CN113501871B - Method for preparing darunavagon by combining solid phase with liquid phase - Google Patents

Method for preparing darunavagon by combining solid phase with liquid phase Download PDF

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CN113501871B
CN113501871B CN202110821997.7A CN202110821997A CN113501871B CN 113501871 B CN113501871 B CN 113501871B CN 202110821997 A CN202110821997 A CN 202110821997A CN 113501871 B CN113501871 B CN 113501871B
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fmoc
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tbu
alkali
otbu
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CN113501871A (en
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徐英军
朱玉
于家懿
杨旭
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Sinopeptide Biomedical Group Co ltd
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Sinopeptide Biomedical Group Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention discloses a method for preparing Daxiglucagon by solid-liquid phase combination, which synthesizes monomers Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH, fmoc-Gly-Thr (tBu) -OH, fmoc-Leu-Asp (OtBu) -OH, fmoc-Ala-Arg (Pbf) -OH, fmoc-Ala-Glu (OtBu) -OH, fmoc-Val-Lys (Boc) -OH and Fmoc-Leu-Glu (OtBu) -OH by a liquid phase method for the first time, and then prepares the Daxiglucagon by taking the monomers as raw materials in a solid phase. The method can effectively improve the purity of the crude peptide, reduce the purification difficulty, improve the yield of the final product, be beneficial to large-scale industrial application of synthesis scale and reduce the production cost.

Description

Method for preparing darcies glucagon by combining solid phase with liquid phase
Technical Field
The invention relates to the field of polypeptide synthesis, in particular to a method for synthesizing darunavagon.
Background
Congenital Hyperinsulinemic Hypoglycemia (CHI) is also called Persistent Hyperinsulinemic Hypoglycemia (PHHI) in infants. Persons suffering from this disease have abnormally high levels of insulin and hypoglycemia often occurs. On 11.5.2018, the disease is listed in the first group of rare diseases catalog jointly formulated by 5 departments such as the national health committee of China. The disease is mainly treated by medicines, and the disease can be treated by surgery (laparopancreatectomy) if the medicines are ineffective.
Severe hypoglycemia is an acute, life-threatening disease that is caused by a severe drop in blood glucose levels, mainly due to insulin therapy, and is one of the most feared complications in the treatment of diabetes. The most common causes of hypoglycemia are insulin therapy and sulfonylureas, which occur at about 20% and especially the first generation of sulfonylureas, chlorpropamide, is most likely to cause hypoglycemia.
Zealand Pharma A/S Biotechnology corporation, 03-23.2021, announced that Zegalogue (Dasiglucagen) injection has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of severe hypoglycemia in children and adults aged 6 years and older. Dasiglucagon is currently the first and only glucagon analog used to treat severe hypoglycemia in children and adults aged 6 years and older, and in diabetic patients. The darcies glucagon consists of 29 amino acid residues, has a molecular weight of 3185.57 and has the following amino acid sequence:
H- 1 His-Ser-Gln-Gly- 5 Thr-Phe-Thr-Ser-Asp- 10 Tyr-Ser-Lys-Tyr-Leu- 15 Asp-Aib-Ala-Arg-Ala- 20 Glu-Glu-Phe-Val-Lys- 25 Trp-Leu-Glu-Ser-Thr-OH
the original patent EP2875043 (CN 104662038) synthesized darcies using standard Fmoc chemical solid phase synthesis method using pseudoproline Fmoc-Phe-Thr (Ψ, me, me, pro) -OH, fmoc-Asp-Ser (Ψ, me, me, pro) -OH and Fmoc-Glu-Ser (Ψ, me, me, pro) -OH as starting materials. The synthetic method of the patent has the main defects that the peptide chain is long, the purity of crude peptide is low due to solid phase stepwise synthesis, amino acid residues with simple structures such as Gly, ala and the like are rich in peptide sequences, gly or Ala impurity peptide is easy to lack, so that the purification difficulty is high, and the total yield is low.
In order to improve the purity of the crude peptide and reduce the generation of peptides with Gly or Ala impurities, the applicant researches a synthesis method of the dapoxetine, and the technical scheme of the invention is obtained by a fragment synthesis method.
Disclosure of Invention
The invention aims to provide a method for preparing darunavagons by a fragment method. The invention reduces the synthesis difficulty and improves the purity of the crude peptide, thereby reducing the purification difficulty, improving the yield, reducing the production cost and being beneficial to large-scale industrial production.
In order to achieve the purpose, the invention provides the following technical scheme:
(a) Liquid phase synthesis of monomers Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH, fmoc-Gly-Thr (tBu) -OH, fmoc-Leu-Asp (OtBu) -OH, fmoc-Ala-Arg (Pbf) -OH, fmoc-Ala-Glu (OtBu) -OH, fmoc-Val-Lys (Boc) -OH, fmoc-Leu-Glu (OtBu) -OH;
(b) Wang resins or 2-CTC resins are adopted as solid phase carriersFmoc-Thr (tBu) -OH is coupled with the Fmoc-Thr (tBu) -OH to prepare Fmoc-Thr (tBu) -resin, and then Fmoc-Ser (tBu) -OH, fmoc-Leu-Glu (OtBu) -OH, fmoc-Trp (Boc) -OH, fmoc-Val-Lys (Boc) -OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Arg (Pbf) -OH, fmoc-Aib-OH, fmoc-Leu-Asp (OtBu) -OH are coupled in sequence, fmoc-Tyr (tBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Ser (tBu) -OH, fmoc-Tyr (tBu) -OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH, fmoc-Gly-Thr (tBu) -OH, boc- 1 His (Trt) -Ser (tBu) -Gln (Trt) -OH to give Dassiglucagon peptide resin:
Boc- 1 His(Trt)-Ser(tBu)-Gln(Trt)-Gly- 5 Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- 10 Tyr(tBu)-Ser(tBu)-Lys-Tyr(tBu)-Leu- 15 Asp(OtBu)-Aib–Ala-Arg(Pbf)-Ala- 20 Glu(OtBu)-Glu(OtBu)-Phe-Val-Lys(Boc)- 25 trp (Boc) -Leu-Glu (OtBu) -Ser (tBu) -Thr (tBu) -resin;
(c) The peptide resin is cracked, purified and freeze-dried to obtain the darcy glucagon peptide.
Preferably, the specific operation steps for synthesizing the monomer Fmoc-Val-Lys (Boc) -OH in the step (a) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Lys (Boc) -OH in a prepared alkali solution in a low-temperature bath, wherein the solution is still alkaline after dissolution; and adding Fmoc-Val-OSu solution into the reaction solution at low temperature, heating to continue stirring for reaction after dropwise addition, monitoring the reaction end point by TLC, and obtaining the monomer Fmoc-Val-Lys (Boc) -OH through concentration, acid adjustment, crystallization and recrystallization.
Preferably, the specific operation steps for synthesizing the monomer Fmoc-Gly-Thr (tBu) -OH in the step (a) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Thr (tBu) -OH in a prepared alkali solution at a low temperature, wherein the solution is still alkaline after the solution is dissolved; adding Fmoc-Gly-OSu solution into the reaction solution at low temperature, heating to continue stirring for reaction after dropwise adding is finished, monitoring the reaction end point by TLC, and obtaining the monomer Fmoc-Gly-Thr (tBu) -OH through concentration, acid adjustment, crystallization and recrystallization.
Preferably, the specific operation steps for synthesizing the monomer Fmoc-Leu-Asp (OtBu) -OH in the step (a) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Asp (OtBu) -OH in a prepared alkali solution at a low temperature, wherein the solution is still alkaline after being dissolved; and (2) adding Fmoc-Leu-OSu solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding, monitoring the reaction end point by TLC, and obtaining the monomer Fmoc-Leu-Asp (OtBu) -OH through concentration, acid adjustment, crystallization and recrystallization.
Preferably, the step (a) of synthesizing the monomer Fmoc-Ala-Arg (Pbf) -OH comprises the following specific operation steps: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Arg (Pbf) -OH in a prepared alkali solution at a low temperature bath, wherein the solution is still alkaline after the solution is dissolved; and adding Fmoc-Ala-OSu solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding, monitoring the reaction end point by TLC, and obtaining the monomer Fmoc-Ala-Arg (Pbf) -OH through concentration, acid adjustment, crystallization and recrystallization.
Preferably, the specific operation steps for synthesizing the monomer Fmoc-Leu-Glu (OtBu) -OH in the step (a) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Glu (OtBu) -OH in a prepared alkali solution under low-temperature bath, wherein the solution is still alkaline after being dissolved; adding Fmoc-Leu-OSu solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding is finished, monitoring the reaction end point by TLC, and obtaining the monomer Fmoc-Leu-Glu (OtBu) -OH through concentration, acid adjustment, crystallization and recrystallization.
Preferably, the specific operation steps for synthesizing the monomer Fmoc-Ala-Glu (OtBu) -OH in the step (a) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Glu (OtBu) -OH in a prepared alkali solution under low-temperature bath, wherein the solution is still alkaline after being dissolved; and adding Fmoc-Ala-OSu solution into the reaction solution at low temperature, heating after dropwise adding, continuously stirring for reaction, monitoring the reaction end point by TLC, and obtaining the monomer Fmoc-Ala-Glu (OtBu) -OH through concentration, acid adjustment, crystallization and recrystallization.
Preferably, the specific operation steps for synthesizing the monomer Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH in the step (a) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Ser (tBu) -OH in a prepared alkali solution at a low temperature, wherein the solution is still alkaline after being dissolved; adding a Boc-His (Trt) -OSu solution into the reaction solution at a low temperature, heating and continuously stirring for reaction after dropwise adding is finished, monitoring the reaction end point by TLC, and obtaining a monomer Boc-His (Trt) -Ser (tBu) -OH through concentration, acid regulation, crystallization and recrystallization; then the mixture is dissolved and grafted in an organic solvent, a certain amount of DCC/HOSu is added for reaction for 3h, and the Boc-His (Trt) -Ser (tBu) -OSu is obtained after filtration, concentration and crystallization; dissolving certain alkali in the solvent to prepare an alkaline solution; dissolving H-Gln (Trt) -OH in a prepared alkali solution at a low temperature bath, wherein the solution is still alkaline after being dissolved; adding Boc-His (Trt) -Ser (tBu) -OSu solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding, monitoring the reaction end point by TLC, and obtaining a monomer Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH through concentration, acid adjustment, crystallization and recrystallization.
The alkaline solution prepared by dissolving the alkali A in the solvent B can be aqueous solution of ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate and the like or mixed solution of organic solvents which are mutually soluble with water; meanwhile, the organic base can also be organic solution of organic bases such as triethylamine, diethylamine, N-diisopropylethylamine and the like;
preferably, in the step (c), the cleavage reagent is a TFA solution added with 1-5% by volume of a scavenger, and the scavenger is one or more of anisole, thioanisole, dithioglycol, mercaptoethanol, phenol, water and TIS.
Compared with the prior art, the invention has the beneficial effects that:
the method adopts monomers Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH, fmoc-Gly-Thr (tBu) -OH, fmoc-Leu-Asp (OtBu) -OH, fmoc-Ala-Arg (Pbf) -OH, fmoc-Ala-Glu (OtBu) -OH, fmoc-Val-Lys (Boc) -OH and Fmoc-Leu-Glu (OtBu) -OH as raw materials for the first time, and realizes the solid phase synthesis of the glucagon.
Drawings
Figure 1 is a dapoxetine peak profile.
Figure 2 is a graph of the dapoxetine peak area results.
Detailed Description
The present invention will be described in detail with reference to the following specific examples, which are not intended to limit the scope of the present invention; it is within the scope of the present invention to vary the raw material feed ratio, the reaction solvent, the condensing agent, etc. according to the present invention.
Abbreviations used in the specification and claims have the following meanings:
fmoc: 9-fluorenylmethoxycarbonyl;
tBu: a tertiary butyl group;
pbf:2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl;
trt: a trityl group;
DCM: dichloromethane;
DMF: n, N-dimethylformamide;
DIPEA: n, N-diisopropylethylamine;
DIC: n, N-diisopropylcarbodiimide;
HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate;
HATU:2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate;
TBTU: O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate;
HOBT: 1-hydroxybenzotriazole;
HOAT: 1-hydroxy-7-azobenzotriazol;
TFA: trifluoroacetic acid;
and (3) TIS: triisopropylsilane;
boc: a tert-butoxycarbonyl group;
su: a succinimide group;
DTT: dithiothreitol.
Example 1: synthesis of Fmoc-Val-Lys (Boc) -OH
Accurately weighing 5.92kg (20 mol) of H-Lys (Boc) -OH in a 100L reaction kettle, adding 24L of 10% sodium carbonate aqueous solution, and dissolving under stirring; after the solution is dissolved clearly, fmoc-Val-OSu 8.72kg (20 mol)/24L tetrahydrofuran solution is added dropwise at low temperature, the mixture is stirred for reaction, and the TLC is used for monitoring the end point; after decompression concentration, acid adjustment, ethyl acetate extraction, drying and crystallization, dipeptide monomer Fmoc-Val-Lys (Boc) -OH10.36kg with the purity of 99.2 percent is obtained, and the yield is 91.28 percent.
Example 2: synthesis of Fmoc-Leu-Glu (OtBu) -OH
Accurately weighing 4.06kg (20 mol) of H-Glu (OtBu) -OH in a 100L reaction kettle, adding 24L of 10% sodium carbonate aqueous solution, and dissolving under stirring; after the solution is dissolved clearly, fmoc-Leu-OSu 9.01kg (20 mol)/24L tetrahydrofuran solution is added dropwise at low temperature, the mixture is stirred for reaction, and the TLC is used for monitoring the end point; after decompression concentration, acid adjustment, ethyl acetate extraction, drying and crystallization, the dipeptide monomer Fmoc-Leu-Glu (OtBu) -OH9.28kg with the purity of 99.3 percent is obtained, and the yield is 86.21 percent.
Example 3: synthesis of Fmoc-Leu-Asp (OtBu) -OH
Accurately weighing 3.78kg (20 mol) of H-Asp (OtBu) -OH into a 100L reaction kettle, adding 24L of 10 percent sodium carbonate aqueous solution, and dissolving under stirring; after the solution is dissolved clearly, fmoc-Leu-OSu 9.01kg (20 mol)/24L tetrahydrofuran solution is added dropwise at low temperature, the mixture is stirred for reaction, and the TLC is used for monitoring the end point; after decompression concentration, acid adjustment, ethyl acetate extraction, drying and crystallization, the dipeptide monomer Fmoc-Leu-Glu (OtBu) -OH8.61kg with the purity of 99.2 percent is obtained, and the yield is 82.12 percent.
Example 4: synthesis of Fmoc-Ala-Arg (Pbf) -OH
Accurately weighing 8.53kg (20 mol) of H-Arg (Pbf) -OH in a 100L reaction kettle, adding 24L of 10% sodium carbonate aqueous solution and 3L of THF, and dissolving under stirring; after the mixture is dissolved and cleared, 8.17kg (20 mol) of Fmoc-Ala-OSu/24L tetrahydrofuran solution is added dropwise at low temperature, the mixture is stirred for reaction, and the end point is monitored by TLC; after decompression concentration, acid adjustment, ethyl acetate extraction, drying and crystallization, 98.7 percent purity dipeptide monomer Fmoc-Ala-Arg (Pbf) -OH11.90kg is obtained, and the yield is 82.62 percent.
Example 5: synthesis of Fmoc-Ala-Glu (OtBu) -OH
Accurately weighing 4.06kg (20 mol) of H-Glu (OtBu) -OH in a 100L reaction kettle, adding 24L of 10% sodium carbonate aqueous solution, and dissolving under stirring; after the solution is dissolved clearly, 8.17kg (20 mol) of Fmoc-Ala-OSu/24L tetrahydrofuran solution is added dropwise at low temperature, the mixture is stirred for reaction, and the end point is monitored by TLC; after decompression concentration, acid adjustment, ethyl acetate extraction, drying and crystallization, 8.30kg of dipeptide monomer Fmoc-Ala-Glu (OtBu) -OH with the purity of 98.6 percent is obtained, and the yield is 83.62 percent.
Example 6: synthesis of Fmoc-Gly-Thr (tBu) -OH
Accurately weighing 3.51kg (20 mol) of H-Thr (tBu) -OH into a 100L reaction kettle, adding 24L of 10% sodium carbonate aqueous solution, and dissolving under stirring; after the mixture is dissolved and cleared, 7.89kg (20 mol) of Fmoc-Gly-OSu/24L tetrahydrofuran solution is added dropwise at low temperature, the mixture is stirred for reaction, and the end point is monitored by TLC; after decompression concentration, acid adjustment, ethyl acetate extraction, drying and crystallization, the dipeptide monomer Fmoc-Gly-Thr (tBu) -OH7.83kg with the purity of 99.1 percent is obtained, and the yield is 86.16 percent.
Example 7: synthesis of Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH
Accurately weighing 3.22kg (20 mol) of H-Ser (tBu) -OH in a 100L reaction kettle, adding 24L of 10% sodium carbonate aqueous solution, and dissolving under stirring; after the mixture is dissolved clearly, boc-His (Trt) -OSu11.89kg (20 mol)/24L tetrahydrofuran solution is added dropwise at low temperature, the reaction is stirred, and the end point is monitored by TLC; after decompression concentration, acid adjustment, ethyl acetate extraction, drying and crystallization, 10.56kg of dipeptide monomer Boc-His (Trt) -Ser (tBu) -OH with the purity of 98.6 percent is obtained, and the yield is 82.38 percent.
Accurately weighing 10.56kg (16.5 mol) of Fmoc-Gly-Thr (tBu) -OH and 2.07kg (18 mol) of HOSu in a 100L reaction kettle, adding 24L tetrahydrofuran, and dissolving under stirring; after the solution is dissolved clearly, dropping DCC 3.71kg (18 mol)/18L tetrahydrofuran solution at low temperature, stirring for reaction for 3h, filtering, concentrating and crystallizing to obtain Boc-His (Trt) -Ser (tBu) -OSu solid, and redissolving the solid in 24L tetrahydrofuran for later use.
Accurately weighing 6.40kg (16.5 mol) of H-Gln (Trt) -OH in a 100L reaction kettle, adding 24L of 10% sodium carbonate aqueous solution and 3L of THF, and dissolving under stirring; after the mixture is dissolved clearly, dropwise adding Boc-His (Trt) -Ser (tBu) -OSu/24L tetrahydrofuran solution at low temperature, stirring for reaction, and monitoring the end point by TLC; after decompression concentration, acid regulation, ethyl acetate extraction, drying and crystallization, 13.63kg of dipeptide monomer Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH with the purity of 99.2 percent is obtained, and the yield is 81.66 percent.
Example 8: synthesis of Fmoc-Thr (tBu) -Wang Resins
The carrier Wang resin 400.0g (sub =0.47 mmol/g) was placed in a synthesis column, washed twice with 2400mL DMF, swollen for 30min with 2400mL DCM; after DCM was filtered off with suction, a mixed DCM solution of Fmoc-Thr (tBu) -OH/DIC/HOBT was added [ 79.5g (200 mmol) of Fmoc-Thr (tBu) -OH and 32.4g (240 mmol) of HOBT were weighed in a glycine activation flask, 2000mL of a mixed solution of DMF and DCM in a volume ratio of 1: 1 was added and dissolved with stirring, 38.2mL (240 mmol) of DIC was added at low temperature (0 ℃ C.) and activated for 5 minutes ], 2.4g (2 mmol) of DMAP was added after 10min of reaction; reacting for 2h, pumping out the reaction liquid, washing twice with 2400mL of DMF, adding 2400mL of end-capping reagent (480 mL of acetic anhydride and 408mL of pyridine are dissolved in 1512mL of DMF), reacting for 2h, filtering the reaction liquid, washing for 2 times with DMF, DCM and methanol respectively, and drying in vacuum to obtain Fmoc-Thr (tBu) -Wang resins471.0g; a sample was taken and the degree of substitution was 0.38mmol/g.
Example 9: synthesis of Fmoc-Thr (tBu) -CTC Resins
Weighing 50.0g (sub =0.41 mmol/g) of CTC resin, placing the CTC resin in a synthetic column, washing the CTC resin twice by 240mL of DMF, and adding 240mL of DCM to swell for 30min; after DCM was filtered off with suction, 150ml of a DCM/DMF (3/1, volume ratio) solution containing 11.92g (30 mmol) of Fmoc-Thr (tBu) -OH was added, and after stirring, 9.9ml (60 mmol) of DIPEA, drum N, was added 2 Reacting for 60min, draining the reaction solution, and adding DCM/CH 3 OH/DIPEA (volume ratio 17; then washing with DMF, DCM and methanol for 2 times respectively, and drying in vacuum to obtain 57.20g of Fmoc-Thr (tBu) -CTC Resins. The degree of substitution was measured to be 0.35mmol/g.
Example 10: preparation of Darcy glucagon peptide resin
Fmoc-Thr (tBu) -Wang resins131.6g (50 mmol in synthesis scale) with substitution degree of 0.38mmol/g in example 8 are accurately weighed and placed in a synthesis column, 1000ml of DCM is added for swelling for 30min; after DCM was filtered off by suction, 1000ml of DMF was washed 2 times, 1000ml of 20% piperidine/DMF solution was added for deprotection 2 times, and the reaction was carried out for 10min and 10min, respectively; then washing with 1000ml DMF, DCM, DMF respectively for 2 times; 500ml of a DMF solution of Fmoc-Ser (tBu) -OH38.4g (100 mmol), HOBT13.5g (100 mmol) and DIC 15.5ml (100 mmol) was added and N was bubbled 2 Stirring and reacting for 2h, taking the detection result of a Kaiser reagent as the standard of the reaction end point, after the reaction end point is reached, pumping out the reaction liquid, and washing with 1000ml of DMF, DCM and DMF for 2 times respectively; followed by deprotection. Repeating the above operation in a cycle according to the darcy glucagon peptideSequentially coupling with protected amino acid; the sequentially connected protected amino acids are: fmoc-Leu-Glu (OtBu) -OH, fmoc-Trp (Boc) -OH, fmoc-Val-Lys (Boc) -OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Arg (Pbf) -OH, fmoc-Aib-OH, fmoc-Leu-Asp (OtBu) -OH, fmoc-Tyr (tBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Ser (tBu) -OH, fmoc-Tyr (tBu) -OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH, fmoc-Gly-Thr (tBu) -OH, boc-1His (Trt) -Ser (tBu) -Gln (Trbu) -OH, and texzft-78 zj 8978 g was obtained.
Example 11: preparation of Darcy glucagon peptide resin
Fmoc-Thr (tBu) -CTC Resins 57.1g (20 mmol in synthesis scale) with substitution degree of 0.35mmol/g in example 8 were weighed accurately and placed in a synthesis column, and 500ml DCM was added to swell for 30min; after DCM was filtered off by suction, 500ml of DMF was washed 2 times, and 500ml of 20% piperidine/DMF solution was added for deprotection 2 times, and the reaction was carried out for 10min and 10min, respectively; then washing with 500ml DMF, DCM, DMF respectively 2 times; 500ml of a DMF solution of 15.4g (40 mmol) of Fmoc-Ser (tBu) -OH, 5.4g (40 mmol) of HOBT and 6.2ml (40 mmol) of DIC were added and N was poured in a drum 2 Stirring and reacting for 2h, taking the detection result of a Kaiser reagent as the standard of the reaction end point, after the reaction end point is reached, pumping out the reaction liquid, and washing with 500ml of DMF, DCM and DMF for 2 times respectively; followed by deprotection. Repeating the operation repeatedly, and coupling with the protected amino acids one by one according to the peptide sequence of the darcy glucagon; the sequentially connected protected amino acids are: fmoc-Leu-Glu (OtBu) -OH, fmoc-Trp (Boc) -OH, fmoc-Val-Lys (Boc) -OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Arg (Pbf) -OH, fmoc-Aib-OH, fmoc-Leu-Asp (OtBu) -OH, fmoc-Tyr (tBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Ser (tBu) -OH, fmoc-Tyr (tBu) -OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH, fmoc-Gly-Thr (tBu) -OH, boc-1His (Trt) -Ser (tBu) -Gln (Trt) -OH, david-2 g of glucagon resin was obtained.
Example 12: cleavage of Darcy glucagon peptide resin
378.0g liraglutide peptide resin obtained in example 10 was added to frozen 3800ml lysate (TFA/TIS/H ratio by volume) 2 0=95/25/2.5), stirring and reacting for 3h at room temperature; after the cleavage reaction is finished, filtering the resin, washing the resin for 2 times by 400ml of TFA, combining the filtrate and the washing liquid, carrying out rotary evaporation and concentration to 2000ml, pouring into 20L of frozen methyl tert-ether, and separating out a white precipitate; standing for 30min, filtering, washing with methyl tert-ether for 6 times, and vacuum drying to obtain crude peptide 168.6g with crude peptide yield of 99.7% and purity of 76.8%.
Example 13: cleavage of Darcy glucagon peptide resin
158.0g liraglutide peptide resin from example 11 was added to frozen 1600ml lysate (TFA/TIS/H ratio by volume) 2 0= 95/2.5/2.5), stirring and reacting for 3h at room temperature; after the cleavage reaction is finished, filtering the resin, washing the resin for 2 times by 150ml TFA, combining the filtrate and the washing liquid, carrying out rotary evaporation and concentration to 1000ml, pouring into 10L of frozen methyl tert-ether, and separating out a white precipitate; standing for 30min, filtering, washing with methyl tert-ether for 6 times, and vacuum drying to obtain crude peptide 63.8g, with a crude peptide yield of 94.7% and a purity of 78.2%.
Example 14: purification of crude dapoxetine peptide
168.0g of the crude peptide obtained in example 12 was dissolved in 400ml of acetic acid, and after complete dissolution, the solution was diluted to 10L with water and filtered through a 0.45um filter.
A C18 preparation column with the inner diameter of 150mm, a mobile phase of a 50mM ammonium acetate/water-1.0% acetic acid/acetonitrile system, the loading amount of 30 g/time, the flow rate of 500ml/min, gradient elution; and performing cyclic sample injection before and after peaks to obtain fine peptide solution with qualified central control analysis purity, desalting and freeze-drying to obtain 96.2g of fine peptide, wherein the total yield is 56.9%, the purity is more than 99.62%, and the single impurity content is less than 0.2%.
Example 15: purification of crude dapoxetine peptide
60.0g of the crude peptide obtained in example 13 was dissolved in 400ml of acetic acid, and after complete dissolution, the solution was diluted to 10L with water and filtered through a 0.45um filter for further use.
A C18 preparation column with the inner diameter of 150mm, a mobile phase of a 50mM ammonium acetate/water-1.0% acetic acid/acetonitrile system, the loading amount of 30 g/time, the flow rate of 500ml/min, gradient elution; and performing circulating sample injection before and after peaks to obtain refined peptide solution with qualified central control analysis purity, desalting, and freeze-drying to obtain refined peptide 38.9g, wherein the total yield is 61.2%, the purity is more than 99.79%, and the single impurity is less than 0.2%.

Claims (9)

1. A method for preparing darunavir by solid-liquid phase combination is characterized by comprising the following steps:
(a) Liquid phase synthesis of monomers Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH, fmoc-Gly-Thr (tBu) -OH, fmoc-Leu-Asp (OtBu) -OH, fmoc-Ala-Arg (Pbf) -OH, fmoc-Ala-Glu (OtBu) -OH, fmoc-Val-Lys (Boc) -OH, fmoc-Leu-Glu (OtBu) -OH;
(b) Using Wang resins or 2-CTC resins as solid phase carriers, coupling Fmoc-Thr (tBu) -OH with Fmoc-Thr (tBu) -OH to prepare Fmoc-Thr (tBu) -resin, and sequentially coupling Fmoc-Ser (tBu) -OH, fmoc-Leu-Glu (OtBu) -OH, fmoc-Trp (Boc) -OH, fmoc-Val-Lys (Boc) -OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Arg (Pbf) -OH, fmoc-Aib-OH, fmoc-Leu-Asp (OtBu) -OH, fmoc-Tyr (tBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Ser (tBu) -OH, fmoc-Tyr (tBu) -OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH, fmoc-Gly-Thr (tBu) -OH, boc- 1 His (Trt) -Ser (tBu) -Gln (Trt) -OH to obtain Daxiglucagon peptide resin Boc- 1 His(Trt)-Ser(tBu)-Gln(Trt)-Gly- 5 Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)- 10 Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu- 15 Asp(OtBu)-Aib-Ala-Arg(Pbf)-Ala- 20 Glu(OtBu)-Glu(OtBu)-Phe-Val-Lys(Boc)- 25 Trp (Boc) -Leu-Glu (OtBu) -Ser (tBu) -Thr (tBu) -resin;
(c) The peptide resin is cracked, purified and freeze-dried to obtain the darunavailability protien peptide.
2. The method of claim 1, wherein the step (a) of synthesizing the monomer Fmoc-Val-Lys (Boc) -OH comprises the following steps: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Lys (Boc) -OH in a prepared alkali solution in a low-temperature bath, wherein the solution is still alkaline after dissolution; adding Fmoc-Val-OSu solution into the reaction solution at low temperature, heating to continue stirring for reaction after dropwise adding is finished, monitoring the reaction end point by TLC, and obtaining monomer Fmoc-Val-Lys (Boc) -OH through concentration, acid adjustment, crystallization and recrystallization; wherein, the alkaline solution prepared by the alkali A and the solvent B is ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate aqueous solution or mixed solution of organic solvents which are mutually soluble with water, or organic solution of organic alkali of triethylamine, diethylamine and N, N-diisopropylethylamine.
3. The method according to claim 1, wherein the step (a) of synthesizing the monomer Fmoc-Gly-Thr (tBu) -OH comprises the following steps: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Thr (tBu) -OH in a prepared alkali solution at a low temperature, wherein the solution is still alkaline after the solution is dissolved; adding Fmoc-Gly-OSu solution into the reaction solution at low temperature, heating to continue stirring for reaction after dropwise adding is finished, monitoring the reaction end point by TLC, and obtaining a monomer Fmoc-Gly-Thr (tBu) -OH through concentration, acid adjustment, crystallization and recrystallization; wherein, the alkaline solution prepared by the alkali A and the solvent B is ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate aqueous solution or mixed solution of organic solvents which are mutually soluble with water, or organic solution of organic alkali of triethylamine, diethylamine and N, N-diisopropylethylamine.
4. The method according to claim 1, wherein the step (a) of synthesizing Fmoc-Leu-Asp (OtBu) -OH comprises the following steps: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Asp (OtBu) -OH in a prepared alkali solution at a low temperature, wherein the solution is still alkaline after being dissolved; adding Fmoc-Leu-OSu solution into the reaction solution at low temperature, heating after dropwise adding, continuously stirring for reaction, monitoring the reaction end point by TLC, and obtaining monomer Fmoc-Leu-Asp (OtBu) -OH through concentration, acid adjustment, crystallization and recrystallization; wherein, the alkaline solution prepared by the alkali A and the solvent B is ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate aqueous solution or mixed solution of organic solvents which are mutually soluble with water, or organic solution of organic alkali of triethylamine, diethylamine and N, N-diisopropylethylamine.
5. The method of claim 1, wherein the step (a) of synthesizing the monomer Fmoc-Ala-Arg (Pbf) -OH comprises the following steps: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Arg (Pbf) -OH in a prepared alkali solution at a low temperature bath, wherein the solution is still alkaline after the solution is dissolved; adding Fmoc-Ala-OSu solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding, monitoring the reaction end point by TLC, and obtaining monomer Fmoc-Ala-Arg (Pbf) -OH through concentration, acid adjustment, crystallization and recrystallization; wherein, the alkaline solution prepared by the alkali A and the solvent B is ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate aqueous solution or mixed solution of organic solvents which are mutually soluble with water, or organic solution of organic alkali of triethylamine, diethylamine and N, N-diisopropylethylamine.
6. The method according to claim 1, wherein the step (a) of synthesizing the monomer Fmoc-Leu-Glu (OtBu) -OH comprises the following steps: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Glu (OtBu) -OH in a prepared alkali solution under low-temperature bath, wherein the solution is still alkaline after being dissolved; adding Fmoc-Leu-OSu solution into the reaction solution at low temperature, heating after dropwise adding, continuously stirring for reaction, monitoring the reaction end point by TLC, and obtaining a monomer Fmoc-Leu-Glu (OtBu) -OH through concentration, acid adjustment, crystallization and recrystallization; wherein, the alkaline solution prepared by the alkali A and the solvent B is ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate aqueous solution or mixed solution of organic solvents which are mutually soluble with water, or organic solution of organic alkali of triethylamine, diethylamine and N, N-diisopropylethylamine.
7. The method of claim 1, wherein the step (a) of synthesizing the monomer Fmoc-Ala-Glu (OtBu) -OH comprises the following steps: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Glu (OtBu) -OH in a prepared alkali solution under low-temperature bath, wherein the solution is still alkaline after being dissolved; adding Fmoc-Ala-OSu solution into the reaction solution at low temperature, heating after dropwise adding, continuously stirring for reaction, monitoring the reaction end point by TLC, and obtaining monomer Fmoc-Ala-Glu (OtBu) -OH through concentration, acid adjustment, crystallization and recrystallization; wherein, the alkaline solution prepared by the alkali A and the solvent B is ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate aqueous solution or mixed solution of organic solvents which are mutually soluble with water, or organic solution of organic alkali of triethylamine, diethylamine and N, N-diisopropylethylamine.
8. The preparation method according to claim 1, wherein the specific operation steps for synthesizing the monomer Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH in the step (a) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Ser (tBu) -OH in a prepared alkali solution at a low temperature, wherein the solution is still alkaline after being dissolved; adding Boc-His (Trt) -OSu solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding, monitoring the reaction end point by TLC, and obtaining a monomer Boc-His (Trt) -Ser (tBu) -OH through concentration, acid regulation, crystallization and recrystallization; then dissolving the Boc-His-Ser (tBu) -OSu in an organic solvent, adding DCC/HOSu for reaction for 3h, filtering, concentrating and crystallizing to obtain Boc-His (Trt) -Ser (tBu) -OSu; then taking an alkali solution prepared from the alkali A and the solvent B; dissolving H-Gln (Trt) -OH in a prepared alkali solution under a low-temperature bath, wherein the solution is still alkaline after being dissolved; adding Boc-His (Trt) -Ser (tBu) -OSu solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding, monitoring the reaction end point by TLC, and obtaining a monomer Boc-His (Trt) -Ser (tBu) -Gln (Trt) -OH through concentration, acid regulation, crystallization and recrystallization; wherein, the alkaline solution prepared by the alkali A and the solvent B is ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate aqueous solution or mixed solution of organic solvents which are mutually soluble with water, or organic solution of organic alkali of triethylamine, diethylamine and N, N-diisopropylethylamine.
9. The method according to claim 1, wherein in step (c), the cleavage reagent is a TFA solution containing 1-5 vol.% of a scavenger selected from the group consisting of anisole, thioanisole, dithioglycol, mercaptoethanol, phenol, water and TIS.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104662038A (en) * 2012-07-23 2015-05-27 西兰制药公司 Glucagon analogues
CN105384809A (en) * 2015-12-30 2016-03-09 济南康和医药科技有限公司 Method for preparing teriparatide by fragment method and solid-liquid combination
CN106749614A (en) * 2017-01-05 2017-05-31 济南康和医药科技有限公司 A kind of fragment method solid-liquid combination is prepared for the method for degree Shandong peptide
CN108059666A (en) * 2018-02-10 2018-05-22 润辉生物技术（威海）有限公司 A kind of method that solid-liquid combination prepares Suo Malu peptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104662038A (en) * 2012-07-23 2015-05-27 西兰制药公司 Glucagon analogues
CN105384809A (en) * 2015-12-30 2016-03-09 济南康和医药科技有限公司 Method for preparing teriparatide by fragment method and solid-liquid combination
CN106749614A (en) * 2017-01-05 2017-05-31 济南康和医药科技有限公司 A kind of fragment method solid-liquid combination is prepared for the method for degree Shandong peptide
CN108059666A (en) * 2018-02-10 2018-05-22 润辉生物技术（威海）有限公司 A kind of method that solid-liquid combination prepares Suo Malu peptides

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