CN1865280A - Solid phase polypeptide synthesis preparation method for leuprorelin - Google Patents

Solid phase polypeptide synthesis preparation method for leuprorelin Download PDF

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CN1865280A
CN1865280A CNA2005100260476A CN200510026047A CN1865280A CN 1865280 A CN1865280 A CN 1865280A CN A2005100260476 A CNA2005100260476 A CN A2005100260476A CN 200510026047 A CN200510026047 A CN 200510026047A CN 1865280 A CN1865280 A CN 1865280A
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leu
fmoc
tbu
pro
arg
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CN1865280B (en
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周达明
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Shanghai Soho Yiming Pharmaceuticals Co Ltd
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周达明
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    • 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

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Abstract

The invention discloses a bright-ala-ruilin preparing method of solid-phase polypeptide, which comprises the following steps: adopting Wang resin or CTC resin as original material to connect amino with protective group to produce protective nonapeptide resin; removing Fmoc-protective group sequently; proceeding side-chain protective group synchronizingly and cutting peptide; connecting ethylamine through ethylamine-to-HOBT to produce crude product; proceeding separation and purifying through C18 (or C8) pillar to produce fine bright-ala-ruilin. The invention avoids the utility of poisonous agent, which improves the purifying, peptide connecting and obtaining rate.

Description

The preparation method of the synthetic Leuprolide of solid-phase polypeptide
Technical field
The present invention relates to the preparation method of Leuprolide, relate in particular to the preparation method of the synthetic Leuprolide of solid-phase polypeptide.
Background technology
Leuprolide, Chinese is a leuprorelin acetate, or is called for short other titles: Leuprolide, English Leuprorelin by name, structural formula is pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHC 2H 5, molecular formula C 59H 84N 16O 12C 2H 4O 2, molecular weight is 1269.48.
The clinical dependency hormone illness that is used for the treatment of of Leuprolide comprises advanced prostate cancer, endometriosis, central precocious puberty etc.
Leuprorelin acetate is a kind of gonadotropin releasing hormone analogues, mainly act on prepituitary gland, heavy dose of application initial stage can cause that the property a crossed lutropin (LH) and follicular stimulating hormone (FSH) release increase, later stage then causes hypophysis susceptibility to reduce, and LH, FSH and sex hormone secretion are reduced.Leuprorelin acetate is used for the treatment of dependency hormone illness, comprising: advanced prostate cancer, endometriosis, central precocious puberty.
The people such as Fujino Masahiko of Japanese military field chemical industry in 1974 at first the nonapeptide amides of invention luteinising hormone-releasing hormo like the synthesis technique of thing.And in national applications patents such as Japan, Germany, the U.S., the patent No. is respectively: JP19740027442, DE2446005, US4008209 (liquid phase method).
Present method all exists certain defective, and each is taken turns and raises one's hat and must use trifluoracetic acid, so it is low to connect the peptide yield, causes production cost higher; Three-waste pollution is many, and uses trifluoracetic acid to carry out purifying, and contaminate environment, purification yield are generally below 25%.Therefore present method has limited the scale operation and the use of leuprorelin acetate.
Summary of the invention
The technical issues that need to address of the present invention are the preparation methods that disclose the synthetic Leuprolide of a kind of solid-phase polypeptide, to overcome the above-mentioned defective that prior art exists.
Method of the present invention comprises the steps:
With .Wang or CTC resin resin starting raw material; method according to solid phase synthesis connects the amino acid with blocking group successively; obtain protection nonapeptide resin; slough the Fmoc-blocking group therebetween successively; take off the side chain protected group synchronously and cut peptide, use ethamine/HOBT to connect ethylamino-at last, obtain crude product; crude product makes the Leuprolide elaboration through C18 (or C8) column separating purification.
According to the present invention, connect amino acid successively with blocking group, obtain protection nonapeptide resin, the method for sloughing the Fmoc-blocking group therebetween successively comprises the steps:
(1) preparation Fmoc-Pro-Wang resin:
Getting the Wang resin soaks with DMF, make the abundant swelling of resin, dry up, adding is dissolved in Fmoc-Pro-OH (FW:337.4), the TBTU/HBTU (FW:321) that connects peptide reagent, the mixture of HOBT (FW:153) then, and 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain the Fmoc-Pro-Wang resin; Add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, dried up, and used DMF and washing with alcohol respectively, dried up.
Said Wang resin replacement amount is: 0.5-1.1mmol/g;
The said peptide reagent that connects is: NMM: DMF=1: 5～15, and volume ratio, down together;
The said reagent of raising one's hat is: PIP: DMF=1: 2～5, and volume ratio, down together;
In the mixture:
The bulking value concentration of Wang resin is 5～20ml/g;
The mole number of TBTU/HBTU is 2～6 times of resin;
The mole number of HOBT=is 2～6 times of resin;
The mole number of Fmoc-Pro-OH is 2～6 times of resin.
The weight of Wang resin is 5～20ml/g with the ratio of the add-on of the reagent of raising one's hat;
(2) preparation Fmoc-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Pro-Wang resin of step (1), add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, and dried up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Arg (Pbf)-OH (FW:648.8), TBTU/HBTU and HOBT, 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Arg (Pbf)-Pro-Wang resin;
In the mixture:
The bulking value concentration of Fmoc-Pro-Wang resin is 5～20ml/g;
The mole number of TBTU/HBTU is 2～6 times of resin.
The HOBT mole number is 2～6 times of resin;
The mole number of Fmoc-Arg (Pbf)-OH is 2～6 times of resin.
The ratio of add-on of the weight of Fmoc-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
(3) preparation Fmoc-Leu-Arg (Pbf)-Pro-Wang resin
In the Fmoc-Arg of step (2) (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, and dried up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Leu-OH (FW:321), TBTU/HBTU and HOBT, 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
The bulking value concentration of Fmoc-Arg (Pbf)-Pro-Wang resin is 5～20ml/g;
The mole number of Fmoc-Leu-OH is 2～6 times of resin.
The ratio of add-on of the weight of Fmoc-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(4) preparation Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Leu-Arg of step (3) (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, and dried up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-D-Leu-OH (FW:354), TBTU/HBTU and HOBT, 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
The bulking value concentration of Fmoc-Leu-Arg (Pbf)-Pro-Wang resin is 5～20ml/g;
The mole number of Fmoc-D-Leu-OH is 2～6 times of resin.
The ratio of add-on of the weight of Fmoc-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(5) preparation Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-D-Leu-Leu-Arg of step (4) (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, dry up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Tyr (tBu)-OH (FW:459.5), TBTU/HBTU and HOBT, 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of Fmoc-Tyr (tBu)-OH is 2～6 times of resin.
The ratio of add-on of the weight of Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(6) preparation Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Tyr of step (5) (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, dry up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Ser (tBu)-OH (FW:383.4), TBTU/HBTU and HOBT, 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of Fmoc-Ser (tBu)-OH is 2～6 times of resin.
The ratio of add-on of the weight of Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(7) preparation Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Ser of step (6) (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, dry up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Trp-OH (FW:426.5), TBTU/HBTU and HOBT, 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of Fmoc-Trp-OH is 2～6 times of resin.
The ratio of add-on of the weight of Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(8) preparation Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Trp-Ser of step (7) (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, dry up, use DMF and washing with alcohol respectively, dry up, add with meeting peptide reagent dissolved Fmoc-His (Trt)-OH (FW:619.7), the mixture of TBTU/HBTU and HOBT, 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of Fmoc-His (Trt)-OH is 2～6 times of resin.
The ratio of add-on of the weight of Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(9) preparation pGlu-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-His of step (8) (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, 20～30 ℃ were reacted 5～30 minutes, dry up, use DMF and washing with alcohol respectively, dry up, add with meeting peptide reagent dissolved pGlu-OH (FW:129), the mixture of TBTU/HBTU and HOBT, 20～30 ℃ were reacted 0.5～1.5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain pGlu-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of pGlu-OH is 2～6 times of resin.
The ratio of add-on of the weight of Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same.
According to the present invention, the said peptide of cutting comprises the steps:
PGlu-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf) that above-mentioned steps (9) is obtained-Pro-Wang resin add be chilled in advance 0～10 ℃ cut peptide reagent (TFA/EDT/H 2O/TIS=680ml/18ml/18ml/3.6ml) in, 20～30 ℃ were reacted 1～3 hour, removed solvent under reduced pressure, added ether sedimentation, and the collecting precipitation thing is washed with ether, P 2O 5Vacuum-drying, the crude product behind the peptide is cut in acquisition;
According to the present invention, the method that said ethamine/HOBT connects ethylamino-comprises the steps:
With the mixture lyophilize of HOBT, water and ethamine, obtain HOBt-ethamine mixture;
HOBt-ethamine mixture is added in the crude product cut behind the peptide, add DIC again, 20～30 ℃ of reactions 8～24 hours, the precipitation that adds diethyl ether, collecting precipitation, with the ether washing, acquisition Leuprolide crude product, yield 80%;
The part by weight of HOBT, water and ethamine is:
HOBT: water: ethamine=1: 50～100: 0.5～2;
According to the present invention, crude product comprises the steps: through the method for C18 (or C8) column separating purification separation and purification
The Leuprolide crude product is dissolved in the acetate, filters, filtrate is through C18 (or C8) column purification, moving phase: 0.1MNH 4Ac: acetonitrile (7.5: 2.5); Flow velocity is 100-650ml/min; The detection wavelength is: 280nm; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph, the sample peak desalts after merging, and freeze-drying obtains finished product (MW:1209), total recovery 27.6% (in the 60mmol of Wang resin).
The weight concentration of acetate is 0.5～10%, and the weight concentration of Leuprolide crude product is 1～50% in the acetate.
Leuprolide operational path of the present invention has following characteristics, possesses the large-scale production ability, process stabilizing; the raw and auxiliary material convenient sources, with short production cycle, production cost is low; the three wastes are few; the yield height, steady quality, production cost is low; has the market competitiveness; connect peptide yield height (per step connects the peptide yield and is 〉=99%), reduced cost, adopt and cut peptide reagent (TFA/EDT/H 2O/TIS) cut peptide, the method that adds the ether sedimentation crude product, avoid using poisonous reagents such as hydrogen fluoride, three-waste pollution is few, and improves yield, adopts C18 (or C8) post to carry out separation and purification, avoid using TFA (trifluoracetic acid) to carry out purifying, reduce the three wastes, purification yield is up to more than 25%, and per step connects the peptide yield all more than 99%; Yield is after cutting peptide: 95.4%, and total recovery is: 21.9%.
Embodiment
Embodiment 1
The raw material that is adopted in embodiment and the aforementioned process is listed as follows:
No The name of an article Production firm
1 The Wang resin Tianjin with become Science and Technology Ltd.
2 Fmoc-Pro-OH Sichuan Sangao Biochemical Co., Ltd
3 Fmoc-Arg(Pbf)-OH Sichuan Sangao Biochemical Co., Ltd
4 Fmoc-Leu-OH Sichuan Sangao Biochemical Co., Ltd
5 Fmoc-D-Leu-OH Sichuan Sangao Biochemical Co., Ltd
6 Fmoc-Tyr(tBu)-OH Sichuan Sangao Biochemical Co., Ltd
7 Fmoc-Ser(tBu)-OH Sichuan Sangao Biochemical Co., Ltd
8 Fmoc-Trp-OH Sichuan Sangao Biochemical Co., Ltd
9 Fmoc-His(Trt)-OH Sichuan Sangao Biochemical Co., Ltd
10 p-Glu-OH Sichuan Sangao Biochemical Co., Ltd
11 1-hydroxy benzo triazole (HOBt) Sichuan Sangao Biochemical Co., Ltd
12 TBTU Sichuan Sangao Biochemical Co., Ltd
13 Trifluoroacetic acid (TFA) Merck company
14 N-methylmorpholine (NMM) Merck company
15 Dimethyl formamide (DMF) Shanghai chemical reagents corporation
16 Dehydrated alcohol Shanghai development chemical industry one factory
17 Hexahydropyridine (PIP) Shanghai chemical reagents corporation
18 Salt of wormwood Shanghai chemical reagents corporation
Among the embodiment:
The said peptide reagent that connects is: NMM: DMF=1: 10, and volume ratio;
The said reagent of raising one's hat is: PIP: DMF=1: 3.5, and volume ratio;
Synthetic peptide chain
Preparation Fmoc-Pro-Wang resin
1) (the 0.8mmol/g resin 60mmol), soaks with 1000mlDMF, makes the abundant swelling of resin, dries up to get the 75gWang resin.
2) (FW:337.4,240mmol) (FW:321,240mmol) (FW:153,240mmol) HOBT connect the peptide reagent dissolving with 750ml, add reaction vessel, and 25 ℃ were reacted 1 hour for TBTU/HBTU, 36.6g for Fmoc-Pro-OH, 77.1g to get 81g.
3) dry up, respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up, get 85g approximately.
Preparation Fmoc-Arg (Pbf)-Pro-Wang resin:
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes dry up, and respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.
2) add 155.7g (FW:648.8,240mmol) Fmoc-Arg (Pbf)-OH, 77.1g (FW:321,240mmol) TBTU/HBTU, 36.6g (FW:153,240mmol) HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) dry up, respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.
Preparation Fmoc-Leu-Arg (Pbf)-Pro-Wang resin:
Add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes dry up, and respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.(FW:354,240mmol) (FW:321,240mmol) (FW:153,240mmol) HOBT connect the peptide reagent dissolving with 750ml, add reaction vessel, and 25 ℃ were reacted 1 hour for TBTU/HBTU, 36.6g for Fmoc-Leu-OH, 77.1g to add 84.9g.All the other operations are the same.
Preparation Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
Add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes dry up, and respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.(FW:354,240mmol) (FW:321,240mmol) (FW:153,240mmol) HOBT connect the peptide reagent dissolving with 750ml, add reaction vessel, and 25 ℃ were reacted 1 hour for TBTU/HBTU, 36.6g for Fmoc-D-Leu-OH, 77.1g to add 84.9g.All the other operations are the same.
Preparation Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
Add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes dry up, and respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.(FW:459.5,240mmol) (FW:321,240mmol) (FW:153,240mmol) HOBT connect the peptide reagent dissolving with 750ml to Fmoc-Tyr (tBu)-OH, 77.1g, add reaction vessel, and 25 ℃ were reacted 1 hour for TBTU/HBTU, 36.6g to add 110.4g.All the other operations are the same.
Preparation Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
Add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes dry up, and respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.(FW:383.4,240mmol) (FW:321,240mmol) (FW:153,240mmol) HOBT connect the peptide reagent dissolving with 750ml to Fmoc-Ser (tBu)-OH, 77.1g, add reaction vessel, and 25 ℃ were reacted 1 hour for TBTU/HBTU, 36.6g to add 92.1g.
Preparation Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
Add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes dry up, and respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.(FW:426.5,240mmol) (FW:321,240mmol) (FW:153,240mmol) HOBT connect the peptide reagent dissolving with 750ml, add reaction vessel, and 25 ℃ were reacted 1 hour for TBTU/HBTU, 36.6g for Fmoc-Trp-OH, 77.1g to add 126.3g.All the other operations are the same.
Preparation Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
Add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes dry up, and respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.(FW:619.7,240mmol) (FW:321,240mmol) (FW:153,240mmol) HOBT connect the peptide reagent dissolving with 750ml to Fmoc-His (Trt)-OH, 77.1g, add reaction vessel, and 25 ℃ were reacted 1 hour for TBTU/HBTU, 36.6g to add 148.7g.Add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 10 minutes.
Preparation pGlu-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin
Add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes dry up, and respectively wash three times with DMF, dehydrated alcohol and DMF respectively, dry up.(FW:129,240mmol) (FW:321,240mmol) (FW:153,240mmol) HOBT connect the peptide reagent dissolving with 750ml, add reaction vessel, and 25 ℃ were reacted 1 hour for TBTU/HBTU, 36.6g for pGlu-OH, 77.1g to add 30.9g.
Cut peptide:
To protect nonapeptide to be transferred in the 1000ml reactor, adding prepares and is chilled in advance about 5 ℃ reagent (TFA/EDT/H in advance 2O/TIS=680ml/18ml/18ml/3.6ml), 25 ℃ were stirred 2 hours.Decompression desolventizes to small volume, adds 1000ml cold diethyl ether precipitation, centrifugal collection, and after washing with anhydrous diethyl ether, P 2O 5Vacuum-drying.
Ethamineization:
The preparation of HOBt-ethamine: 67.5gHOBT adds 6000ml water, adds 67ml ethamine and is stirred to the HOBt dissolving.Lyophilize.HOBt-ethamine is transferred in the crude product of cutting behind the peptide, added DIC 48g again, 25 ℃ of stirrings are spent the night.Ether sedimentation.Filter,, get Leuprolide crude product 58g (48.0mmol), yield 80% with ether washing three times.
Separation and purification:
The Leuprolide crude product is dissolved in 5% acetate, filters, filtrate is through the C18 column purification, moving phase: 0.1MNH 4Ac: acetonitrile (7.5: 2.5); Flow velocity is 500ml/min; The detection wavelength is: 280nm; Follow the tracks of to collect needed effluent liquid with liquid chromatograph, the sample peak desalts after merging, freeze-drying, approximately 20g white block finished product (MW:1209,6.2mmol), total recovery 27.6% (in the 60mmol of Wang resin).

Claims (7)

1. the preparation method of the synthetic Leuprolide of solid-phase polypeptide is characterized in that, comprises the steps:
With .Wang resin or CTC resin starting raw material; method according to solid phase synthesis connects the amino acid with blocking group successively; obtain protection nonapeptide resin; slough the Fmoc-blocking group therebetween successively; take off the side chain protected group synchronously and cut peptide, use ethamine/HOBT to connect ethylamino-at last, obtain crude product; crude product makes the Leuprolide elaboration through C18 (or C8) column separating purification.
2. method according to claim 1 is characterized in that, connects the amino acid with blocking group successively, obtains protection nonapeptide resin, and the method for sloughing the Fmoc-blocking group therebetween successively comprises the steps:
(1) preparation Fmoc-Pro-Wang resin:
Getting the Wang resin soaks with DMF, make the abundant swelling of resin, dry up, add then and be dissolved in Fmoc-Pro-OH (FW:337.4), the TBTU/HBTU (FW:321) that connects peptide reagent, the mixture of HOBT (FW:153), reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain the Fmoc-Pro-Wang resin;
The said peptide reagent that connects is: NMM: DMF=1: 5～15, down together;
The said reagent of raising one's hat is: PIP: DMF=1: 2～5, down together;
In the mixture:
The bulking value concentration of Wang resin is 5～20ml/g;
Fmoc-Pro-OH mole number be 2～5 times of resin;
The weight of Wang resin is 5～20ml/g with the ratio of the add-on of the reagent of raising one's hat
The reagent of raising one's hat;
(2) preparation Fmoc-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Pro-Wang resin of step (1), add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-Arg (Pbf)-OH (FW:648.8), TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-Arg (Pbf)-Pro-Wang resin;
In the mixture:
The bulking value concentration of Fmoc-Pro-Wang resin is 5～20ml/g;
The mole number of Fmoc-Arg (Pbf)-OH is 2～5 times of resin;
The ratio of add-on of the weight of Fmoc-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
(3) preparation Fmoc-Leu-Arg (Pbf)-Pro-Wang resin
In the Fmoc-Arg of step (2) (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-Leu-OH (FW:321), TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
The bulking value concentration of Fmoc-Arg (Pbf)-Pro-Wang resin is 5～20ml/g;
The mole number of Fmoc-Leu-OH is 2～5 times of resin.
The ratio of add-on of the weight of Fmoc-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(4) preparation Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Leu-Arg of step (3) (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-D-Leu-OH (FW:354), TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
The bulking value concentration of Fmoc-Leu-Arg (Pbf)-Pro-Wang resin is 5～20ml/g;
The mole number of Fmoc-D-Leu-OH is 2～5 times of resin;
The ratio of add-on of the weight of Fmoc-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(5) preparation Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-D-Leu-Leu-Arg of step (4) (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, reaction dries up, and uses DMF and washing with alcohol respectively, dries up.Add with the mixture that meets peptide reagent dissolved Fmoc-Tyr (tBu)-OH (FW:459.5), TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of Fmoc-Tyr (tBu)-OH is 2～5 times of resin;
The ratio of add-on of the weight of Fmoc-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(6) preparation Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Tyr of step (5) (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, reaction dries up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Ser (tBu)-OH (FW:383.4), TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of Fmoc-Ser (tBu)-OH is 2～5 times of resin.
The ratio of add-on of the weight of Fmoc-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(7) preparation Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Ser of step (6) (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, reaction dries up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Trp-OH (FW:426.5), TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of Fmoc-Trp-OH is 2～5 times of resin;
The ratio of add-on of the weight of Fmoc-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(8) preparation Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-Trp-Ser of step (7) (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, reaction, dry up, use DMF and washing with alcohol respectively, blow in, add with the mixture that meets peptide reagent dissolved Fmoc-His (Trt)-OH (FW:619.7), TBTU/HBTU and HOBT, reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of Fmoc-His (Trt)-OH is 2～5 times of resin.
The ratio of add-on of the weight of Fmoc-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same;
(9) preparation pGlu-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin:
In the Fmoc-His of step (8) (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin, add the reagent of raising one's hat, reaction, dry up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved pGlu-OH (FW:129), TBTU/HBTU and HOBT, reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain pGlu-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin;
In the mixture:
Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang weight resin volumetric concentration is 5～20ml/g;
The mole number of pGlu-OH is 2～5 times of resin;
The ratio of add-on of the weight of Fmoc-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf)-Pro-Wang resin and the reagent of raising one's hat is 5～20ml/g reagent of raising one's hat;
All the other operations and processing condition are the same.
3. method according to claim 2 is characterized in that, the temperature of reaction of the first step is 20～30 ℃, and the reaction times is 0.5～1.5 hour, and the temperature of reaction in second step is 20～30 ℃, and the reaction times is 5～30 minutes.
4. method according to claim 1 is characterized in that, the said peptide of cutting comprises the steps:
PGlu-His (Trt)-Trp-Ser (tBu)-Tyr (tBu)-D-Leu-Leu-Arg (Pbf) that above-mentioned steps (9) is obtained-Pro-Wang resin add be chilled in advance 0～10 ℃ cut peptide reagent (TFA/EDT/H 2O/TIS=680ml/18ml/18ml/3.6ml) in, 20～30 ℃ were reacted 1～3 hour, removed solvent under reduced pressure, added ether sedimentation, and the collecting precipitation thing is washed with ether, P2O5 vacuum-drying, and the crude product behind the peptide is cut in acquisition.
5. method according to claim 1 is characterized in that the method that said ethamine/HOBT connects ethylamino-comprises the steps:
With the mixture lyophilize of HOBT, water and ethamine, obtain the HOBt-ethylamine compounds;
HOBt-ethamine mixture is added in the crude product cut behind the peptide, add DIC or TBTU, HBTU again, 20～30 ℃ of reactions 8～24 hours, the precipitation that adds diethyl ether, collecting precipitation, with the ether washing, acquisition Leuprolide crude product, yield 80%;
The part by weight of HOBT, water and ethamine is:
HOBT: water: ethamine=1: 50～100: 0.5～2.
6. method according to claim 1 is characterized in that, crude product comprises the steps: through the method for C18 or the separation and purification of C8 column separating purification
The Leuprolide crude product is dissolved in the acetate, filters, filtrate is through C18 or C8 column purification, moving phase: 0.1MNH 4Ac: acetonitrile (7.5: 2.5); Flow velocity is 100-650ml/min; The detection wavelength is: 280nm; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph, the sample peak desalts after merging, and freeze-drying obtains finished product (MW:1209), total recovery 27.6% (in the 60mmol of Wang resin).
7. method according to claim 6 is characterized in that, the weight concentration of ammonium acetate is 0.01～10%.The weight concentration of acetonitrile is 10～40%.
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CN101195653B (en) * 2006-12-08 2010-05-12 吉尔生化（上海）有限公司 solid-liquid synthesizing method for leuprorelin
CN101538315B (en) * 2009-01-13 2012-11-28 深圳翰宇药业股份有限公司 Method for preparing Leuprorelin by combination of solid phase method and liquid phase method
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CN103554229A (en) * 2013-11-11 2014-02-05 宁波市三生药业有限公司 Solid-phase synthesis method for Buserelin
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CN105330726A (en) * 2015-11-05 2016-02-17 江苏诺泰生物制药股份有限公司 Leuprorelin synthesis method
CN106146622A (en) * 2015-04-03 2016-11-23 中肽生化有限公司 A kind of industrialized process for preparing of leuprorelin acetate
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CN109593118A (en) * 2018-12-18 2019-04-09 江苏诺泰澳赛诺生物制药股份有限公司 A kind of liquid-phase synthesis process of Leuprorelin

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CN101195653B (en) * 2006-12-08 2010-05-12 吉尔生化（上海）有限公司 solid-liquid synthesizing method for leuprorelin
CN101357936B (en) * 2007-07-31 2013-07-03 崔颀 Method for synthesizing triptorelin from solid phase polypeptide
CN101538315B (en) * 2009-01-13 2012-11-28 深圳翰宇药业股份有限公司 Method for preparing Leuprorelin by combination of solid phase method and liquid phase method
CN103554229A (en) * 2013-11-11 2014-02-05 宁波市三生药业有限公司 Solid-phase synthesis method for Buserelin
CN103554229B (en) * 2013-11-11 2016-01-06 宁波市三生药业有限公司 The method of solid phase synthesis buserelin
CN104004054A (en) * 2014-05-04 2014-08-27 安徽瀚海博兴生物技术有限公司 Synthetic method for goserelin
CN106146622A (en) * 2015-04-03 2016-11-23 中肽生化有限公司 A kind of industrialized process for preparing of leuprorelin acetate
CN105330726A (en) * 2015-11-05 2016-02-17 江苏诺泰生物制药股份有限公司 Leuprorelin synthesis method
CN106518979A (en) * 2016-10-24 2017-03-22 合肥国肽生物科技有限公司 Leuprorelin synthesis preparation method
CN106518979B (en) * 2016-10-24 2019-08-30 合肥国肽生物科技有限公司 A kind of synthesis preparation method of Leuprorelin
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