Boc) -Gly-RINK AMIDE AM RESIN, and peptide resin is oxidized after being cracked to obtain crude atosiban. In the method, as the tBu protecting group and the Boc protecting group containing the tBu source are used, tBu cations can be generated when the peptide resin is cracked, and the tBu cations which cannot be timely captured by the capturing agent can react with active groups on the peptide again when being cracked, so that the peptide with the protecting group is generated. both-SH and-OH are similar in nature and bind to the tBu cations, SH being more prone to capture the tBu cations than OH during cleavage, and after binding to the two-SH groups on the peptide, the tBu cations cannot be oxidized to become impurities. Therefore, the linear peptide obtained after the cracking is easy to generate impurities with sulfhydryl groups attacking tBu, and is difficult to be converted into products during oxidation, so that the quality and the yield of the products are reduced; the method uses ammonia water for oxidation, and the volume of waste liquid generated by oxidation is large, so that the environmental protection pressure is high. The Chinese patent with the publication number of CN101696236B adopts solid phase gradual coupling to obtain Mpa (Trt) -D-Tyr (Et) -Ile-Thr (tBu) -Asn (Trt) -Cys (Trt) -Pro-Orn (Boc) -Gly-RINK AMIDE AM RESIN, directly solid phase oxidation to generate disulfide bond, and then cracking to obtain atosiban. The method can separate the peptide from the resin only in a stronger acidic environment, and the reaction condition is not mild, which is not beneficial to the stabilization of the peptide; under the condition of high-concentration acid, the resin at the joint of the peptide and the resin is easy to fall off groups to generate impurities, and the peptide resin after solid-phase oxidation is easy to generate dimers during cleavage. The cracking reagents in the prior art comprise sulfur-containing trapping agents EDT or thioanisole, the trapping agents are strong in smell and are difficult to control in the workshop smell due to the large-scale production, and the pressure is formed for industrial mass production and environmental protection.

Therefore, it is highly desirable to invent a synthetic method of atosiban which can reduce the generation of the ditbu impurity and the dimer impurity, has mild cracking conditions, and can improve the product quality and yield.

Disclosure of Invention

Aiming at the problems of high impurity quantity, low crude product purity, low yield and high cost of crude peptide after cracking in the prior art, the invention provides a solid-phase synthesis method of atosiban. Meanwhile, because the Trt protecting group is more sensitive to acid and is easier to fall off, the invention adopts Sieber Resin sensitive to acid as the initial Resin, so that the cracking reagent is simple, the solvent DCM in the cracking reagent has low boiling point and is easy to remove, the subsequent treatment is convenient, the using amount of isopropyl ether is reduced, and the cost is saved.

The invention provides a solid-phase synthesis method of atosiban, which mainly comprises the following steps:

1) Coupling Fmoc-Gly-OH with acid-sensitive amino resin to obtain Fmoc-Gly-resin;

2) Sequentially coupling corresponding protective amino acids or fragments on Fmoc-Gly-resin according to the peptide sequence of atosiban to prepare atosiban linear peptide resin, wherein Fmoc-Orn (Dde) -OH is adopted as the 8 th amino acid;

3) Removing Dde protecting groups on Fmoc-Orn (Dde) -OH;

4) Solid-phase oxidation of the linear peptide resin to obtain atosiban resin;

5) And cracking the atosiban resin to obtain the atosiban.

In a preferred embodiment of the present invention, the acid-sensitive amino Resin in step 1) is selected from Sieber resins, and conventional resins which are acid-sensitive and which can simultaneously provide amino groups to the peptide sequence may be selected from Sieber resins.

Further preferably, the Sieber Resin substitution is 0.8 to 1.0mmol/g. The cost of substitution is increased too low, and the coupling difficulty is increased too high.

In a preferred embodiment of the invention, step 2) is specifically: and sequentially coupling Fmoc- Orn(Dde)-OH、Fmoc-Pro-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asn-OH、Fmoc-Thr-OH、Fmoc-Ile- OH、Fmoc-D-Tyr(Et)-OH、Mpa(Trt)-OH on Fmoc-Gly-resin to obtain atosiban linear peptide resin Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-resin.

In a preferred embodiment of the invention, the corresponding fragment in the atosiban sequence accessed in step 2) is selected from Mpa(Trt)-D-Tyr(Et)-OH、Fmoc-D-Tyr(Et)-Ile-OH、Fmoc-Asn-Cys(Trt)-OH、Fmoc-Cys(Trt)-Pro-OH、Fmoc-Pro-Orn(Dde)-OH.

In a preferred embodiment of the invention, the deprotection reagent used in step 3) is hydrazine hydrate, preferably at a concentration of 1 to 5%. Because hydrazine hydrate is strong in alkalinity, the concentration is too high, so that the peptide is unstable.

In a preferred embodiment of the invention, the oxidation method used in step 4 is selected from I ₂/DMF oxidation. Compared with other reagents, the industrial selection of I ₂ has the advantages of stable property, high oxidation speed, good effect, easy acquisition and price.

In a preferred embodiment of the invention, the cleavage reagent used in step 5 is TFA/DCM, and the cleavage reagent formulation is further preferably TFA: dcm=1: 99 to 20:80 (V: V). The low concentration of TFA can lead the Trt protecting group and Sieber resin to be separated, and simultaneously can ensure the stability of cyclized peptide chains.

According to the invention, acid-sensitive Sieber Resin is used as a starting Resin, fmoc-Orn (Dde) -OH is used for replacing Fmoc-Orn (Boc) -OH, and the atosiban crude peptide solid can be obtained under the conditions of low concentration acid and no sulfur-containing capture agent cracking, so that the stability of cyclized peptide chain is enhanced, and the residual DCM of a cracking reagent is easier to remove during purification, thereby saving the cost. Meanwhile, the problem of generating double tBu impurities is avoided, the total content of generated atosiban cis-dimer and trans-dimer impurities is far lower than 0.1%, and the risk of generating dimer impurities is reduced. The atosiban product obtained by the method has high quality and yield, and little three-waste discharge, thereby reducing the environmental protection pressure and increasing the economic benefit.

Drawings

FIG. 1 is a HPLC profile of a crude atosiban peptide obtained in example 12;

FIG. 2 is a HPLC profile of atosiban peptide obtained in example 16;

FIG. 3 is a HPLC profile of a crude atosiban peptide obtained in comparative example 1;

FIG. 4 is a peak mass spectrum of the double tBu impurity M+1 extracted in comparative example 1;

FIG. 5 is a HPLC profile of a crude atosiban peptide obtained using comparative example 2;

FIG. 6 is a graph of the peak mass spectrum of the trans-dimer 1/2M+1 extracted in comparative example 2;

FIG. 7 is a peak mass spectrum of cis-dimer 1/2M+1 extracted in comparative example 2.

Detailed Description

The following examples illustrate the invention in further detail, but are not intended to limit the invention. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of the present invention.

EXAMPLE 1 Synthesis of Fmoc-Gly-Sieber Resin with substitution degree of 0.76mmol/g

Sieber Resin (125 g,100mmol, substitution: 0.80 mmol/g) was weighed into a solid phase reaction synthesis column. 2000ml of DMF was added and the mixture was swelled for 30min and the DMF was removed by suction. The resin was washed with 3 x 2000ml DMF and the DMF was removed.

2000Ml of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, for 5min first and 15min second. After deprotection, the resin was washed 6 times with 2000ml DMF each time, a few resin was taken with a glass rod after the 4 th wash, and the ninhydrin detection resin was positive, indicating Fmoc was taken off.

89.2G Fmoc-Gly-OH and 48.6g HOBt are weighed, 1000ml DMF is added for dissolution, after complete dissolution, the solution is cooled to below 5 ℃, then 56.8g DIC (precooled to <0 ℃) is added, the mixture is activated for about 3 to 5 minutes in the solution, the activated mixture is controlled to be added into a reaction column, the reaction is carried out for 2 to 3 hours at 20 to 35 ℃, ninhydrin is detected to be negative, 2000ml DMF is added for resin washing for 6 times. After washing, the wash was removed and the resin was washed with 3 x 2000ml of CH ₂Cl₂ and CH ₂Cl₂ was removed. Taking out the Resin, and vacuum drying at 25-35 ℃ to obtain Fmoc-Gly-Sieber Resin, 132g of amino acid Resin is obtained, and the substitution degree is measured to be 0.76mmol/g.

EXAMPLE 2 Synthesis of Fmoc-Gly-Siber Resin with substitution degree of 0.85mmol/g

Siber Resin (111 g,100mmol, substitution: 0.90 mmol/g) was weighed into a solid phase reaction synthesis column. 2000ml of DMF was added and the mixture was swelled for 30min and the DMF was removed by suction. The resin was washed with 3 x 2000ml DMF and the DMF was removed.

2000Ml of DBLK solution (20% piperidine/DMF solution, V/V) was added for deprotection twice, 5min for the first time and 15min for the second time, the resin was washed with 2000ml DMF each time after deprotection, 6 times, a few resin was taken with a glass rod after the 4 th washing, and positive detection of ninhydrin indicated Fmoc was taken off.

89.2G Fmoc-Gly-OH and 48.6g HOBt are weighed, 1000ml DMF is added for dissolution, after complete dissolution, the solution is cooled to below 5 ℃, then 56.8g DIC (precooled to <0 ℃) is added, the mixture is activated for about 3 to 5 minutes in the solution, the activated mixture is controlled to be added into a reaction column, the reaction is carried out for 2 to 3 hours at 20 to 35 ℃, ninhydrin is detected to be negative, 2000ml DMF is added for resin washing for 6 times. After washing, the wash was removed and the resin was washed with 3 x 2000ml of CH ₂Cl₂ and CH ₂Cl₂ was removed. Taking out the Resin, and vacuum drying at 25-35 ℃ to obtain Fmoc-Gly-Siber Resin, 117g of amino acid Resin is obtained, and the substitution degree is measured to be 0.85mmol/g.

EXAMPLE 3 Synthesis of Fmoc-Gly-Sieber Resin with substitution degree of 0.94mmol/g

Sieber Resin (100 g,100mmol, substitution: 1.00 mmol/g) was weighed into a solid phase reaction synthesis column. 2000ml of DMF was added and the mixture was swelled for 30min and the DMF was removed by suction. The resin was washed with 3 x 2000ml DMF and the DMF was removed.

2000Ml of DBLK solution (30% piperidine/DMF solution, V/V) was added, the deprotection was performed twice, the first 5min and the second 15min, the resin was washed with 2000ml DMF each time after deprotection, 6 times, and after the 4 th washing, a few of the resin was taken with a glass rod, and the resin was checked positive with ninhydrin, indicating that Fmoc had been removed.

89.2G Fmoc-Gly-OH and 48.6g HOBt are weighed, 1000ml DMF is added for dissolution, after complete dissolution, the solution is cooled to below 5 ℃, then 56.8g DIC (precooled to <0 ℃) is added, the mixture is activated for about 3 to 5 minutes in the solution, the activated mixture is controlled to be added into a reaction column, the reaction is carried out for 2 to 3 hours at 20 to 35 ℃, ninhydrin is detected to be negative, 2000ml DMF is added for resin washing for 6 times. After washing, the wash was removed and the resin was washed with 3 x 2000ml of CH ₂Cl₂ and CH ₂Cl₂ was removed. Taking out the Resin, and vacuum drying at 25-35 ℃ to obtain Fmoc-Gly-Sieber Resin, 107g of amino acid Resin is obtained, and the substitution degree is measured to be 0.94mmol/g.

EXAMPLE 4 Synthesis of atosiban Linear peptide resin 1

Fmoc-Gly-Sieber Resin (13.2 g) prepared in example 1 was weighed into a solid phase reaction synthesis column. Repeating the peptide reaction step and removing Fmoc protecting group, and coupling Fmoc-Orn(Dde)- OH、Fmoc-Pro-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asn-OH、Fmoc-Thr-OH、Fmoc-Ile-OH、Fmoc-D-Tyr(Et)-OH、Mpa(Trt)-OH sequentially according to atosiban amino acid sequence to obtain Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-Sieber Resin.

EXAMPLE 5 Synthesis of atosiban Linear peptide resin 2

Mpa (Trt) -OH (69.69 g,200 mmol) and N-hydroxysuccinimide (23.00 g,200 mmol) were weighed into 800ml tetrahydrofuran and stirred at room temperature. 2, slowly adding a tetrahydrofuran (160 ml) solution of DCC (45.36 g,220 mmol) at a temperature of about 5 ℃ to stir for 2.5H at room temperature, filtering, concentrating, adding into petroleum ether to recrystallize and separate out solid, washing and drying, dissolving the obtained activated ester solid into 200ml of tetrahydrofuran, slowly dripping H-D-Tyr (Et) -OH (41.85 g,200 mmol) into 150ml of tetrahydrofuran, continuing to react at room temperature, monitoring the reaction of the raw materials to be complete, filtering, concentrating under reduced pressure, adding the concentrated solution into petroleum ether to separate out solid, washing the solid, drying again, recrystallizing and drying with isopropanol to obtain white solid Mpa (Trt) -D-Tyr (Et) -OH 77.98g, and obtaining the yield of 72 percent.

Fmoc-Gly-Sieber Resin (11.76 g) prepared in example 2 was weighed into a solid phase reaction synthesis column. Repeating the peptide reaction step and removing Fmoc protecting group, and coupling Fmoc-Orn(Dde)- OH、Fmoc-Pro-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asn-OH、Fmoc-Thr-OH、Fmoc-Ile-OH、Mpa(Trt)-D-Tyr(Et)-OH sequentially according to the atosiban amino acid sequence to obtain Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-Siber Resin.

EXAMPLE 6 Synthesis of atosiban Linear peptide resin 3

Fmoc-Cys (Trt) -OH (117.14 g,200 mmol) and N-hydroxysuccinimide (23.00 g,200 mmol) were weighed into 800ml of tetrahydrofuran and stirred at room temperature. 2, slowly adding a tetrahydrofuran (160 ml) solution of DCC (45.36 g,220 mmol) at the temperature of about 5 ℃ to stir for 2.5 hours at room temperature, filtering, concentrating, adding into petroleum ether to recrystallize and separate out solid, washing and drying, dissolving the obtained activated ester solid into 200ml of tetrahydrofuran, slowly dripping H-Pro-OH (23.03 g,200 mmol) into 150ml of tetrahydrofuran, continuing to react at the room temperature, monitoring the complete reaction of raw materials, filtering, concentrating under reduced pressure, adding concentrated solution into petroleum ether to separate out solid, washing the solid, drying again by isopropanol recrystallization and drying to obtain 102.43g of white solid, and obtaining 75% yield, and synthesizing Fmoc-Cys (Trt) -Pro-OH. Fmoc-D-Tyr (Et) -OH (86.30 g,200 mmol) was reacted with H-Ile-OH (23.03 g,200 mmol) in the same manner and recrystallized from isopropanol to dryness to give 78.37g of Fmoc-D-Tyr (Et) -Ile-OH as a white solid in 73% yield.

Fmoc-Gly-Siber Resin (10.64 g) prepared in example 3 was weighed into a solid phase reaction synthesis column. Repeating the peptide reaction step and removing Fmoc protecting group, and coupling Fmoc-Orn (Dde) -OH, fmoc-Cys (Trt) -Pro-OH, fmoc-Asn-OH, fmoc-Thr-OH, fmoc-D-Tyr (Et) -Ile-OH and Mpa (Trt) -OH according to the atosiban amino acid sequence to obtain Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-Siber Resin.

EXAMPLE 7 Synthesis of atosiban Linear peptide resin 4

Fmoc-Pro-OH (67.47 g,200 mmol) and N-hydroxysuccinimide (23.00 g,200 mmol) were weighed into 800ml of tetrahydrofuran and stirred at room temperature. 2, slowly adding a tetrahydrofuran (160 ml) solution of DCC (45.36 g,220 mmol) at the temperature of about 5 ℃ to stir for 2.5 hours at room temperature, filtering, concentrating, adding into petroleum ether to recrystallize and separate out solid, washing and drying, dissolving the obtained activated ester solid into 200ml of tetrahydrofuran, slowly dripping H-Orn (Dde) -OH (59.32 g,200 mmol) into 150ml of tetrahydrofuran, continuing to react at room temperature, monitoring the reaction of raw materials to be complete, filtering, concentrating under reduced pressure, adding concentrated solution into petroleum ether to separate out solid, washing the solid, drying again, recrystallizing and drying with isopropanol to obtain white solid Fmoc-Pro-Orn (Dde) -OH 87.47g, and obtaining the yield 71 percent. Fmoc-Asn-OH (70.87 g,200 mmol) was reacted with H-Cys (Trt) -OH (72.69 g,200 mmol) by the same method, and recrystallized from isopropanol and dried to give Fmoc-Asn-Cys (Trt) -OH as a white solid 70.88g in 69% yield.

Fmoc-Gly-Siber Resin (10.64 g) prepared in example 3 was weighed into a solid phase reaction synthesis column. Repeating the peptide reaction step and removing Fmoc protecting group, and coupling Fmoc-Pro-Orn (Dde) -OH, fmoc-Asn-Cys (Trt) -OH, fmoc-Thr-OH, fmoc-Ile-OH, fmoc-D-Tyr (Et) -OH and Mpa (Trt) -OH according to the atosiban amino acid sequence to obtain Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-Siber Resin.

Example 8 Synthesis of atosiban resin 1

To the Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-Sieber Resin prepared in example 4 was added 200ml of a 1% hydrazine hydrate/DMF solution to wash 2 times, 3 min/time, the reaction solution was withdrawn, 200ml DMF wash Resin was added, and the Resin was washed 6 times. After washing, the washing solution was removed to remove Dde protecting group on Fmoc-Orn (Dde) -OH.

To the resulting Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn-Gly-Sieber Resin was added I ₂/DMF at 1g/10ml, iodine at 2 times the amino acid equivalent for 2 hours, the reaction solution was withdrawn, 200ml DMF was added to wash the Resin, and 6 washes were performed. After washing, the washing liquid was pumped out.

The resin was then washed 4 times with 200ml each time of DCM, the DCM was pumped off 5 min/time and the resin was vacuum dried at 20-35℃until it became a quicksand. The peptide resin was 21.46g after drying, and the weight gain of the resin was 88%.

EXAMPLE 9 Synthesis of atosiban resin 2

To the Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-Sieber Resin prepared in example 5 was added 200ml of a 5% hydrazine hydrate/DMF solution to wash 2 times, 3 min/time, the reaction solution was withdrawn, 200ml DMF wash Resin was added, and washing was performed 6 times. After washing, the washing solution was removed to remove Dde protecting group on Fmoc-Orn (Dde) -OH.

The resin was then washed 4 times with 200ml each time of DCM, the DCM was pumped off 5 min/time and the resin was vacuum dried at 20-35℃until it became a quicksand. The peptide resin was 19.20g after drying, and the weight gain of the resin was 90%.

EXAMPLE 10 Synthesis of atosiban resin 3

To the Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-Sieber Resin prepared in example 6 was added 200ml of 3% hydrazine hydrate/DMF solution to wash 2 times, 3 min/time, the reaction solution was withdrawn, 200ml DMF wash Resin was added, and washing was performed 6 times. After washing, the washing solution was removed to remove Dde protecting group on Fmoc-Orn (Dde) -OH.

The resin was then washed 4 times with 200ml each time of DCM, the DCM was pumped off 5 min/time and the resin was vacuum dried at 20-35℃until it became a quicksand. The peptide resin was 18.99g after drying, and the weight gain of the resin was 89%.

EXAMPLE 11 Synthesis of atosiban resin 4

To the Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-Sieber Resin prepared in example 7 was added 200ml of a 4% hydrazine hydrate/DMF solution to wash 2 times, 3 min/time, the reaction solution was withdrawn, 200ml DMF wash Resin was added, and the Resin was washed 6 times. After washing, the washing solution was removed to remove Dde protecting group on Fmoc-Orn (Dde) -OH.

EXAMPLE 12 Synthesis of crude atosiban peptide 1

214.6Ml of 1% TFA/DCM (V/V) lysate was prepared, cooled to 5-10 ℃, atosiban resin obtained in example 8 was added to the lysate, reacted for 5 hours at room temperature (20-35 ℃), filtered, washed 2 times with DCM, 25 ml/time, combined into filtrate, the filtrate was dried by spin-drying to obtain a solid. Drying under reduced pressure at 20-35 ℃ until the weight is constant at 9.20g, and obtaining 83 percent of yield. Thus obtaining the atosiban crude peptide. Through detection, the purity of the crude peptide reaches 92.75%, the content of the impurities of the double tBu is 0%, the content of the impurities of the cis-dimer and the trans-dimer of atosiban is lower than 0.1%, and the HPLC spectrogram of the obtained atosiban crude peptide is shown in figure 1.

EXAMPLE 13 Synthesis of crude atosiban peptide 2

192Ml of 20% TFA/DCM (V/V) lysate was prepared, cooled to 5-10 ℃, the atosiban resin obtained in example 9 was added to the lysate, reacted for 2 hours at room temperature (20-35 ℃), filtered, washed 2 times with DCM, 25 ml/time, combined into filtrate, the filtrate was dried by spin-drying, and the solid obtained after drying was dried under reduced pressure to constant weight 9.31g at 20-35 ℃ in 84% yield. Thus obtaining the atosiban crude peptide. Through detection, the purity of the crude peptide reaches 93.13%, the content of the double tBu impurities is 0%, the content of the impurities of the cis-dimer and the trans-dimer of atosiban is lower than 0.1%, and the obtained HPLC spectrogram of the crude peptide of atosiban is similar to that of FIG. 1.

EXAMPLE 14 Synthesis of crude atosiban peptide 3

Preparing 189.9ml of 5% TFA/DCM (V/V) lysate, cooling to 5-10 ℃, adding the atosiban resin obtained in example 10 into the lysate, reacting for 3 hours at room temperature (20-35 ℃), filtering, washing 2 times with DCM, 25 ml/time, merging into filtrate, spin-drying the filtrate, drying to obtain solid, decompressing and drying to constant weight 9.31g at 20-35 ℃, and obtaining the atosiban crude peptide with the yield of 84%. Through detection, the purity of the crude peptide reaches 93.40%, the content of the double tBu impurities is 0%, the content of the impurities of the cis-dimer and the trans-dimer of atosiban is lower than 0.1%, and the obtained HPLC spectrogram of the crude peptide of atosiban is similar to that of FIG. 1.

EXAMPLE 15 Synthesis of crude atosiban peptide 4

Preparing 189.9ml of 10% TFA/DCM (V/V) lysate, cooling to 5-10 ℃, adding the atosiban resin obtained in example 11 into the lysate, reacting for 3h at room temperature (20-35 ℃), filtering, washing 2 times with DCM, 25 ml/time, merging into filtrate, spin-drying the filtrate, drying to obtain solid, drying under reduced pressure at 20-35 ℃ until the constant weight is 9.20g, and obtaining 83% yield, thus obtaining the atosiban crude peptide. Through detection, the purity of the crude peptide reaches 93.38%, the content of the double tBu impurities is 0%, the content of the impurities of the cis-dimer and the trans-dimer of atosiban is lower than 0.1%, and the obtained HPLC spectrogram of the crude peptide of atosiban is similar to that of FIG. 1.

EXAMPLE 16 purification of crude atosiban peptide 1

The crude atosiban peptide of example 12 was dissolved in 15% acetonitrile in water and filtered, purified by preparative reverse phase HPLC (C18 column), converted to salt, and the fraction was collected to more than 99%, concentrated and lyophilized to give 6.96g, yield 66%, purity 99% and the resulting atosiban peptide HPLC profile as shown in fig. 2.

EXAMPLE 17 purification of crude atosiban peptide 2

The crude atosiban peptide of example 13 was purified by preparative reverse phase HPLC (C18 column), trans-salified, and the fraction was collected to more than 99% after dissolution and filtration with 15% acetonitrile in water, concentrated and lyophilized to give 6.85g, 65% yield, 99% purity, and the resulting atosiban peptide HPLC profile was similar to fig. 2.

EXAMPLE 18 purification of crude atosiban peptide 3

The crude atosiban peptide of example 14 was purified by preparative reverse phase HPLC (C18 column), trans-salified, and the fraction was collected to more than 99% after dissolution and filtration with 15% acetonitrile in water, concentrated and lyophilized to give 6.96g, yield 66%, purity 99% and the resulting atosiban peptide HPLC profile was similar to fig. 2.

EXAMPLE 19 purification of crude atosiban peptide 4

The crude atosiban peptide of example 15 was filtered by dissolution in 15% acetonitrile in water, purified by preparative reverse phase HPLC (C18 column), converted to salt, and the fraction was collected to more than 99%, concentrated and lyophilized to give 6.75g, 64% yield, 99% purity, and the resulting atosiban peptide HPLC profile was similar to fig. 2.

Comparative example 1 liquid phase Synthesis of atosiban crude peptide Using amino acid with tBu Source protecting group

RINK AMIDE AM resin is used as a starting material, fmoc-protected amino acid is used as a carrier, TBTU, HBTU and NMM are used as condensing agents and alkali according to a solid phase coupling method, Fmoc-Gly-OH、Fmoc- Orn(Boc)-OH、Fmoc-Pro-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、 Fmoc-Ile-OH、Fmoc-D-Tyr(Et)-OH and Mpa (Trt) -OH are sequentially coupled according to an atosiban peptide sequence, and the fully-protected reduced atosiban peptide resin is obtained. The cracking reagent adopts TFA/HBr/HAc/Tis/EDT=8.70/0.35/0.15/0.55/0.25, the dosage of the cracking reagent is 15ml/g based on the weight of the fully protected reduced atosiban Ban Tai resin, after cracking, isopropyl ether is used for sedimentation, the sedimentation volume is 10 times of that of the cracking reagent, and the crude product of reduced atosiban is obtained after centrifugal washing and drying. Dissolving reduced atosiban Ban Cupin in purified water at a concentration of 1mmol/L, adjusting pH to about 8 with 1mmol/L ammonia water, stirring overnight, adding acetic acid to adjust pH to acidity, and stopping reaction to obtain atosiban crude peptide, wherein HPLC chart is shown in figure 3. Wherein rt= 13.101min is a bis-tBu impurity, and fig. 4 is a peak mass spectrum of the extracted bis-tBu impurity m+1, analyzed as impurity Mpa (tBu) -D-Tyr (Et) -lie-Thr-Asn-Cys (tBu) -Pro-Orn-Gly-NH ₂.

Comparative example 2 solid phase Synthesis of atosiban crude peptide Using amino acid with tBu Source protecting group

Taking RINK AMIDE AM resin as a carrier, taking 30% piperidine/DMF solution as a deprotection reagent, and removing Fmoc-protection to obtain H ₂ N-RINK AMIDE resin. And (3) taking HOBt and DIC as condensing agents, and connecting carboxyl of Fmoc-Gly-OH with amino of the resin to obtain Fmoc-Gly (resin). Adopting Fmoc strategy, and sequentially carrying out solid phase coupling on the sequence Fmoc-Orn(Boc)-OH、Fmoc- Pro-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ile-OH、Fmoc- D-Tyr(Et)-OH and Mpa (Trt) -OH to obtain the full-protection reduced atosiban peptide resin of 9 peptides. Solid-phase cyclization was carried out with iodine in DMF (1 mmol/10ml, 1.5 eq. Used) to give oxidized atosiban peptide resin. Cleavage with a cleavage reagent (TFA/thioanisole/1, 2-ethanethiol/water=90/5/3/2), 10ml/g of the cleavage reagent was used based on the weight of the oxidized atosiban Ban Tai resin, a small amount of TFA was washed and combined into the cleavage solution, and the cleavage solution was added to isopropyl ether of 10 times the volume of the cleavage solution for sedimentation, and the washing and drying by centrifugation gave a crude atosiban peptide, the HPLC profile of which is shown in fig. 5. Wherein at rt= 10.937min and 11.442min are two dimer impurities, fig. 6 is a 1/2m+1 peak mass spectrum of the extracted trans-dimer, analyzed as impuritiesFIG. 7 is a 1/2M+1 peak mass spectrum of the extracted cis dimer, analyzed as impuritiesThe impurity content was large, 6.57% and 6.79%, respectively, and the total of the two dimer impurities was 13.36%.

Claims

1. The solid phase synthesis method of atosiban is characterized by mainly comprising the following steps:

1) Coupling the Fmoc-Gly-OH with acid-sensitive amino Resin Sieber Resin to obtain Fmoc-Gly-OH Resin;

2) Sequentially coupling corresponding protected amino acids or fragments on Fmoc-Gly-resin according to the peptide sequence of atosiban to prepare the atosiban linear peptide resin

Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (Dde) -Gly-resin, wherein Fmoc-Orn (Dde) -OH is adopted as amino acid at 8 th position;

Wherein the protective amino acid which is coupled in turn is specifically: fmoc-Orn (Dde) -OH, fmoc-Pro-OH, fmoc-Cys (Trt) -OH, fmoc-Asn-OH, fmoc-Thr-OH, fmoc-Ile-OH,

Fmoc-D-Tyr(Et)-OH、Mpa(Trt)-OH；

Wherein the corresponding fragments in the sequence of atosiban which are coupled in sequence are selected from

Mpa(Trt)-D-Tyr(Et)-OH、Fmoc-D-Tyr(Et)-Ile-OH、Fmoc-Asn-Cys(Trt)-OH、Fmoc-Cys(Trt)-Pro-OH、Fmoc-Pro-Orn(Dde)-OH;

3) Removing Dde protecting groups on Fmoc-Orn (Dde) -OH;

4) Solid-phase oxidation of the linear peptide resin to obtain atosiban resin;

5) And cracking the atosiban resin to obtain the atosiban.

2. The method according to claim 1, wherein the resin substitution degree in the step 1) is 0.8 to 1.0mmol/g.

3. The method according to claim 1, wherein the deprotection reagent used in step 3) is hydrazine hydrate.

4. A method according to claim 3, characterized in that the hydrazine hydrate concentration is 1-5%.

5. The method according to claim 1, wherein the oxidation method used in step 4) is selected from the group consisting of I ₂/DMF oxidation.

6. The method according to claim 1, wherein the cleavage reagent used in step 5) is TFA/DCM in a volume ratio of TFA: dcm=1: 99 to 20:80.