CN110922453B - Method for synthesizing goserelin - Google Patents
Method for synthesizing goserelin Download PDFInfo
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- CN110922453B CN110922453B CN201811101461.2A CN201811101461A CN110922453B CN 110922453 B CN110922453 B CN 110922453B CN 201811101461 A CN201811101461 A CN 201811101461A CN 110922453 B CN110922453 B CN 110922453B
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- 238000000034 method Methods 0.000 title claims abstract description 31
- 108010069236 Goserelin Proteins 0.000 title claims abstract description 28
- BLCLNMBMMGCOAS-URPVMXJPSA-N Goserelin Chemical compound C([C@@H](C(=O)N[C@H](COC(C)(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(=O)NNC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 BLCLNMBMMGCOAS-URPVMXJPSA-N 0.000 title claims abstract description 27
- 229960002913 goserelin Drugs 0.000 title claims abstract description 26
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 12
- 239000011347 resin Substances 0.000 claims abstract description 60
- 229920005989 resin Polymers 0.000 claims abstract description 60
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 53
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims abstract description 32
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 230000008878 coupling Effects 0.000 claims abstract description 27
- 238000010168 coupling process Methods 0.000 claims abstract description 27
- 238000005859 coupling reaction Methods 0.000 claims abstract description 27
- 229910052763 palladium Inorganic materials 0.000 claims abstract description 14
- 125000006239 protecting group Chemical group 0.000 claims abstract description 14
- USFPINLPPFWTJW-UHFFFAOYSA-N tetraphenylphosphonium Chemical compound C1=CC=CC=C1[P+](C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 USFPINLPPFWTJW-UHFFFAOYSA-N 0.000 claims abstract description 9
- 230000008569 process Effects 0.000 claims abstract description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 4
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- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 7
- 239000007822 coupling agent Substances 0.000 claims description 7
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 claims description 7
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 7
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- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 3
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- YDJXDYKQMRNUSA-UHFFFAOYSA-N tri(propan-2-yl)silane Chemical compound CC(C)[SiH](C(C)C)C(C)C YDJXDYKQMRNUSA-UHFFFAOYSA-N 0.000 claims 1
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- REITVGIIZHFVGU-LJQANCHMSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-LJQANCHMSA-N 0.000 description 5
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- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 5
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- UKVIEHSSVKSQBA-UHFFFAOYSA-N methane;palladium Chemical compound C.[Pd] UKVIEHSSVKSQBA-UHFFFAOYSA-N 0.000 description 5
- 239000007858 starting material Substances 0.000 description 5
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 4
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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Abstract
The invention relates to the technical field of polypeptide synthesis, in particular to a method for synthesizing goserelin. The synthesis method comprises the following steps: coupling Azaply with the resin; sequentially coupling amino acids at the amino terminus of the Azaply-resin; b, removing Arg guanidine protecting group in the peptide resin obtained in the step B by adopting a remover containing triphenylphosphine palladium and morpholine; and C, cracking the peptide resin obtained in the step C by adopting a cracking solution to obtain goserelin. The Sieber Resin and the protective amino acid combination can realize coupling, deprotection and modification on the Resin without C-terminal modification, and the process is simple in operation, convenient in post-treatment and good in reproducibility; the removal adopts tetraphenylphosphine palladium/morpholine removal, and avoids the liquid phase palladium hydrocarbon reduction step; the side chain protecting group is consistent with the cracking condition of the resin, so that the resin cracking and protecting group removal can be completed in one step.
Description
Technical Field
The invention relates to the technical field of polypeptide synthesis, in particular to a method for synthesizing goserelin.
Background
Goserelin acetate is a Luteinizing Hormone Releasing Hormone Analogue (LHRHA) with amino acid sequence Pyr-His-Trp-Ser-Tyr-D-Ser (tBu) -Leu-Arg-Pro-Azagly-NH 2 . It can inhibit secretion of sex hormone (testosterone and estradiol), thereby atrophy of hormone sensitive tumor; is a subcutaneous implant, belongs to a long-acting hormone preparation, and can maintain the action time for 1 month. The original manufacturer is Aspirin (AstraZeneca plc), the trade name is Zoladex 'Norider', and the research and development is completed in the United kingdom for the first time in 3 months of 1987; market in the united states in 1990, month 5; the application of the medicine in treating breast cancer in 1996 is on the market in China. The current sales of this drug is second to worldwide rank in LHRHa formulations.
The product synthesis method mainly comprises two main types, one type is liquid phase synthesis; the other is solid phase synthesis. The strategy of liquid phase synthesis mainly adopts a fragment condensation method, namely, different fragments are synthesized firstly, and then the fragments are condensed; such as "4+6", "5+5", "3+7", etc. The liquid phase synthesis method mainly comprises the following patents and documents:
J.Med.chem 121 (10), 1018-1024,1978, by condensing the "4+6" fragment, were synthesized first by liquid phase synthesis to protect the hexapeptide Z-Tyr (Bzl) -D-Ser-Arg (NO) 2 )-Pro-AzaGly-NH 2 Synthesizing a tetrapeptide pGlu-His-Trp-Ser-OMe; and synthesizing goserelin from the synthesized tetrapeptide fragment and hexapeptide fragment by adopting an azide method. US4024248 PL194997 et al employ a "3+7" fragment or a "5+5" condensation method. Firstly, liquid phase synthesis of a protective tripeptide pGlu-His-Trp-OMe, and then synthesis of a heptapeptide Z-Ser-Tyr-D-Ser (tBu) -Leu-Arg-Pro-Azaply-NH 2 . Then, through hydrazinolysis, deprotection and azide condensation, goserelin is obtained. However, the liquid phase fragment condensation has the steps of long synthesis period, complex synthesis operation, complex post-treatment and purification, low purity of crude peptide, hydrazinolysis, azide, removal of protective groups from palladium carbon and the like. Is not easy to enlarge production.
While the strategy of solid phase synthesis is mainly directed to protecting group strategies and deprotection methods. The common strategy is to synthesize the full-protection peptide first and then to react with NH 2 -Azagly-NH 2 Condensing and finally removing the protecting group in different modes. The solid-liquid phase combination is adopted, and the synthesis method is mainly to synthesize the full-protection peptide, then carry out C-terminal modification and then remove the protecting group. Only the choice of protecting groups is different, and the removal modes adopted are also different. The main protecting group site is the side chain of His, ser, tyr, arg. For example: patent 201010256054.6 uses His (Trt), ser (Trt), arg (HCl) protection, 5% TFA-DCM removal; patent 201210155366.7 uses His (Trt), tyr (Bzl), arg (NO) 2 ) Protecting and removing palladium hydrocarbon in a transferring mode; patent 201310557778.8 uses His (Trt), ser (Bzl), tyr (Bzl), arg (NO) 2 ) Removing palladium-carbon catalytic hydrogenolysis in methanol or ethanol solution; patent 201510005951.2 uses His (Trt), tyr (Bzl), arg (NO) 2 ) Protection, two-step removal, first pickingTrt was removed with 20% TFA-DCM and Bzl and NO were removed by hydrogen transfer 2 . However, most of the solid-liquid phase condensation methods adopt Bn and NO 2 The protecting group needs to be removed by liquid, and the common removing mode is palladium hydrocarbon reduction or palladium carbon/acetic acid/60 ℃ heating removal, the racemization and the damage of polypeptide three-dimensional structure can be caused by high temperature, and the palladium hydrocarbon reduction of the high-pressure reaction kettle has higher equipment requirement. And the C-terminal modification is carried out in a liquid phase, and side reactions such as self-condensation of the full-protection peptide exist, so that the yield is reduced, and under the technical scheme disclosed by the four patents, the yield of the goserelin crude peptide only reaches 30% -40%.
Therefore, development of a new synthesis process is urgently needed to solve the disadvantages of complicated liquid phase operation and unfavorable purification, and avoid a series of problems such as palladium-carbon reduction.
Disclosure of Invention
In view of this, the present invention provides a method for synthesizing goserelin. The goserelin obtained by the synthetic method has high yield, is simple to operate, is beneficial to purification, and avoids palladium-carbon reduction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for synthesizing goserelin, which comprises the following steps:
step A: coupling the Azagly with resin to obtain Azagly-resin;
and (B) step (B): pro, arg (Alloc) are sequentially coupled to the amino terminus of Azaply-resin 2 Leu, D-Ser (tBu), tyr (Trt), ser (Trt), trp, his (Mmt), pyr, resulting in Pyr-His (Mmt) -Trp-Ser (Trt) -Tyr (Trt) -D-Ser (tBu) -Leu-Arg (Alloc) 2 -Pro-Azagly-resin;
step C: removing Arg guanidyl protecting group Alloc in the peptide resin obtained in the step B by adopting a remover containing tetraphenylphosphine palladium and morpholine to obtain Pyr-His (Mmt) -Trp-Ser (Trt) -Tyr (Trt) -D-Ser (tBu) -Leu-Arg-Pro-Azagly-resin;
step D: c, cracking the peptide resin obtained in the step C by adopting a cracking solution to obtain Pyr-His-Trp-Ser-Tyr-D-Ser (tBu) -Leu-Arg-Pro-Azaply-NH 2 The amino acid sequence of the polypeptide is shown in SEQ ID NO:1, namely goserelin.
In the present invention, in step A and step B, the amino acid required for coupling is Fmoc-amino acid.
Preferably, azagly, pro, arg (Alloc) 2 Leu, D-Ser (tBu), tyr (Trt), ser (Trt), trp, his (Mmt) are Fmoc-Azaply-OH or Fmoc-Azaply-OMe, fmoc-Pro, fmoc-Arg (Alloc), respectively 2 、Fmoc-Leu、Fmoc-D-Ser(tBu)、Fmoc-Tyr(Trt)、Fmoc-Ser(Trt)、Fmoc-Trp、Fmoc-His(Mmt)。
Preferably, the Resin is a Sieber Resin.
Preferably, the substitution degree of Sieber Resin is 0.5 to 1.0mmol/g.
Preferably, the substitution degree of Sieber Resin is 0.9-1.0 mmol/g.
More preferably, the degree of substitution of the Sieber Resin is 1.0mmol/g.
Preferably, the coupling agent used in step A is HOAt/DIC or pyridine.
Preferably, the coupling agent used in step A is HOAt/DIC.
Preferably, the equivalent ratio of HOAt to DIC in the coupling agent HOAt/DIC is (1-2): (1-2).
Preferably, the equivalent ratio of HOAt to DIC in the coupling agent HOAt/DIC is 1:1.
preferably, the coupling agent used in step B is HOBt/DIC.
Preferably, the coupling time is 2 to 4 hours.
Preferably, the equivalent ratio of the tetraphenylphosphine palladium to the morpholine is (0.2 to 0.8): (15-25).
Preferably, the equivalent ratio of palladium tetraphenyl phosphine to morpholine is 0.5:20.
preferably, the removal agent containing the tetraphenylphosphine palladium and the morpholine further comprises a solvent DCM.
Preferably, the cleavage solution used for cleavage is a mixture of DCM, TFA and an organic capturing agent, or a mixture of DCM, TFA, an organic capturing agent and water.
Preferably, the organic capturing agent is one or a combination of more of TIS, ethylene Dithiol (EDT), anisole sulfide or phenyl sulfide.
Preferably, the removal is performed under nitrogen bubbling.
Preferably, the removal time is 2 to 3 hours.
Preferably, the volume ratio of DCM, TFA, organic capturing agent and water in the mixed solution of DCM, TFA, capturing agent and water is (70-90): (5-30): (2-10): (2-10).
Preferably, in the lysate, the volume ratio of DCM, TFA, organic capturing agent and water is (80-90): (8-12): (2-3): (2-3).
Preferably, the volume ratio of DCM, TFA and organic capturing agent in the mixed solution of DCM, TFA and organic capturing agent is (70-90): (5-30): (2-10).
Preferably, in the lysate, the volume ratio of DCM, TFA and organic capturing agent is (80-90): (8-12): (2-3).
Preferably, in the lysate, DCM: TFA: anisole: phenyl sulfide: the TIS has a volume ratio of 85:10:2:1:2; or DCM: TFA: TIS: h 2 The volume ratio of O is 85:10:2.5:2.5; or DCM: TFA: anisole: phenyl sulfide: the volume ratio of water is 85:10:2:1:2; or DCM: TFA: anisole: phenyl sulfide: the volume ratio of EDT was 85:10:2:1:2.
preferably, the time of cleavage is 1 to 3 hours.
Preferably, the method further comprises the steps of diethyl ether precipitation and centrifugation after the cleavage.
Preferably, the diethyl ether precipitation is carried out at freezing temperature.
Preferably, the centrifugation further comprises a step of purifying the goserelin crude peptide.
The invention provides a method for synthesizing goserelin. The synthesis method comprises the following steps: coupling the Azagly with resin to obtain Azagly-resin; amino acid is sequentially coupled at the amino terminal of Azaply-resin to obtain Pyr-His (Mmt) -Trp-Ser (Trt) -Tyr (Trt) -D-Ser (tBu) -Leu-Arg (Alloc) 2 -Pro-Azaply-resin; removing Arg guanidyl protecting group Alloc in the peptide resin obtained in the step B by adopting a remover containing tetraphenylphosphine palladium and morpholine to obtain Pyr-His (Mmt) -Trp-Ser (Trt) -Tyr (Trt) -D-Ser (tBu) -Leu-Arg-Pro-Azagly-resin; and C, cracking the peptide resin obtained in the step C by adopting a cracking solution to obtain goserelin. The invention has the technical effects that:
1. the Sieber Resin and the protective amino acid combination can realize coupling, deprotection and modification on the Resin without C-terminal modification, and the process has simple operation, convenient post-treatment and good reproducibility.
2. Introducing guanidine protecting group Alloc, removing with tetraphenylphosphine palladium/morpholine, and avoiding guanidine protecting group (NO 2 ) And the like, avoiding the liquid phase palladium hydrocarbon reduction step, and the hydrogenation step has high equipment requirement and is inconvenient for large-scale production.
3. The weak acid protecting groups of the side chains of His (Mmt), ser (Trt) and Tyr (Trt) are consistent with the cleavage conditions of Sieber Resin, so that the Resin cleavage and protecting group removal can be completed in one step. The operation is simple and convenient.
4. After the reaction, diethyl ether was directly precipitated instead of the concentrated TFA full-protected peptide mentioned in the literature, followed by C-terminal modification. During TFA concentration, the acid concentration increased, which tends to result in removal of the Ser (tBu) side chain protecting group tBu. The impurity is similar to the structure of the target product, and is difficult to remove.
Drawings
FIG. 1 shows an HPLC chromatogram of the crude peptide in example 5;
FIG. 2 shows an HPLC chromatogram of the refined peptide of example 5;
FIG. 3 shows an HPLC chromatogram of the crude peptide in example 6;
FIG. 4 shows an HPLC chromatogram of the crude peptide in example 7;
FIG. 5 shows an HPLC chromatogram of the crude peptide in comparative example 1;
FIG. 6 shows an HPLC chromatogram of the crude peptide in comparative example 2;
FIG. 7 shows an HPLC chromatogram of the crude peptide of comparative example 3;
FIG. 8 shows a flow chart of the synthesis of the polypeptide of the present invention.
Detailed Description
The invention discloses a method for synthesizing goserelin, which can be realized by appropriately improving process parameters by a person skilled in the art by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The synthesis flow chart of the polypeptide is shown in figure 8.
Abbreviations and English meanings are as follows:
the reagent or instrument used in the method for synthesizing goserelin provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 Synthesis of Fmoc-Azaply-NH-Sieb Resin
Sieber Resin with substitution rate of sub=0.76 mmol/g is weighed, swelled for 10min by DCM, drained, washed for 3 times by DMF, 20% piperidine-DMF (Fmoc removal) is added after draining, 5 minutes at a time, 7 minutes at a time, washed for 5 times by DMF, 3 times of Fmoc-Azaply-OH, 1.2eqHOAt/1.2eqDIC is added, and the coupling is carried out for 2 hours, so that ninhydrin detection is transparent. Then the resin was blocked with anhydride/pyridine, washed with DMF, DCM and shrunk with methanol to give the degree of substitution sub=0.50 mmol/g.
EXAMPLE 2 Synthesis of Fmoc-Azaply-NH-Sieb Resin
Weighing Sieber Resin with substitution rate of sub=0.5 mmol/g, swelling with DCM for 10min, draining, washing with DMF for 3 times, adding 20% piperidine-DMF (Fmoc removal) after draining, washing with DMF for 5 times once for 5min and 7 min once for 5min, adding 3 times of Fmoc-Azaply-OH, 1.2eqHOAt/1.2eqDIC, coupling for 2 hours, and detecting transparent ninhydrin. Then the resin was blocked with anhydride/pyridine, washed with DMF, DCM and shrunk with methanol to give the degree of substitution sub=0.32 mmol/g.
EXAMPLE 3 Synthesis of Fmoc-Azaply-NH-Sieb Resin
Weighing Sieber Resin with substitution rate of sub=1.0 mmol/g, swelling with DCM for 10min, draining, washing with DMF for 3 times, adding 20% piperidine-DMF (Fmoc removal) after draining, washing with DMF for 5 times for 5 minutes and 7 minutes, adding 3 times of Fmoc-Azaply-OH, 1.2eqHOAt/1.2eqDIC, coupling for 2 hours, and detecting transparent ninhydrin. Then the resin was blocked with anhydride/pyridine, washed with DMF, DCM and shrunk with methanol to give the degree of substitution sub=0.58 mmol/g.
EXAMPLE 4 Synthesis of Fmoc-Azaply-NH-Sieb Resin
Weighing Sieber Resin with substitution rate of sub=1.0 mmol/g, swelling with DCM for 10min, draining, washing with DMF for 3 times, adding 20% piperidine-DMF (Fmoc removal) after draining, washing with DMF for 5 times once for 5min and 7 min once, adding 3 times of Fmoc-Azaply-OMe, 3.5eq pyridine, coupling for 4 hours, and detecting with ninhydrin to be transparent. Then the resin was blocked with anhydride/pyridine, washed with DMF, DCM and shrunk with methanol to give the degree of substitution sub=0.34 mmol/g.
EXAMPLE 5 preparation of goserelin crude and refined peptides
10g (5.8 mmol) of Fmoc-Azagly-NH-Sieber Resin with substitution degree of sub=0.58 mmol/g obtained in example 3 is weighed, the mixture is swelled for 10min by DCM, the mixture is dried by suction, washed 3 times by DMF, 20% piperidine-DMF (Fmoc removal) is added after the dried mixture is dried by suction, one time is 5 minutes, one time is 7 minutes, the mixture is washed 5 times by DMF, 3 times of Fmoc-Pro-OH,1.2eqHOBt/1.2eqDIC is added, and the mixture is coupled for 2 hours, so that the ninhydrin is detected to be transparent; washing 3 times with DMF, adding 20% piperidine-DMF (Fmoc removed), 5min once, 7 min once, washing 5 times with DMF, and coupling Fmoc-Arg (Alloc) sequentially with reference to the above solid phase synthesis method 2 -OH、Fmoc-Leu-OH、Fmoc-D-Ser(tBu)-OH、Fmoc-Tyr(Trt)-OH、Fmoc-Ser(Trt)-OH、Fmoc-Trp-OH. Fmoc-His (Mmt) -OH, pyr-OH, after coupling, washed 3 times with DMF, 3 times with DCM and drained.
0.5eq of tetrakis triphenylphosphine palladium, 20eq of morpholine, dissolved in DCM, N2-pulsed, reacted for 2 hours, drained, 1% piperidine-DMF washed 3 times, DMF washed 5 times, DCM washed 5 times, and methanol shrunk to give approximately 21.02g of peptide resin.
DCM 340mL, TFA 40mL, organic capture TIS 10mL, H 2 O10 mL, formulation 85:10:2.5:2.5 lysate 400L. 21.0g of peptide resin was taken and added to 210mL of the above-mentioned lysate, followed by cleavage for 2 hours. The filtrate was filtered and precipitated in diethyl ether, centrifuged to obtain a crude peptide, which was dried with nitrogen to obtain 5.66g (4.46 mmol) of the crude peptide with a chromatographic purity of about 81.9% and a yield of 76.9%. The HPLC purity of the crude peptide is shown in FIG. 1.
The refined peptide was obtained after two preparation using a 0.3% ammonium acetate solution (pH 7.0) -0.1% acetic acid solution-acetonitrile system, kromasil C18 packed column, with a purity of about 99.41% and HPLC as shown in FIG. 2.
EXAMPLE 6 preparation of goserelin crude peptide
Weighing 10g (0.58 mmol) of Fmoc-Azagly-NH-Sieber Resin with substitution degree of sub=0.32 mmol/g, swelling for 10min by DCM, draining, washing 3 times by DMF, adding 20% piperidine-DMF (removing Fmoc) after draining, once for 5min, once for 7 min and washing 5 times by DMF, adding 3 times of Fmoc-Pro-OH,1.2eqHOBt/1.2eqDIC, coupling for 2 hours, and detecting transparency by ninhydrin; washing 3 times with DMF, adding 20% piperidine-DMF (Fmoc removed), 5min once, 7 min once, washing 5 times with DMF, and coupling Fmoc-Arg (Alloc) sequentially with reference to the above solid phase synthesis method 2 -OH, fmoc-Leu-OH, fmoc-D-Ser (tBu) -OH, fmoc-Tyr (Trt) -OH, fmoc-Ser (Trt) -OH, fmoc-Trp-OH, fmoc-His (Mmt) -OH, pyr-OH, after coupling, washing 3 times with DMF, washing 3 times with DCM, and draining.
0.5eq of tetrakis triphenylphosphine palladium, 20eq of morpholine, dissolved in DCM, N2 driven, reacted for 1.5 hours, drained, 1% piperidine-DMF washed 3 times, DMF washed 5 times, DCM washed 5 times, and methanol shrunk to give approximately 16.02g of peptide resin.
Taking 170mL of DCM and 20mL of TFA, and preparing an organic capturing agent comprising 4mL of TIS, 4mL of anisole and 2mL of phenylmethyl sulfide into 85:10:2:2:1, 200L of lysate. 16.0g of peptide resin was taken, 160mL (10-fold) of the above-mentioned lysate was added thereto, and the mixture was lysed for 2 hours. Filtering, precipitating the filtrate with diethyl ether, centrifuging to obtain crude peptide, and blow-drying with nitrogen to obtain crude peptide 3.19g with chromatographic purity of 72.4% and yield 80.44%, wherein the crude peptide HPLC is shown in figure 3.
EXAMPLE 7 preparation of goserelin crude peptide
Weighing 10g (0.58 mmol) of Fmoc-Azagly-NH-Sieber Resin with substitution degree of sub=0.32 mmol/g, swelling for 10min by DCM, draining, washing 3 times by DMF, adding 20% piperidine-DMF (removing Fmoc) after draining, once for 5min, once for 7 min and washing 5 times by DMF, adding 3 times of Fmoc-Pro-OH,1.2eqHOBt/1.2eqDIC, coupling for 2 hours, and detecting transparency by ninhydrin; washing 3 times with DMF, adding 20% piperidine-DMF (Fmoc removed), 5min once, 7 min once, washing 5 times with DMF, and coupling Fmoc-Arg (Alloc) sequentially with reference to the above solid phase synthesis method 2 -OH, fmoc-Leu-OH, fmoc-D-Ser (tBu) -OH, fmoc-Tyr (Trt) -OH, fmoc-Ser (Trt) -OH, fmoc-Trp-OH, fmoc-His (Mmt) -OH, pyr-OH, after coupling, washing 3 times with DMF, washing 3 times with DCM, and draining.
0.5eq of tetrakis triphenylphosphine palladium, 20eq of morpholine, dissolved in DCM, N2-pulsed, reacted for 2 hours, drained, 1% piperidine-DMF washed 3 times, DMF washed 5 times, DCM washed 5 times, and methanol shrunk to give about 15.87g of peptide resin.
170mL of DCM and 20mL of TFA are taken, and the organic capturing agent comprises 4mL of anisole, 2mL of phenylsulfide and 4mL of water to prepare 85: 200L of lysate of 10:2:1:2. 15.5g of peptide resin was taken, 124mL (8-fold) of the above lysate was added thereto, and the mixture was cleaved for 2 hours. Filtering, precipitating the filtrate with diethyl ether, centrifuging to obtain crude peptide, and blow-drying with nitrogen to obtain crude peptide 3.29g with chromatographic purity of about 84.12% and yield of 81.0%. The crude peptide HPLC is shown in FIG. 4.
Comparative example 1 preparation of goserelin crude peptide (conventional protocol)
Weighing 2-CTC Resin with substitution rate of sub=1.0 mmol/g, swelling with DCM for 10min, pumping, washing with DMF for 3 times, pumping, adding Fmoc-Pro-OH with 3 times equivalent weight and adding about 200ml of DMF for dissolution, coupling for 2 hours, cooling with ice-water bath, adding 8.7ml of DIPEA for activation for 10min, adding amino acid activation liquid into a reaction column for reaction, adding 17.5ml of DIPEA into the reactor for continuous reaction for 2 hours after 5min, adding DMF and DCM for washing after reaction, sealing with sealing liquid DCM/MEOH/DIPEA (17:2:1 volume ratio) for 2 times, washing with DMF for shrinkage after each 10min, sampling to measure substitution rate of 0.52mmol/g
19.2g (10 mmol) of the Fmoc-Pro-CTC Resin described above were taken, swollen for 10min with DCM, drained and washed 3 times with DMF, after draining 20% piperidine-DMF (Fmoc removed) was added, once for 5min, once for 7 min, washed 5 times with DMF and 3 times the amount of Fmoc-Arg (NO) 2 ) -OH,1.2 equbt/1.2 equdic, coupling for 2 hours, ninhydrin detection transparent; washing 3 times with DMF, adding 20% piperidine-DMF (Fmoc-removed), 5min once, 7 min once, and 5 times with DMF, coupling Fmoc-Leu-OH, fmoc-D-Ser (tBu) -OH, fmoc-Tyr (Bzl) -OH, fmoc-Ser (Trt) -OH, fmoc-Trp-OH, fmoc-His (Trt) -OH, and Pyr-OH, washing 3 times with DMF, and washing 3 times with DCM, and draining. The methanol shrinks.
The peptide resin was added to 1% TFA-DCM (V/V) solution and stirred for 2 hours, and concentrated to dryness to give a white solid. (m= 1533.40) Pyr-His (Trt) -Trp-Ser (Trt) -Tyr (Bzl) -D-Ser (tBu) -Leu-Arg (NO) 2 )-Pro-COOH。
Dissolving the solid with DCM, adding HOBt\DIC, stirring for dissolving, and adding NH 2 -Azagly-CONH 2 The reaction was stirred for 2 hours. Washing with 5% citric acid aqueous solution for 2 times, collecting DCM layer, and adding 10% Na 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid.
Pyr-His(Trt)-Trp-Ser(Trt)-Tyr(Bzl)-D-Ser(tBu)-Leu-Arg(NO 2 )-Pro-Azagly-NH 2
The above-mentioned compound was dissolved in methanol, 1.1 times equivalent of 10% Pd/C was added thereto, the mixture was pressurized to 10 atm, reacted at room temperature for 5 hours, palladium on carbon was filtered off, and the reaction mixture was added to frozen diethyl ether to precipitate a precipitate. The total yield was about 32%, HPLC 69.07% (fig. 5).
Comparative example 2 preparation of goserelin crude peptide (7+2)
Weighing 2-CTC Resin with substitution rate of sub=1.0 mmol/g, swelling with DCM for 10min, pumping, washing with DMF for 3 times, pumping, adding Fmoc-Leu-OH with 3 times equivalent weight and adding about 200ml of DMF for dissolution, coupling for 2 hours, cooling with ice-water bath, adding 8.7ml of DIPEA for activation for 10min, adding amino acid activation liquid into a reaction column for reaction, adding 17.5ml of DIPEA into the reactor for 2 hours after 5min, adding DMF and DCM for washing after reaction, sealing for 2 times with sealing liquid DCM/MEOH/DIPEA (17:2:1 volume ratio), sealing for 10min each time, washing with DMF for MeOH for shrinkage after sealing, and sampling to measure substitution rate of 0.52mmol/g
19.2g (10 mmol) of Fmoc-Leu-CTC Resin described above was taken, swollen for 10min with DCM, dried with suction, washed 3 times with DMF, after dried with 20% piperidine-DMF (Fmoc removed), once for 5min, once for 7 min, washed 5 times with DMF, 3 times the amount of Fmoc-D-Ser (tBu) -OH,1.2eqHOBt/1.2eqDIC was added, and the coupling was carried out for 2 hours, and ninhydrin detection was transparent; washing 3 times with DMF, after which 20% piperidine-DMF (Fmoc-removed) was added, once for 5 minutes, once for 7 minutes, washing 5 times with DMF, and then, referring to the above method of solid phase synthesis, fmoc-Tyr (Bzl) -OH, fmoc-Ser (Trt) -OH, fmoc-Trp-OH, fmoc-His (Trt) -OH, pyr-OH were coupled in sequence, washing 3 times with DMF, washing 3 times with DCM, and draining. The methanol shrinks.
The peptide resin was added to 1% TFA-DCM (V/V) solution and stirred for 2 hours, and concentrated to dryness to give a white solid. (m= 1533.40) Pyr-His (Trt) -Trp-Ser (Trt) -Tyr (Bzl) -D-Ser (tBu) -Leu-COOH.
Boc-Pro-OH and DCM were dissolved with stirring and 1.2 equivalents of NH was added 2 -Azagly-CONH 2 1.2 times equivalent of DCC was then added, the reaction was continued overnight at room temperature, the spot-plating was monitored, the starting material Boc-Pro-OH disappeared, and the mixture was washed 2 times with 5% aqueous citric acid to give a DCM layer and 10% Na 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. Boc-Pro-NH-Azaply-CONH 2 。
The above-mentioned solid is subjected to a reaction,dissolving in ethyl acetate solution of hydrogen chloride, stirring for 2 hr, removing Boc, concentrating to dryness after reaction, adding DCM for dissolving, and adding 10% Na 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. NH (NH) 2 -Pro-NH-Azagly-CONH 2 。
Boc-Arg(NO 2 ) Stirring to dissolve the-OH and DCM, adding 1.2 times of equivalent NH 2 -Pro-NH-Azagly-CONH 2 1.2 times equivalent of DCC was then added, the reaction was continued overnight at room temperature, the starting material Boc-Arg (NO 2) -OH was spotted, washed 2 times with 5% aqueous citric acid, the DCM layer was taken and 10% Na was then used 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. Boc-Arg (NO) 2 )-Pro-NH-Azagly-CONH 2 。
Dissolving the solid in ethyl acetate solution of hydrogen chloride, stirring for 2 hr, removing Boc, concentrating to dryness after the reaction, adding DCM for dissolving, and adding 10% Na 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. NH (NH) 2 -Arg(NO 2 )-Pro-NH-Azagly-CONH 2 。
Adding the Pyr-His (Trt) -Trp-Ser (Trt) -Tyr (Bzl) -D-Ser (tBu) -Leu-COOH into DCM, stirring for dissolving, adding 1.1 times of equivalent NH 2 -Arg(NO 2 )-Pro-NH-Azagly-CONH 2 1.1 times equivalent of DCC was then added, the reaction was performed at room temperature, the HPLC was monitored, the starting material Pyr-His (Trt) -Trp-Ser (Trt) -Tyr (Bzl) -D-Ser (tBu) -Leu-COOH disappeared, and 2 times the washing with 5% aqueous citric acid was performed to give a DCM layer, and 10% Na was further used 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. Pyr-His (Trt) -Trp-Ser (Trt) -Tyr (Bzl) -D-Ser (tBu) -Leu-Arg (NO) 2 )-Pro-NH-Azagly-CONH 2 。
The above-mentioned compound was dissolved in methanol, 1.1 times equivalent of 10% Pd/C was added thereto, the mixture was pressurized to 10 atm, reacted at room temperature for 5 hours, palladium on carbon was filtered off, and the reaction mixture was added to frozen diethyl ether to precipitate a precipitate. The overall yield was about 34%, HPLC 74.7% (fig. 6).
Comparative example 3 preparation of goserelin crude peptide (liquid phase Synthesis)
Boc-Pro-OH and DCM were dissolved with stirring and 1.2 equivalents of NH was added 2 -Azagly-CONH 2 1.2 times equivalent of DCC was then added, the reaction was continued overnight at room temperature, the spot-plating was monitored, the starting material Boc-Pro-OH disappeared, and the mixture was washed 2 times with 5% aqueous citric acid to give a DCM layer and 10% Na 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. Boc-Pro-NH-Azaply-CONH 2 。
Dissolving the solid in ethyl acetate solution of hydrogen chloride, stirring for 2 hr, removing Boc, concentrating to dryness after the reaction, adding DCM for dissolving, and adding 10% Na 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. NH (NH) 2 -Pro-NH-Azagly-CONH 2 。
Boc-Arg(NO 2 ) Stirring to dissolve the-OH and DCM, adding 1.2 times of equivalent NH 2 -Pro-NH-Azagly-CONH 2 1.2 times equivalent of DCC was then added, the reaction was continued overnight at room temperature, the starting material Boc-Arg (NO 2) -OH was spotted, washed 2 times with 5% aqueous citric acid, the DCM layer was taken and 10% Na was then used 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. Boc-Arg (NO) 2 )-Pro-NH-Azagly-CONH 2 。
Dissolving the solid in ethyl acetate solution of hydrogen chloride, stirring for 2 hr, removing Boc, concentrating to dryness after the reaction, adding DCM for dissolving, and adding 10% Na 2 Washing with CO water solution for 2 times, washing with saturated sodium chloride water solution for 2 times, collecting DCM layer, drying with anhydrous sodium sulfide, and concentrating to obtain white solid. NH (NH) 2 -Arg(NO 2 )-Pro-NH-Azagly-CONH 2 。
According to the upper partThe coupling and removing method sequentially carries out liquid phase condensation on Leu, D-Ser (tBu), tyr (Bzl), ser (Trt), trp, his (Trt) and Pyr. Obtaining Pyr-His (Trt) -Trp-Ser (Trt) -Tyr (Bzl) -D-Ser (tBu) -Leu-Arg (NO) 2 )-Pro-NH-Azagly-CONH 2 。
The above-mentioned compound was dissolved in methanol, 1.1 times equivalent of 10% Pd/C was added thereto, the mixture was pressurized to 10 atm, reacted at room temperature for 5 hours, palladium on carbon was filtered off, and the reaction mixture was added to frozen diethyl ether to precipitate a precipitate. The total yield was about 19%, HPLC 62.94% (fig. 7).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. A method for synthesizing goserelin, which is characterized by comprising the following steps:
step A: coupling Fmoc-Azagly-OH with the resin to obtain Azagly-resin; the Resin is Sieber Resin, and the substitution degree of the Sieber Resin is 0.5-1.0 mmol/g; the coupling agent adopted in the step A is HOAt/DIC or pyridine;
and (B) step (B): pro, arg (Alloc) are sequentially coupled to the amino terminus of Azaply-resin 2 Leu, D-Ser (tBu), tyr (Trt), ser (Trt), trp, his (Mmt), pyr, resulting in Pyr-His (Mmt) -Trp-Ser (Trt) -Tyr (Trt) -D-Ser (tBu) -Leu-Arg (Alloc) 2 -Pro-Azagly-resin; the coupling agent adopted in the step B is HOBt/DIC;
step C: removing Arg guanidyl protecting group Alloc in the peptide resin obtained in the step B by adopting a remover containing tetraphenylphosphine palladium and morpholine to obtain Pyr-His (Mmt) -Trp-Ser (Trt) -Tyr (Trt) -D-Ser (tBu) -Leu-Arg-Pro-Azagly-resin; the equivalent ratio of the tetraphenylphosphine palladium to the morpholine is (0.2-0.8): (15-25), wherein the remover containing the triphenylphosphine palladium and the morpholine further comprises a solvent DCM;
step D: adopting the lysate to carry out the process of the peptide resin obtained in the step CCleavage to obtain Pyr-His-Trp-Ser-Tyr-D-Ser (tBu) -Leu-Arg-Pro-Azaply-NH 2 I.e. goserelin;
the lysate adopted in the cleavage is a mixed solution of DCM, TFA and an organic capturing agent or a mixed solution of DCM, TFA, an organic capturing agent and water; the organic capturing agent is one or a combination of more of TIS, anisole sulfide or phenyl sulfide;
the volume ratio of DCM, TFA, organic capturing agent and water in the mixed solution of DCM, TFA, organic capturing agent and water is (70-90): (5-30): (2-10): (2-10), wherein the volume ratio of DCM, TFA and organic capturing agent in the mixed solution of DCM, TFA and organic capturing agent is (70-90): (5-30): (2-10).
2. The synthetic method of claim 1, wherein in the lysate, DCM: TFA: anisole: phenyl sulfide: the TIS has a volume ratio of 85:10:2:1:2; or DCM: TFA: TIS: h 2 The volume ratio of O is 85:10:2.5:2.5; or DCM: TFA: anisole: phenyl sulfide: the volume ratio of water is 85:10:2:1:2.
3. the method according to claim 1, wherein the step of precipitating diethyl ether and centrifuging is further included after the cleavage.
4. The method of claim 3, further comprising the step of refining the goserelin crude peptide after centrifugation.
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| WO2000071570A1 (en) * | 1999-05-20 | 2000-11-30 | Lipotec, S.A. | Solid phase peptide synthesis method |
| CN102653555A (en) * | 2012-05-18 | 2012-09-05 | 深圳翰宇药业股份有限公司 | Method for preparing goserelin by solid phase |
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| WO2000071570A1 (en) * | 1999-05-20 | 2000-11-30 | Lipotec, S.A. | Solid phase peptide synthesis method |
| US6897289B1 (en) * | 1999-05-20 | 2005-05-24 | Lipotec, S.A. | Peptide synthesis procedure in solid phase |
| CN102653555A (en) * | 2012-05-18 | 2012-09-05 | 深圳翰宇药业股份有限公司 | Method for preparing goserelin by solid phase |
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