CN115038711B - Synthesis method of atosiban - Google Patents
Synthesis method of atosiban Download PDFInfo
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- CN115038711B CN115038711B CN202180001434.1A CN202180001434A CN115038711B CN 115038711 B CN115038711 B CN 115038711B CN 202180001434 A CN202180001434 A CN 202180001434A CN 115038711 B CN115038711 B CN 115038711B
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Abstract
The invention belongs to the field of polypeptide synthesis, and discloses a synthesis method of atosiban, which comprises the following steps: synthesis of Fmoc-Pro-Orn-Gly-NH 2 Tripeptide, fmoc-Pro-Orn (trityl resin) -Gly-NH is prepared by taking trityl resin as starting resin 2 The method comprises the steps of carrying out a first treatment on the surface of the Sequentially accessing corresponding protective amino acid or fragment in the atosiban sequence by adopting a condensing agent to prepare atosiban linear peptide resin; and obtaining atosiban through pyrolysis and oxidation. The method effectively reduces the generation of tBu impurities and the problem of Pro missing peptide impurities, is simple and convenient to operate, has easily controlled quality, and is favorable for purifying and improving the product quality.
Description
Technical Field
The invention relates to the field of polypeptide synthesis, in particular to a synthesis method of atosiban.
Background
Atosiban injection (AtosibanAcetate Injection) is first marketed in australia at 3/23/2000 under the trade name:
atosiban (Atosiban), a novel anti-premature drug developed by Ferring Gmbh, is an oxytocin analog, is a competitive antagonist of oxytocin receptors in utero and on decidua and fetal membranes, and is a european doctorFirst line medication recommended by the society; it can inhibit the combination of oxytocin and oxytocin receptor, thereby directly inhibiting oxytocin from acting on uterus and inhibiting uterine contraction; can also inhibit hydrolysis of phosphatidylinositol.
Atosiban is a cyclic nonapeptide of formula C 43 H 67 N 11 O 12 S 2 The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight 994.19; CAS registry number 90779-69-4; the peptide sequence is shown in the following formula:
Cyclo[Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH 2
in Chinese patent publication No. CN101314613B and CN101696236B, atosiban is subjected to solid-phase stepwise coupling by using Rink Amide AM Resin Resin to obtain Mpa (Trt) -D-Tyr (Et) -Ile-Thr (tBu) -Asn (Trt) -Cys (Trt) -Pro-Orn (Boc) -Gly-Resin, directly performing solid-phase oxidation to generate disulfide bond, and then performing cleavage to obtain atosiban. Rink Amide AM Resin resin adopted in the prior art can be cracked under a strong acid environment, which is unfavorable for product stability and has high operation danger; both Mpr and Cys have sulfhydryl, the sulfhydryl has the capability of capturing tBu to generate double tBu impurities, and when the peptide resin subjected to solid phase oxidation is subjected to cracking to remove protective groups and resin, the requirement on the capturing agent is high due to the existence of the tBu or the Boc protective groups of the tBu source, the product quality control is not facilitated, and the product yield is reduced.
Chinese patent publication No. CN105408344B discloses a method for preparing Fmoc-Orn-Gly-NH 2 Method for the initial synthesis of atosiban, wherein Fmoc-Orn-Gly-NH 2 The ornithine side chain is connected to the trityl resin, so that impurities can be effectively controlled. However, the dipeptide is coupled with trityl resin, and the resin grafted on the dipeptide Orn side chain leads to the increase of steric hindrance of the subsequent Pro coupling, and the coupling time is prolonged, so that the missing peptide impurities are easy to cause.
Therefore, a synthetic method of atosiban, which is convenient for coupling and cracking, is favorable for stable product quality, convenient and safe operation, controllable product quality and improved product yield, needs to be found.
Disclosure of Invention
Aiming at the problems of the prior art that the impurity is captured by an isomer, an insertion peptide and tBu cations, and the Pro lacks the peptide impurity due to steric hindrance and overlong reaction time, the invention provides a synthesis method of atosiban, which comprises the following steps:
1) Synthesizing Fmoc-Pro-Orn-Gly-NH 2 Tripeptides, then reacted with resin to give Fmoc-Pro-Orn (resin) -Gly-NH 2 A peptide resin;
2) Removal of Fmoc-Pro-Orn (resin) -Gly-NH 2 Sequentially accessing corresponding protected amino acid or fragment in the atosiban sequence by adopting a condensing agent to prepare the atosiban linear peptide resin;
3) Cracking and oxidizing to obtain atosiban.
In a preferred embodiment of the invention, the resin in step 1) is a polymeric resin capable of binding to the pendant amino functions of Orn. Further preferably, the polymer resin is selected from trityl type resins. Wherein the trityl resin is specifically trityl resin, 2-chloro-trityl resin, 4-methyl-trityl resin, 4-methoxy-trityl resin.
The process involves both the ability to couple tripeptides to resins and the easier cleavage of peptides. The trityl resin is resin containing triphenylmethyl structure, is easy to react with amino, can be cracked under mild conditions, is easy to remove and meets the conditions. Tripeptides may be coupled to triphenylmethyl groups using trityl-type resins.
In a preferred embodiment of the invention, the degree of substitution of the resin in step 1) is from 0.8 to 1.0mmol/g, the substitution being increased at too low a cost and the substitution being too difficult to couple.
In a preferred embodiment of the invention, the tripeptide of step 1) is reacted with a resin under DIEA to form a tripeptide resin. DIEA as a base helps to drive the overall reaction.
In a preferred embodiment of the invention, step 2) is specifically: in Fmoc-Pro-Orn (resin) -Gly-NH 2 Fmoc-Cys (Trt) -OH, fmoc-Asn-OH, fmoc-Thr-OH, fmoc-Ile-OH, fmoc-D-Tyr (Et) -OH, mpa (Trt) -OH were coupled sequentially to give Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (trityl resin) -Gly-NH 2 。
In a preferred embodiment of the invention, the corresponding fragment in the atosiban sequence accessed in step 2) is selected from the group consisting of Mpa (Trt) -D-Tyr (Et) -OH, fmoc-D-Tyr (Et) -lie-OH, fmoc-Asn-Cys (Trt) -OH.
In a preferred embodiment of the present invention, the condensing agent in step 2) is selected from any one of HOBt/DIC, HCTU/DIEA, HBTU/DIEA, HATU/HOAt/DIEA, HBTU/HOBt/DIEA. More preferably, the condensing agent is HOBt/DIC.
In a preferred embodiment of the invention, step 3) the cleavage reagent is selected from TFA/DCM, and the cleavage reagent ratio is further preferably TFA: dcm=1: 99 to 20:80. the low concentration of TFA allows separation of the peptide from the resin, ensures stability of the cyclized peptide chain, and eliminates the need for high concentrations of acid for Trt cleavage.
In a preferred embodiment of the invention, the oxidizing agent in step 3) is selected from iodine, hydrogen peroxide.
The invention synthesizes Fmoc-Pro-Orn-Gly-NH 2 The tripeptide is coupled with resin, so that the problem of Pro missing peptide impurities caused by steric hindrance and prolonged reaction time can be solved; with trityl resin as the starting resin, fmoc-Pro-Orn (trityl resin) -Gly-NH 2 As an intermediate, the active group-NH of Orn in tripeptide 2 The method has the advantages that one Boc protecting group is reduced when the method is hung on resin for coupling, and Fmoc-Thr-OH is adopted, so that the generation of tBu impurities is reduced, the purity of a main peak is improved, and the yield is increased; the acid-sensitive resin is used for cutting peptide under weak acid condition, and the protecting group can be completely removed by only one time of cleavage, so that the operation is convenient and safe. The atosiban product obtained by the method has high quality controllable yield, is beneficial to purifying and improving the product quality, is safe and convenient to operate, simple in post-treatment, recoverable in solvent, reduces the production cost, and is suitable for industrial production.
Drawings
FIG. 1 is a HPLC chart of a crude atosiban peptide prepared in example 10.
FIG. 2 is a HPLC profile of atosiban peptide obtained in example 14.
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 Fmoc-Pro-Orn-Gly-NH 2 Synthesis of tripeptides
Fmoc-Pro-OH (134.94 g,400 mmol) and N-hydroxysuccinimide (46.00 g,400 mmol) were weighed into 1600ml of tetrahydrofuran and stirred at room temperature. Slowly adding DCC (90.72 g,440 mmol) tetrahydrofuran (320 ml) solution at about 5deg.C, stirring at room temperature for 2.5 hr, filtering, concentrating, adding into petroleum ether, recrystallizing to obtain solid, washing, drying, dissolving the obtained activated ester solid in tetrahydrofuran 400ml, dissolving H-Orn (Boc) -NH 2 (92.92 g,400 mmol) dissolved in tetrahydrofuran 300ml was slowly added dropwise to the above solution, the reaction was continued at room temperature, the completion of the reaction of the starting materials was monitored, filtered, the filtrate was concentrated to dryness under reduced pressure, N-hydroxysuccinimide (46.00 g,400 mmol) and 1600ml of tetrahydrofuran were added for dissolution, and stirring was carried out at room temperature. Slowly adding DCC (90.72 g,440 mmol) tetrahydrofuran (320 ml) solution at about 5deg.C, stirring at room temperature for 2.5 hr, filtering, concentrating, adding into petroleum ether, recrystallizing to obtain solid, washing, drying, dissolving the obtained activated ester solid in tetrahydrofuran 400ml, dissolving H-Gly-NH 2 (29.64 g,400 mmol) dissolved in tetrahydrofuran 300mL was slowly added dropwise to the above solution, the reaction was continued at room temperature, the starting material was monitored for completion, filtered, the filtrate was concentrated to dryness under reduced pressure, 1000mL of 5% TFA/DCM solution was added to the reaction solution, the reaction was continued for 1 hour, and concentrated to dryness to give a yellow oil, which was recrystallized from isopropanol to give 171.56g of a white solid in 69% yield.
EXAMPLE 2 Fmoc-Pro-Orn (trityl resin) -Gly-NH having a degree of substitution of 0.42mmol/g 2 Synthesis of peptide resins
Trityl resin (37.5 g,30mmol, substitution: 0.80 mmol/g) was weighed into a solid phase reaction synthesis column. Swelling with addition of 400mL of dried DMFThe DMF was removed by pumping for 30 min. The resin was washed with 3 x 400ml dry DMF and the DMF was removed. Fmoc-Pro-Orn-Gly-NH prepared in example 1 was added 2 (37.30 g,60 mmol), DIEA (11.63 g,90 mmol), was dissolved and clarified in 100mL dry DMF, reacted in resin for 2h, methanol (9.61 g,300 mmol) was added for 20min, drained and dried with 3X 400mL CH 2 Cl 2 Washing the resin and pumping out CH 2 Cl 2 . Taking out the resin, and vacuum drying at 25-35 ℃ to obtain Fmoc-Pro-Orn (trityl resin) -Gly-NH 2 52.14g of resin, and a substitution degree of 0.42mmol/g was measured.
EXAMPLE 3 Fmoc-Pro-Orn (2-CTC Resin) -Gly-NH with degree of substitution of 0.50mmol/g 2 Synthesis of peptide resins
2-CTC Resin (30.0 g,30mmol, substitution: 1.00 mmol/g) was weighed into a solid phase reaction synthesis column. 400mL of dry DMF was added and the mixture was swelled for 30min, and the DMF was removed by suction. The resin was washed with 3 x 400ml dry DMF and the DMF was removed. Fmoc-Pro-Orn-Gly-NH prepared in example 1 was added 2 (37.30 g,60 mmol), DIEA (11.63 g,90 mmol), was dissolved and clarified in 100mL dry DMF, reacted in resin for 2h, methanol (9.61 g,300 mmol) was added for 20min, drained and dried with 3X 400mL CH 2 Cl 2 Washing the resin and pumping out CH 2 Cl 2 . Taking out the Resin, and vacuum drying at 25-35 ℃ to obtain Fmoc-Pro-Orn (2-CTC Resin) -Gly-NH 2 43.80g of the resin was found to have a substitution of 0.50mmol/g.
EXAMPLE 4 Fmoc-Pro-Orn (4-methyl-trityl resin) -Gly-NH having a degree of substitution of 0.50mmol/g 2 Synthesis of peptide resins
4-methyl-trityl resin (33.33 g,30mmol, substitution: 0.90 mmol/g) was weighed into a solid phase reaction synthesis column. 400mL of dry DMF was added and the mixture was swelled for 30min, and the DMF was removed by suction. The resin was washed with 3 x 400ml dry DMF and the DMF was removed. Fmoc-Pro-Orn-Gly-NH prepared in example 1 was added 2 (37.30 g,60 mmol), DIEA (11.63 g,90 mmol), was dissolved and clarified in 100mL dry DMF, reacted in resin for 2h, methanol (9.61 g,300 mmol) was added for 20min, drained and dried with 3X 400mL CH 2 Cl 2 Washing the resin and pumping out CH 2 Cl 2 . Taking out the resin at 25-35 DEG CVacuum drying to obtain Fmoc-Pro-Orn (4-methyl-trityl resin) -Gly-NH 2 43.89g of resin, and the degree of substitution was measured to be 0.50mmol/g.
EXAMPLE 5 Fmoc-Pro-Orn (4-methoxy-trityl resin) -Gly-NH having a degree of substitution of 0.50mmol/g 2 Synthesis of peptide resins
4-methoxy-trityl resin (30.0 g,30mmol, substitution: 1.00 mmol/g) was weighed into a solid phase reaction synthesis column. 400mL of dry DMF was added and the mixture was swelled for 30min, and the DMF was removed by suction. The resin was washed with 3 x 400ml dry DMF and the DMF was removed. Fmoc-Pro-Orn-Gly-NH prepared in example 1 was added 2 (37.30 g,60 mmol), DIEA (11.63 g,90 mmol), was dissolved and clarified in 100mL dry DMF, reacted in resin for 2h, methanol (9.61 g,300 mmol) was added for 20min, drained and dried with 3X 400mL CH 2 Cl 2 Washing the resin and pumping out CH 2 Cl 2 . Taking out the resin, and vacuum drying at 25-35 ℃ to obtain Fmoc-Pro-Orn (4-methoxy-trityl resin) -Gly-NH 2 43.69g of resin, and the degree of substitution was measured to be 0.50mmol/g.
EXAMPLE 6 Synthesis of atosiban Linear peptide resin 1
Fmoc-Pro-Orn (trityl resin) -Gly-NH prepared in example 2 was weighed 2 (35.71 g) in a solid phase reaction synthesis column. 400mL of DMF was added and the mixture was swelled for 30min, and the DMF was removed by pumping. The resin was washed with 3 x 200ml dry DMF and the DMF was removed. 200mL 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 200mL DMF each time, a few resin was taken with a glass rod after the 4 th washing, and the ninhydrin test was positive, indicating Fmoc was taken off.
17.57g Fmoc-Cys (Trt) -OH and 4.86g HOBt were weighed, dissolved in 100mL DMF, after complete dissolution the solution was cooled to below 5℃and then 5.68g DIC was added (precooled to<0 ℃ and activating for about 3-5 min in the solution, adding the activated solution into a reaction column, reacting for 2-3 h at 20-35 ℃, detecting that ninhydrin is negative, pumping out the reaction solution, adding 200mL of DMF to wash the resin, and washing for 6 times. After washing, the washing solution was removed to obtain Fmoc-Cys (Trt) -Pro-Orn (trityl resin) -Gly-NH 2 。
Repeating the peptide reaction step and removing Fmoc protecting group, and performing Fmoc-Cys (Trt) -Pro-Orn (trityl resin) -Gly-NH according to the amino acid sequence of atosiban 2 Fmoc-Asn-OH, fmoc-Thr-OH, fmoc-Ile-OH, fmoc-D-Tyr (Et) -OH, mpa (Trt) -OH were coupled sequentially to give Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (trityl resin) -Gly-NH 2 . After DMF washing, the washing liquid was removed. 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 dried under vacuum at room temperature (20-35 ℃) until it had a quicksand shape. The peptide resin was 48.72g after drying, and the weight gain of the resin was 89.0%.
EXAMPLE 7 Synthesis of atosiban Linear peptide resin 2
Fmoc-Pro-Orn (2-CTC Resin) -Gly-NH prepared in example 3 was weighed 2 (30.00 g) in a solid phase reaction synthesis column. 400mL of DMF was added and the mixture was swelled for 30min, and the DMF was removed by pumping. The resin was washed with 3 x 200ml dry DMF and the DMF was removed. 200mL 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 200mL DMF each time, a few resin was taken with a glass rod after the 4 th washing, and the ninhydrin test was positive, indicating Fmoc was taken off.
17.57g Fmoc-Cys (Trt) -OH and 13.65g HBTU were weighed out and dissolved in 100mL DMF, after complete dissolution the solution was cooled to below 5℃and then 5.82g DIEA was added (pre-chilled to<0 ℃ and activating for about 3-5 min in the solution, adding the activated solution into a reaction column, reacting for 2-3 h at 20-35 ℃, detecting that ninhydrin is negative, pumping out the reaction solution, adding 200mL of DMF to wash the resin, and washing for 6 times. After washing, the washing solution was removed to obtain Fmoc-Cys (Trt) -Pro-Orn (2-CTC Resin) -Gly-NH 2 。
Fmoc-D-Tyr (Et) -OH (86.30 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 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-Ile-OH (26.24 g,200 mmol) into 150ml of tetrahydrofuran, continuing to react at room temperature, monitoring that the raw materials react completely, filtering, concentrating under reduced pressure, adding the concentrated solution into petroleum ether to separate out solid, washing the solid, then drying again, recrystallizing and drying with isopropanol to obtain Fmoc-D-Tyr (Et) -Ile-OH 75.60g, and obtaining the yield of 75%.
Repeating the peptide reaction step and removing Fmoc protecting group, and performing Fmoc-Cys (Trt) -Pro-Orn (2-CTC Resin) -Gly-NH according to the amino acid sequence of atosiban 2 Fmoc-Asn-OH, fmoc-Thr-OH, fmoc-D-Tyr (Et) -Ile-OH, and Mpa (Trt) -OH were coupled sequentially to give Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (2-CTC Resin) -Gly-NH 2 . After DMF washing, the washing liquid was removed. 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 dried under vacuum at room temperature (20-35 ℃) until it had a quicksand shape. The peptide resin was 42.77g after drying, and the weight gain of the resin was 87.4%.
EXAMPLE 8 Synthesis of atosiban Linear peptide resin 3
Fmoc-Pro-Orn (4-methyl-trityl resin) -Gly-NH prepared in example 4 was weighed 2 (30.00 g) in a solid phase reaction synthesis column. 400mL of DMF was added and the mixture was swelled for 30min, and the DMF was removed by pumping. The resin was washed with 3 x 200ml dry DMF and the DMF was removed. 200mL 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 200mL DMF each time, a few resin was taken with a glass rod after the 4 th washing, and the ninhydrin test was positive, indicating Fmoc was taken off.
17.57g Fmoc-Cys (Trt) -OH, 13.65g HBTU and 4.05g HOBt were weighed and dissolved in 100mL DMF and after complete dissolution the solution was cooled to below 5℃and then 5.82g DIEA (pre-chilled to<0 ℃ and activating for about 3-5 min in the solution, adding the activated solution into a reaction column, reacting for 2-3 h at 20-35 ℃, detecting that ninhydrin is negative, pumping out the reaction solution, adding 200mL of DMF to wash the resin, and washing for 6 times. After washing, the washing solution was removed to give Fmoc-Cys (Trt) -Pro-Orn (4-methyl-trityl resin) -Gly-NH 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 Mpa (Trt) -D-Tyr (Et) -OH 77.98g, and obtaining the yield of 72%.
Repeating the peptide reaction step and removing Fmoc protecting group, and performing Fmoc-Cys (Trt) -Pro-Orn (4-methyl-trityl resin) -Gly-NH according to the amino acid sequence of atosiban 2 Fmoc-Asn-OH, fmoc-Thr-OH, fmoc-Ile-OH, mpa (Trt) -D-Tyr (Et) -OH were coupled sequentially to give Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (4-methyl-trityl resin) -Gly-NH 2 . After DMF washing, the washing liquid was removed. 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 dried under vacuum at room temperature (20-35 ℃) until it had a quicksand shape. The peptide resin was 42.91g after drying, and the weight gain of the resin was 88.3%.
EXAMPLE 9 Synthesis of atosiban Linear peptide resin 4
Fmoc-Pro-Orn (4-methoxy-trityl resin) -Gly-NH prepared in example 5 was weighed 2 (30.00 g) in a solid phase reaction synthesis column. 400mL of DMF was added and the mixture was swelled for 30min, and the DMF was removed by pumping. The resin was washed with 3 x 200ml dry DMF and the DMF was removed. 200mL 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 200mL DMF each time, a few resin was taken with a glass rod after the 4 th washing, and the ninhydrin test was positive, indicating Fmoc was taken off.
Fmoc-Asn-OH (70.87 g,200 mmol) and N-hydroxysuccinimide (23.00 g,200 mmol) were weighed into 800ml of tetrahydrofuran and stirred at room temperature. Slowly adding a tetrahydrofuran (160 ml) solution of DCC (45.36 g,220 mmol) at about 5 ℃ under stirring for 2.5H at room temperature, filtering, concentrating, adding into petroleum ether, recrystallizing to separate out solid, washing, drying, dissolving the obtained activated ester solid in 200ml tetrahydrofuran, slowly dripping H-Cys (Trt) -OH (79.96 g,200 mmol) in 150ml tetrahydrofuran, continuing to react at room temperature, monitoring the reaction of raw materials completely, filtering, concentrating under reduced pressure, adding the concentrated solution into petroleum ether to separate out solid, washing the solid, drying again, recrystallizing with isopropanol, and drying to obtain Fmoc-Asn-Cys (Trt) -OH 102.17g, and obtaining 73% yield.
20.99g Fmoc-Asn-Cys (Trt) -OH and 13.65g HCTU were weighed out and dissolved in 100mL DMF, after complete dissolution the solution was cooled to below 5℃and then 5.82g DIEA (pre-chilled to<0 ℃ and activating for about 3-5 min in the solution, adding the activated solution into a reaction column, reacting for 2-3 h at 20-35 ℃, detecting that ninhydrin is negative, pumping out the reaction solution, adding 200mL of DMF to wash the resin, and washing for 6 times. After washing, the washing solution was removed to obtain Fmoc-Asn-Cys (Trt) -Pro-Orn (4-methoxy-trityl resin) -Gly-NH 2 。
Repeating the peptide reaction step and removing Fmoc protecting group, and according to the amino acid sequence of atosiban, performing the reaction on Fmoc-Asn-Cys (Trt) -Pro-Orn (4-methoxy-trityl resin) -Gly-NH 2 Sequentially coupling Fmoc-Thr-OH, fmoc-Ile-OH, fmoc-D-Tyr (Et) -OH, and Mpa (Trt) -OH to obtain Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (4-methoxy-trityl resin) -Gly-NH 2 . After DMF washing, the washing liquid was removed. 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 dried under vacuum at room temperature (20-35 ℃) until it had a quicksand shape. The peptide resin was 42.28g after drying, and the weight gain of the resin was 84.0%.
EXAMPLE 10 Synthesis of crude atosiban peptide 1
Preparing 487.2ml of TFA/DCM=2/98 (V/V) lysate, cooling to 5-10 ℃, adding 48.72g of peptide resin prepared in example 6 into the lysate, reacting for 5h at room temperature (20-35 ℃), filtering, washing the peptide resin 2 times with acetonitrile, 50 ml/time, combining the peptide resin into filtrate, spin-drying the filtrate to obtain solid, washing with isopropyl ether, filtering, drying under reduced pressure at 20-35 ℃ until the weight is constant to obtain 14.77g of atosiban Ban Xianxing peptide, dissolving 14.30g of atosiban Ban Xianxing peptide in 0.75L glacial acetic acid, adding 6.75L of water for dilution, dripping 0.1M/L of iodoethanol solution until the solution changes color, reacting for 1.0h at room temperature, and obtaining the atosiban crude peptide, wherein the HPLC spectrum is shown in figure 1.
EXAMPLE 11 Synthesis of crude atosiban peptide 2
Preparing 448.6ml of TFA/DCM=5/95 (V/V) lysate, cooling to 5-10 ℃, adding 42.77g of the peptide resin prepared in example 7 into the lysate, reacting for 3 hours at room temperature (20-35 ℃), filtering, washing the peptide resin 2 times with acetonitrile, 50 ml/time, combining the peptide resin into filtrate, spin-drying the filtrate to obtain solid, washing with isopropyl ether, filtering, drying under reduced pressure at 20-35 ℃ until the constant weight is obtained, dissolving 14.21g of atosiban Ban Xianxing peptide in 1.5L glacial acetic acid, diluting with 6L of water, dripping 0.1M/L of iodoethanol solution until the solution changes color, reacting for 1.0 hour at room temperature, and obtaining the atosiban crude peptide, wherein the HPLC chart is similar to that of FIG. 1.
EXAMPLE 12 Synthesis of crude atosiban peptide 3
Preparing 450.5ml of TFA/DCM=20/80 (V/V) lysate, cooling to 5-10 ℃, adding 45.05g of the peptide resin prepared in the example 8 into the lysate, reacting for 2 hours at room temperature (20-35 ℃), filtering, washing the peptide resin 2 times with acetonitrile, 50 ml/time, combining the peptide resin into filtrate, spin-drying the filtrate to obtain solid, washing with isopropyl ether, filtering, drying under reduced pressure to constant weight at 20-35 ℃ to obtain 14.63g of atosiban Ban Xianxing peptide, dissolving 14.63g of atosiban Ban Xianxing peptide in 1.5L of glacial acetic acid, diluting with 6L of water, adding 10% hydrogen peroxide solution, reacting for 1.0 hour at room temperature, and obtaining the atosiban crude peptide, wherein the HPLC spectrum of the atosiban is similar to that of FIG. 1.
EXAMPLE 13 Synthesis of crude atosiban peptide 4
Preparing 442.7ml of TFA/DCM=1/99 (V/V) lysate, cooling to 5-10 ℃, adding 44.27g of peptide resin prepared in example 9 into the lysate, reacting for 5h at room temperature (20-35 ℃), filtering, washing the peptide resin 2 times with acetonitrile, 50 ml/time, combining the peptide resin with the filtrate, spin-drying the filtrate to obtain solid, washing with isopropyl ether, filtering, drying under reduced pressure at 20-35 ℃ until the constant weight is obtained to obtain 14.13g of atosiban Ban Xianxing peptide, dissolving 14.13g of atosiban Ban Xianxing peptide in 1.5L of glacial acetic acid, adding 6L of water for dilution, adding 30% hydrogen peroxide solution, reacting for 1.0h at room temperature, and obtaining the atosiban crude peptide, wherein the HPLC spectrum of the atosiban is similar to that of FIG. 1.
EXAMPLE 14 purification of crude atosiban peptide 1
The atosiban crude peptide prepared in example 10 was dissolved in 15% acetonitrile aqueous solution and filtered, and then purified by preparative reverse phase HPLC (C18 column), converted to salt, and the fraction was collected to more than 99%, concentrated and lyophilized to obtain 10.12g, with a yield of 64%, and a purity of 99%, and the obtained atosiban refined peptide HPLC spectrum was shown in fig. 2.
EXAMPLE 15 purification of crude atosiban peptide 2
The crude atosiban peptide obtained in example 11 was dissolved in 15% acetonitrile and filtered, and then purified by preparative reverse phase HPLC (C18 column), converted to salt, and the fraction was collected to more than 99%, concentrated and lyophilized to obtain 9.80g, with a yield of 62%, and a purity of 99%, and the resulting atosiban peptide HPLC profile was similar to fig. 2.
EXAMPLE 16 purification of crude atosiban peptide 3
The crude atosiban peptide obtained in example 12 was dissolved in 15% acetonitrile and filtered, and then purified by preparative reverse phase HPLC (C18 column), converted to salt, and the fraction was collected to more than 99%, concentrated and lyophilized to obtain 10.28g, and the obtained atosiban peptide was purified to 99% in a yield of 65%, and the HPLC profile of the obtained atosiban peptide was similar to that of fig. 2.
EXAMPLE 17 purification of crude atosiban peptide 4
The crude atosiban peptide obtained in example 13 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 10.27g, with a yield of 65%, and a purity of 99%, and the resulting atosiban peptide HPLC profile was similar to that of fig. 2.
Claims (10)
1. The solid phase synthesis method of atosiban is characterized by mainly comprising the following steps:
1) Synthesizing Fmoc-Pro-Orn-Gly-NH 2 Tripeptides, then reacted with resin to give Fmoc-Pro-Orn (resin) -Gly-NH 2 ；
2) Removal of Fmoc-Pro-Orn (resin) -Gly-NH 2 Sequentially accessing corresponding protected amino acid or fragment in the atosiban sequence by adopting a condensing agent to prepare the atosiban linear peptide resin;
3) Cracking and oxidizing to obtain atosiban.
2. The method according to claim 1, wherein the resin in step 1) is selected from polymer resins capable of binding with the pendant amino functions of Orn.
3. The method according to claim 2, wherein the polymer resin in the step is selected from trityl type resins.
4. The method according to claim 3, wherein the degree of substitution of the resin is 0.8 to 1.0mmol/g.
5. The method of claim 1, wherein the tripeptide of step 1) is reacted with a resin under DIEA to form a tripeptide resin.
6. The method according to claim 1, wherein the step 2) is specifically: in Fmoc-Pro-Orn (resin) -Gly-NH 2 Fmoc-Cys (Trt) -OH, fmoc-Asn-OH, fmoc-Thr-OH, fmoc-Ile-OH, fmoc-D-Tyr (Et) -OH, and Mpa (Trt) -OH were coupled sequentially to give Mpa (Trt) -D-Tyr (Et) -Ile-Thr-Asn-Cys (Trt) -Pro-Orn (trityl resin) -Gly-NH 2 。
7. The method according to claim 1, wherein the corresponding fragment in the sequence of atosiban accessed in step 2) is selected from the group consisting of Mpa (Trt) -D-Tyr (Et) -OH, fmoc-D-Tyr (Et) -lie-OH, fmoc-Asn-Cys (Trt) -OH.
8. The method according to claim 1, wherein the condensing agent in step 2) is selected from any one of HOBt/DIC, HCTU/DIEA, HBTU/DIEA, HATU/HOAt/DIEA, HBTU/HOBt/DIEA.
9. The method according to claim 1, wherein the cleavage reagent in step 3) is selected from TFA/DCM in a volume ratio of TFA: dcm=1: 99 to 20:80.
10. the method according to claim 1, wherein the oxidizing agent in step 3) is selected from the group consisting of iodine, hydrogen peroxide.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005014640A1 (en) * | 2003-08-02 | 2005-02-17 | Css-Albachem Limited | Method of peptide synthesis |
| CN105408344A (en) * | 2013-06-19 | 2016-03-16 | 佩特雷化学和生物制药学实验室有限公司 | Peptide-resin conjugate and use thereof |
| CN105713073A (en) * | 2016-03-31 | 2016-06-29 | 济南康和医药科技有限公司 | Method for liquid phase preparation of atosiban |
| CN110759972A (en) * | 2019-10-31 | 2020-02-07 | 成都圣诺生物制药有限公司 | Preparation method of atosiban |
| CN111393508A (en) * | 2020-03-26 | 2020-07-10 | 上海苏豪逸明制药有限公司 | Preparation method of atosiban |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005014640A1 (en) * | 2003-08-02 | 2005-02-17 | Css-Albachem Limited | Method of peptide synthesis |
| CN105408344A (en) * | 2013-06-19 | 2016-03-16 | 佩特雷化学和生物制药学实验室有限公司 | Peptide-resin conjugate and use thereof |
| CN105713073A (en) * | 2016-03-31 | 2016-06-29 | 济南康和医药科技有限公司 | Method for liquid phase preparation of atosiban |
| CN110759972A (en) * | 2019-10-31 | 2020-02-07 | 成都圣诺生物制药有限公司 | Preparation method of atosiban |
| CN111393508A (en) * | 2020-03-26 | 2020-07-10 | 上海苏豪逸明制药有限公司 | Preparation method of atosiban |
Non-Patent Citations (2)
| Title |
|---|
| STYMIEST，J.L. 等.Synthesis of Oxytocin Analogues with Replacement of Sulfur by Carbon Gives Potent Antagonists with Increased Stability.THE JOURNAL OF ORGANIC CHEMISTRY.2005,第70卷(第20期),第7799-7809页. * |
| 钟祥龙 等.醋酸阿托西班制备工艺的研究.中国当代医药.2021,第21卷(第20期),第17-21页. * |
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