CN101357937B - Method for synthesizing atosiban acetate from solid phase polypeptide - Google Patents

Method for synthesizing atosiban acetate from solid phase polypeptide Download PDF

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CN101357937B
CN101357937B CN200710044420XA CN200710044420A CN101357937B CN 101357937 B CN101357937 B CN 101357937B CN 200710044420X A CN200710044420X A CN 200710044420XA CN 200710044420 A CN200710044420 A CN 200710044420A CN 101357937 B CN101357937 B CN 101357937B
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resin
fmoc
trt
dmf
gly
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CN101357937A (en
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崔颀
周逸明
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Shanghai Soho Yiming Pharmaceuticals Co Ltd
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Shanghai Soho Yiming Pharmaceuticals Co Ltd
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Abstract

The invention discloses a preparation method of solid phase peptide synthesis atosiban which includes the following steps: taking Rink Amide resins, Rink Amide MBHA resins or Rink Amide AM resins as starting materials and taking Fmoc amino acids as monomers, amino acids are grafted one by one and mercaptopropionic acids (Map(SX)) are protected by the last peptide chain; after protected nonapeptide resins are obtained, the acellular side-chain protective groups and cutting peptides are synchronized; then cutting peptides is carried out, and reduced crude atosiban is collected; the pH value is adjusted to 7.5 to 10.0, and oxidized crude atosiban is collected; target products are obtained by the separation and purification by preparative HPLC(C18 or C8 column). The preparation method is convenient in material source, simplifies technology and reduces cost; and the preparation method is low in the pollution of three wastes and is high in yield, and the preparation method is convenient for being industrialized and has good industrialization prospect.

Description

Preparing method as the synthetic atropic west of solid-phase polypeptide
Technical field
The present invention relates to the preparation method as a kind of atropic west, be specifically related to the preparation method as the synthetic atropic west of solid-phase polypeptide.
Background technology
Chinese name: as the atropic west, as the acetic acid atropic west, RWJ-22164, acetic acid RWJ-22164.
English name: Atosiban, Atosiban Acetate.
Structural formula is:
Figure RE-S07144420X20070828D000011
Molecular formula is: C 43H 67N 11O 12S 2
Molecular weight: 994.2
Be the Uteracon competitive antagonist of acceptor on a kind of intrauterine and decidua, the fetal membrane that can quiet notes clinically as the atropic west.But dosage correlation ground suppresses uterine contraction behind these article of use, and the prostaglandin(PG) secretion of Uteracon mediation is reduced, and reaches the purpose of preventing miscarriage, and can be used for more than 18 years old pregnant 24-33 weeks of age, the normal pregnant woman of fetal heart frequency.In external pregnant woman's the clinical experiment; These article respectively with β2Ji Dongji (ritodrine, salbutamol, terbutaline) in a kind ofly carried out the double blinding comparative studies; The synthesis result that the treatment intention is analyzed shows; These article suppress uterine contraction effectively, reach the effect of preventing miscarriage, and untoward reaction and drug withdrawal person are all less.Two groups pregnant age of average production and fetus or baby's index (baby weight, survival rate, respiratory distress syndrome or the incidence of intracranialing hemorrhage) is not seen significant difference in the analysis-by-synthesis.
USP 4,504,469th adopts the Boc route, is starting raw material with the chloromethyl resin, accomplishes connecing peptide through condensation reaction, cuts peptide with exsiccant liquefied ammonia, after oxidizing reaction and separation and purification.The major defect that this patent exists is, the production cycle is longer, complicated operation, and cost of labor is higher, and it is more to produce the three wastes, can not satisfy the needs of the parties concerned.
Summary of the invention
The objective of the invention is to disclose the preparation method as the synthetic atropic west of a kind of solid-phase polypeptide, to overcome the defective that prior art exists.
Method of the present invention comprises the steps:
(1) be starting raw material with Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin; Amino acid with the Fmoc protection is monomer; Use TBTU/HOBt, HBTU/HOBt, BOP/HOBt, TBTU/HOAt, HBTU/HOAt or BOP/HOAt to connect reactive polypeptide as condensing agent; Connect amino acid one by one, last peptide chain uses the thiohydracrylic acid (Map (X)) of protection; Behind the nonapeptide resin that must protect, take off the side chain protected group synchronously and cut peptide.
Said Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin are a kind of aminoresin commonly used, can adopt the commercially available prod, are the product of HC RAm04-1 like Tianjin Nankai Hecheng S&T Co., Ltd.'s trade mark;
(2) adding is cut peptide reagent (TFA/HBr/HAc/TIS/EDT) and is cut peptide then;
(3) with the ether be solvent, deposition is also collected bullion as the reduced form atropic west;
(4) bullion as the reduced form atropic west is soluble in water, regulate PH to 7.5-10.0 with ammoniacal liquor, bullion as the collection oxidized form atropic west;
(5) bullion of collecting is passed through preparation HPLC (C 18Or C 8Post) separation and purification obtains title product, and total recovery can reach more than 18.5%.
Amino acid with the Fmoc protection is monomer, and the temperature of reaction when connecting amino acid one by one is 10～60 ℃, and cutting the peptide temperature is 10～60 ℃;
Preferably, method of the present invention comprises the steps:
Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin and Fmoc-Gly-OH, TBTU/HOBT and hexahydropyridine reaction obtain the Fmoc-Gly-resin;
With Fmoc-Om (Boc)-OH, TBTU/HOBT and hexahydropyridine reaction, obtain Fmoc-Orn (Boc)-Gly-resin then;
With Fmoc-Pro-OH, TBTU/HOBT and hexahydropyridine reaction, obtain Fmoc-Pro-Orn (Boc)-Gly-resin then;
With Fmoc-Cys (Trt)-OH, TBTU/HOBT and hexahydropyridine reaction, obtain Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With Fmoc-Asn (Trt)-OH, TBTU/HOBT and hexahydropyridine reaction, obtain Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc--Thr (tBu)-OH/TBTU/HOBt reaction, obtain Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc-Ile-OH/TBTU/HOBt reaction, obtain Fmoc-Ile--Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc-D-Tyr (OEt)-OH/TBTU/HOBt reaction, obtain Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmap (X)/TBTU/HOBt reaction, obtain Map (SX)-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With the TFA/HBr/HAc/TIS/EDT reaction, obtain Map-D-Tyr (OEt)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH then 2(reduced form RWJ-22164);
Carry out then: 1. oxidation; 2. purifying; 3. desalt; 4. freeze-drying obtains:
Figure DEST_PATH_S07144420X20071115D000021
More concrete comprises the steps:
(1) pre-treatment of resin: with Rink Amide resin, Rink Amide mbha resin or Rink AmideAM resin, soaked 0.5～2 hour, make the abundant swelling of resin with DMF; Nitrogen dries up; The DMF solution that adds hexahydropyridine, 10～80 ℃ were reacted 10～60 minutes, and nitrogen blows elimination and removes hexahydropyridine; The DMF washing, nitrogen dries up.
In the DMF solution of hexahydropyridine, hexahydropyridine: DMF=1: 3～5 volume ratios;
In the DMF solution of hexahydropyridine, the concentration ratio of Rink Amide mbha resin is: 1 gram/5～15ml.
(2) preparation of Fmoc-Gly-resin:
The pretreated resin of step (1) is mixed with Fmoc-Gly-OH (MW:297.3), TBTU (MW:321), HOBT (MW:135.1), NMM (MW=101.2) and DMF; Mixture was reacted under 10～60 ℃ condition 0.5～3.0 hour; Nitrogen dries up, the DMF washing, and nitrogen dries up;
NMM: DMF=1: 5～15, volume ratio;
The mole number of Fmoc-Gly-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
The DMF solution that adds hexahydropyridine then, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, and with the DMF washing, nitrogen dries up, and obtains the Fmoc-Gly-resin;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml
Reaction expression is:
Answer general formula to be:
(3) preparation of Fmoc-Orn (Boc)-Gly-resin:
In the Fmoc-Gly-resin of step (2), add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Orn (Boc)-OH (MW:454.5), TBTU (MW:321), HOBT (MW:135.1), NMM (MW=101.2) and DMF and mix, 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Orn (Boc)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
Reaction expression is:
Figure RE-S07144420X20070828D000042
(4) preparation of Fmoc-Pro-Orn (Boc)-Gly-resin:
In the Fmoc-Orn of step (3) (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Pro-OH (MW337.4), TBTU (MW:321), HOBT (MW:135.1), NMM (MW=101.2) and DMF and mix, 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Pro-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
Reaction expression is:
Figure RE-S07144420X20070828D000051
(5) preparation of Fmoc-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Pro-Orn of step (4) (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Cys (Trt)-OH (MW:585.7), TBTU MW:321), HOBT (MW:135.1), NMM (MW=101.2) mix with DMF, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, DMF washs, nitrogen dries up; Obtain Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Cys (Trt)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
Reaction expression is:
Figure RE-S07144420X20070828D000052
(6) preparation of Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Cys of step (5) (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Asn (Trt)-OH (MW:596.7), TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Asn (Trt)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
Reaction expression is:
Figure RE-S07144420X20070828D000061
(7) preparation of Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Asn of step (6) (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Thr (tBu)-OH (MW:397.5), TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Thr (tBu)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
Reaction expression is:
Figure RE-S07144420X20070828D000071
(8) preparation of Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Thr of step (7) (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Ile-OH (MW:353.4), TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Ile-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
Reaction expression is:
Figure RE-S07144420X20070828D000072
(9) preparation of Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Ile-Thr of step (8) (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-D-Tyr (OEt)-OH (MW:413), TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-D-Tyr (OEt)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration that six hydrogen adjoin pyridine is 10～40%;
Six hydrogen adjoin in the DMF solution of pyridine, and the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
Reaction expression is:
Figure RE-S07144420X20070828D000081
(10) preparation of Map (SX)-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-D-Tyr of step (9) (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Map (SX)-OH, TBTU, HOBT, NMM and DMF, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Map (SX) is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
Methanol wash is drained, and nitrogen dries up, and must protect resin as the reduced form atropic west;
Reaction expression is:
Figure RE-S07144420X20070828D000091
(11) cut peptide
Add resin as the reduced form atropic west of protection with cutting peptide reagent, 10～60 ℃ of reactions 2～6 hours are filtered, drain, and the filtrating deposition that adds diethyl ether, the filtration collecting precipitation gets bullion (MW=833.96) as the reduced form atropic west, and yield is 96%;
The said peptide reagent of cutting is:
TFA/HBr/HAc/TIS/EDT＝(800-900ml/25-40ml/5-20ml/40-60ml/20-30ml)；
Reaction expression is:
Figure RE-S07144420X20070828D000092
In the weight of resin as the reduced form atropic west, the add-on of cutting peptide reagent is 5～20ml/g;
(12) oxidation (formation of disulfide linkage)
Bullion as the reduced form atropic west is soluble in water, and adding concentration is the ammoniacal liquor of 0.1～1.5mol/L, regulates pH to 7.5-10.0, blowing air stir about 15～30 hours, filtration;
Reaction expression is:
Figure RE-S07144420X20070828D000093
(13) purifying
Filtrate flow is through preparation HPLC (C 18Or C 8) post carries out purifying:
Flow velocity is: 200-800ml/min;
Follow the tracks of the needed effluent of collection with analysis mode HPLC.After the sample peak merges, adopt conventional method to desalt, remove acetonitrile, freeze-drying, get white loose block finished product approximately;
Moving phase is:
0.1-0.9M acetate buffer: acetonitrile (70-90: 30-10), volume ratio;
Or 0.1-0.9M phosphate buffered saline buffer: acetonitrile (70-90: 30-10), volume ratio;
Or 0.05%-0.1% trifluoracetic acid damping fluid: acetonitrile (70-90: 30-10), volume ratio;
Visible by above-mentioned disclosed technical scheme; Method of the present invention, adopting Rink Amide resin, Rink Amide AM resin or Rink Amide mbha resin is starting raw material, the amino acid of protecting with Fmoc is monomer; Connect amino acid whose method one by one, it is high to connect the peptide yield.Select Fmoc-amino acid for use, raw material sources are convenient, and last peptide chain uses the thiohydracrylic acid (Map (SX)) of protection, has simplified technology, has reduced cost; Employing is cut peptide reagent (TFA/HBr/HAc/TIS/EDT) and is cut peptide, adds the method for ether sedimentation bullion, avoids the use of poisonous reagents such as hydrogen fluoride; Three-waste pollution is few, and improves yield, adopts preparation HPLC (C18 or C8) post to carry out separation and purification; Avoid the use of TFA (trifluoracetic acid) and 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: 18.5%.This shows that method of the present invention is convenient to industrializing implementation, have bigger industrialization prospect.
Description of drawings:
Fig. 1 is a schema of the present invention.
Embodiment:
The raw material tabulation of being adopted in embodiment and the aforementioned process as follows
No The name of an article Production firm
1 Fmoc-Gly-OH Suzhou heavenly steed medicine group
2 Fmoc-Orn(Boc)-OH Suzhou heavenly steed medicine group
3 Fmoc-Pro-OH Suzhou heavenly steed medicine group
4 Fmoc-Cys(Trt)-OH Suzhou heavenly steed medicine group
5 Fmoc-Asn(Trt)-OH Suzhou heavenly steed medicine group
6 Fmoc-Thr(tBu)-OH Suzhou heavenly steed medicine group
7 Fmoc-Ile-OH Suzhou heavenly steed medicine group
8 Fmoc-D-Tyr(OEt)-OH Suzhou heavenly steed medicine group
9 Map(SX)-OH Merck company
10 Rink Amide AM resin Tianjin with become scientific & technical corporation
10 Rink Amide mbha resin Tianjin with become scientific & technical corporation
11 1-hydroxy benzo triazole (HOBt) Suzhou heavenly steed medicine group
12 1-pyridone and triazole (HOAt) Suzhou heavenly steed medicine group
13 Benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid ester (TBTU) Suzhou heavenly steed medicine group
14 Benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester (HBTU) Suzhou heavenly steed medicine group
15 Block special condensing agent (BOP) Suzhou heavenly steed medicine group
16 Trifluoroacetic acid (TFA) Merck company
17 Tri isopropyl silane (TIS) Merck company
18 N-methylmorpholine (NMM) Sail company of Shanghai section
19 N (DMF) Merck company
20 Hexahydropyridine (PIP) Merck company
Embodiment 1
The peptide chain preparation:
Taking by weighing Rink Amide AM resin is equivalent to the amount of 720mmol (200 orders 0.80mmol/g), in the Peptide synthesizer of the SHYM-25 that packs into, soaks with 6000mlDMF, make resin swelling, drain.Add the DMF solution that 6400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with DMF, washing for several times, drain respectively.
The preparation of Fmoc-Gly-resin:
Add Fmoc-Gly-OH (MW:297.3,1440mmol) 428g, HOBT (MW:135.1,1440mmol) 195g, TBTU (MW:321,1440mmol) 462g, NMM320ml (MW=101.2), 4000mlDMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
The preparation of Fmoc-Orn (Boc)-Gly-resin:
Add the DMF solution that 6400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with the DMF washing for several times, drain.
Fmoc-Orn (Boc)-OH (MW:454.5,1440mmol) 654g, TBTU (MW:321,1440mmol) 462g, HOBT (MW:135.1,1440mmol) 195g, NMM320ml (MW=101.2), 4000mlDMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
The preparation of Fmoc-Pro-Orn (Boc)-Gly-resin:
Add the DMF solution that 6400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with the DMF washing for several times, drain.
Add Fmoc-Pro-OH (MW:337.4,1440mmol) 486g, TBTU (MW:321,1440mmol) 462g, HOBT (MW:135.1,1440mmol) 195g, NMM320ml (MW=101.2), 4000ml DMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
The preparation of Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin:
Add the DMF solution that 6400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with the DMF washing for several times, drain.
Add Fmoc-Cys (Trt)-OH (MW:585.7,1440mmol) 843g, TBTU (MW:321,1440mmol) 486g, HOBT (MW:135.1,1440mmol) 195g, NMM320ml (MW=101.2), 4000mlDMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
The preparation of Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin:
Add the DMF solution that 6400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with the DMF washing for several times, drain.
Add Fmoc-Asn (Trt)-OH (MW:596.7,1440mmol) 859g, TBTU (MW:321,1440mmol) 486g, HOBT (MW:135.1,1440mmol) 195g, NMM320ml (MW=101.2), 4000mlDMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
The preparation of Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin:
Add the DMF solution that 6400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with the DMF washing for several times, drain.
Add Fmoc-Thr (tBu)-OH (MW:397.5,1440mmol) 572g, TBTU (MW:321,1440mmol) 486g, HOBT (MW:135.1,1440mmol) 195g, NMM 320ml (MW=101.2), 4000mlDMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
The preparation of Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin:
Add the DMF solution that 6400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with the DMF washing for several times, drain.
Add Fmoc-Ile-OH (MW:353.4,1440mmol) 509g, TBTU (MW:321,1440mmol) 486g, HOBT (MW:135.1,1440mmol) 195g, NMM320ml (MW=101.2), 4000mlDMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
The preparation of Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin:
Add the DMF solution that 6400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with the DMF washing for several times, drain.
Add Fmoc-D-Tyr (OEt)-OH (MW:413,1440mmol) 595g, TBTU (MW:321,1440mmol) 486g, HOBT (MW:135.1,1440mmol) 195g, NMM320ml (MW=101.2), 4000mlDMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
The preparation of Map-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin:
Add the DMF solution that 400 ml volumes concentration are 25% hexahydropyridine, 25 ℃ of joltings 30 minutes.Drain, with the DMF washing for several times, drain.
Add Map (MW:196,1440mmol) 282g, TBTU (MW:321,1440mmol) 486g, HOBT (MW:135.1,1440mmol) 195g, NMM320ml (MW=101.2), 4000mlDMF was with 25 ℃ of joltings of mixture 1.5 hours.Drain, DMF washs for several times, drains.
Again for several times with the anhydrous methanol washing.After draining, put into vacuum and drain device and drain, weigh, must protect the about 700g of resin as the atropic west.
Cut peptide:
Get that resin transfer is to cutting in the peptide bottle as the atropic west of 700g protection, cooling adds while stirring and cuts peptide reagent down: (TFA/HBr/HAc/TIS/EDT=850ml/34ml/11ml/51ml/25ml), 25 ℃ were stirred 3 hours.Filter, drain, filtrating adds the anhydrous diethyl ether deposition, filter collecting precipitation must about 37g reduced form atropic west as bullion (MW=833.96,44.4mmol), yield is 95.4%.
Oxidation (formation of disulfide linkage):
Bullion as the 37g reduced form atropic west is dissolved in the 40L purified water, under agitation adds 1mol/L ammoniacal liquor, regulate pH to 8, stirred overnight is filtered.
Get 700g reduced form RWJ-22164 bullion and be dissolved in the 5000ml purified water, transfer pH7.5 with NaOH, under agitation add ydrogen peroxide 50, room temperature reaction, liquid chromatograph are followed the tracks of and are detected, and confirm reaction end.After adding HAc to PH4.5 ± 0.5, filter.
Purifying:
Filtrating is through preparation HPLC (C 18Post) carries out purifying;
Moving phase: 0.5M phosphoric acid buffer: acetonitrile (80:20); Flow velocity is: 550ml/min; The detection wavelength is: 220nm; Follow the tracks of the needed effluent of collection with analysis mode HPLC.Desalt, remove acetonitrile, freeze-drying after the sample peak merges, approximately 132g white loose block finished product (MW:993.4,133mmol); About 18.5% (in the 720mmol of Rink Amide AM-resin) of total recovery.

Claims (5)

1. the preparation method as the synthetic atropic west of solid-phase polypeptide is characterized in that, comprises the steps:
(1) be starting raw material with Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin; Amino acid with the Fmoc protection is monomer; Use TBTU/HOBt, HBTU/HOBt, BOP/HOBt, TBTU/HOAt, HBTU/HOAt or BOP/HOAt to connect reactive polypeptide as condensing agent; Connect amino acid one by one, connect thiohydracrylic acid Mpa at last; Behind the nonapeptide resin that must protect, take off the side chain protected group synchronously and cut peptide;
Concrete comprise following content:
1. the pre-treatment of resin: with Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin, soaked 0.5～2 hour, make the abundant swelling of resin with DMF; Nitrogen dries up; The DMF solution that adds hexahydropyridine, 10～80 ℃ were reacted 10～60 minutes, and nitrogen blows elimination and removes hexahydropyridine; The DMF washing, nitrogen dries up;
In the DMF solution of hexahydropyridine, hexahydropyridine: DMF=1: 3～5 volume ratios;
In the DMF solution of hexahydropyridine, the concentration ratio of Rink Amide mbha resin is: 1 gram/5～15ml;
2. the preparation of Fmoc-Gly-resin
The 1. pretreated resin of step is mixed with Fmoc-Gly-OH, TBTU, HOBT, NMM and DMF, mixture was reacted under 10～60 ℃ condition 0.5～3.0 hour, nitrogen dries up, the DMF washing, and nitrogen dries up;
NMM: DMF=1: 5～15, volume ratio;
The mole number of Fmoc-Gly-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
The DMF solution that adds hexahydropyridine then, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, and with the DMF washing, nitrogen dries up, and obtains the Fmoc-Gly-resin;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%; The volumetric concentration of the weight of resin is: 1 gram/5～15ml;
3. the preparation of Fmoc-Orn (Boc)-Gly-resin:
In step Fmoc-Gly-resin 2., add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up; Add Fmoc-Orn (Boc)-OH, TBTU, HOBT, NMM and DMF and mix, 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Orn (Boc)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
4. the preparation of Fmoc-Pro-Orn (Boc)-Gly-resin
In step Fmoc-Orn (Boc)-Gly-resin 3., add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up; Add Fmoc-Pro-OH, TBTU, HOBT, NMM and DMF and mix, 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Pro-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
5. the preparation of Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In step Fmoc-Pro-Orn (Boc)-Gly-resin 4., add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up; Add Fmoc-Cys (Trt)-OH, TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Cys (Trt)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
6. the preparation of Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In step Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin 5., add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up; Add Fmoc-Asn (Trt)-OH, TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Asn (Trt)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
7. the preparation of Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In step Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin 6., add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up; Add Fmoc-Thr (tBu)-OH, TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Thr (tBu)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
8. the preparation of Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In step Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin 7., add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes; Nitrogen dries up, the DMF washing, and nitrogen dries up; Add Fmoc-Ile-OH, TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up; The DMF washing, nitrogen dries up; Obtain Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Ile-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml
9. the preparation of Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In step Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin 8., add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up; Add Fmoc-D-Tyr (OEt)-OH, TBTU, HOBT, NMM and DMF and mix, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-D-Tyr (OEt)-OH is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
In the DMF solution of hexahydropyridine, the volumetric concentration of hexahydropyridine is 10～40%;
In the DMF solution of hexahydropyridine, the volumetric concentration of the weight of resin is: 1 gram/5～15ml;
10. the preparation of Mpa-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In step Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin 9., add the DMF solution of hexahydropyridine, 10～60 ℃ were reacted 10～60 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up; Add Mpa, TBTU, HOBT, NMM and DMF, with 10～60 ℃ of reactions of mixture 0.5～3.0 hour, nitrogen dried up, the DMF washing, and nitrogen dries up;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Mpa is 2-6 a times of resin mole number;
The TBTU mole number is 2-6 a times of resin mole number;
The HOBT mole number is 2-6 a times of resin mole number;
Methanol wash is drained, and nitrogen dries up, and must protect resin as the reduced form atropic west;
(2) adding is cut peptide reagent TFA/HBr/HAc/TIS/EDT and is cut peptide then; Concrete proportioning is 800-900ml/25-40ml/5-20ml/40-60ml/20-30ml; 10～60 ℃ were reacted 2-6 hour;
(3) with the ether be solvent, deposition is also collected bullion as the reduced form atropic west;
(4) bullion as the reduced form atropic west is soluble in water, regulate PH to 7.5-10.0 with ammoniacal liquor, bullion as the collection oxidized form atropic west;
(5) bullion of collecting is passed through preparation HPLC, filler is C 18Separation and purification obtains title product; Moving phase is:
0.1-0.9M acetate buffer: acetonitrile equals 70-90: 30-10, volume ratio;
Or 0.1-0.9M phosphate buffered saline buffer: acetonitrile equals 70-90: 30-10, volume ratio;
Or 0.05%-0.1% trifluoracetic acid damping fluid: acetonitrile equals 70-90: 30-10, volume ratio.
2. method according to claim 1; It is characterized in that; Comprise the steps: Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin and hexahydropyridine, Fmoc-Gly-OH, TBTU and HOBt reaction, obtain the Fmoc-Gly-resin;
With hexahydropyridine, Fmoc-Orn (Boc)-OH, TBTU and HOBt, obtain Fmoc-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc-Pro-OH, TBTU and HOBt reaction, obtain Fmoc-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc-Cys (Trt)-OH, TBTU and HOBt reaction, obtain Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc-Asn (Trt)-OH, TBTU and HOBt reaction, obtain Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc-Thr (tBu)-OH, TBTU and HOBt reaction, obtain Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc-Ile-OH, TBTU and HOBt reaction, obtain Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Fmoc-D-Tyr (OEt)-OH, TBTU and HOBt reaction, obtain Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With hexahydropyridine, Mpa, TBTU and HOBt reaction, obtain Mpa-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin then;
With the TFA/HBr/HAc/TIS/EDT reaction, obtain then
Mpa-D-Tyr (OEt)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH2 (as the reduced form atropic west)
Carry out 1. oxidations then; 2. purifying; 3. desalt; 4. freeze-drying obtains:
Figure FSB00000887598800061
(as the atropic west).
3. method according to claim 2 is characterized in that, comprises the steps:
(1) pre-treatment of resin: with Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin, soak, make resin swelling with DMF; Nitrogen dries up; The DMF solution that adds hexahydropyridine, 10～80 ℃ were reacted 10～60 minutes, and nitrogen blows elimination and removes hexahydropyridine; The DMF washing, nitrogen dries up;
(2) preparation of Fmoc-Gly-resin:
With the pretreated resin of step (1) with the Fmoc-Gly-OH molecular weight be: 297.3, the TBTU molecular weight is: 321, the HOBT molecular weight is: 135.1, the NMM molecular weight be 101.2 and DMF mix, reaction, nitrogen dries up, DMF washing, nitrogen dries up;
The DMF solution that adds hexahydropyridine then, 10-60 ℃ was reacted 10-60 minute, and nitrogen dries up, and with the DMF washing, nitrogen dries up, and obtains the Fmoc-Gly-resin;
Reaction formula is:
Figure FSB00000887598800062
(3) preparation of Fmoc-Orn (Boc)-Gly-resin:
In the Fmoc-Gly-resin of step (2), add the DMF solution of hexahydropyridine, reaction, nitrogen dries up, the DMF washing, nitrogen dries up.Adding Fmoc-Orn (Boc)-OH molecular weight is: 454.5, TBTU, HOBT, NMM and DMF mix, and 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Orn (Boc)-Gly-resin;
Reaction formula is:
Figure FSB00000887598800063
(4) preparation of Fmoc-Pro-Orn (Boc)-Gly-resin:
In the Fmoc-Orn of step (3) (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10-60 ℃ was reacted 10-60 minute; Nitrogen dries up, the DMF washing, and nitrogen dries up; Add the Fmoc-Pro-OH molecular weight and be 337.4, TBTU, HOBT, NMM and DMF mix, 10～60 ℃ of reactions 0.5～3.0 hour, nitrogen dries up; The DMF washing, nitrogen dries up; Obtain Fmoc-Pro-Orn (Boc)-Gly-resin;
Reaction formula is:
Figure FSB00000887598800071
(5) preparation of Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Pro-Orn of step (4) (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10-60 ℃ was reacted 10-60 minute, and nitrogen dries up, the DMF washing, nitrogen dries up.Adding Fmoc-Cys (Trt)-OH molecular weight is: 585.7, TBTU, HOBT, NMM and DMF mix, and 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
Reaction formula is:
Figure FSB00000887598800072
(6) preparation of Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Cys of step (5) (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10-60 ℃ was reacted 10-60 minute; Nitrogen dries up, the DMF washing, and nitrogen dries up; Adding Fmoc-Asn (Trt)-OH molecular weight is: 596.7, TBTU, HOBT, NMM and DMF mix, and 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up; The DMF washing, nitrogen dries up; Obtain Fmoc-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
Reaction formula is:
Figure FSB00000887598800073
(7) preparation of Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Asn of step (6) (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10-60 ℃ was reacted 10-60 minute, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Thr (tBu)-OH molecular weight and be 397.5, TBTU, HOBT, NMM and DMF mix, 10～60 ℃ of reactions 0.5～3.0 hour, nitrogen dries up, the DMF washing, nitrogen dries up; Obtain Fmoc-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
Reaction formula is:
Figure FSB00000887598800081
(8) preparation of Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Thr of step (7) (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10-60 ℃ was reacted 10-60 minute; Nitrogen dries up, the DMF washing, and nitrogen dries up; Adding the Fmoc-Ile-OH molecular weight is: 353.4, TBTU, HOBT, NMM and DMF mix, and 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up; The DMF washing, nitrogen dries up; Obtain Fmoc-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
Reaction formula is:
Figure FSB00000887598800082
(9) preparation of Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-Ile-Thr of step (8) (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, 10-60 ℃ was reacted 10-60 minute; Nitrogen dries up, the DMF washing, and nitrogen dries up; Adding Fmoc-D-Tyr (OEt)-OH molecular weight is: 413, TBTU, HOBT, NMM and DMF mix, and 10～60 ℃ were reacted 0.5～3.0 hour, and nitrogen dries up; The DMF washing, nitrogen dries up; Obtain Fmoc-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin;
Reaction formula is:
Figure FSB00000887598800091
(10) preparation of Mpa-D-Tyr (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin
In the Fmoc-D-Tyr of step (9) (OEt)-Ile-Thr (tBu)-Asn (Trt)-Cys (Trt)-Pro-Orn (Boc)-Gly-resin, add the DMF solution of hexahydropyridine, reaction, nitrogen dries up; The DMF washing, nitrogen dries up, and adds Mpa, TBTU, HOBT, NMM and DMF, and 10～60 ℃ were reacted 0.5～3.0 hour; Nitrogen dries up, the DMF washing, and nitrogen dries up, washing; Drain, nitrogen dries up, and must protect resin as the reduced form atropic west;
Reaction formula is:
Figure FSB00000887598800092
(11) cut peptide
Add resin as the reduced form atropic west of protection with cutting peptide reagent, 10～60 ℃ of reactions 2～6 hours are filtered, drain, and the filtrating deposition that adds diethyl ether, the filtration collecting precipitation gets bullion as the reduced form atropic west, and molecular weight is 833.96, and yield is 96%;
The said peptide reagent of cutting is:
TFA/HBr/HAc/TIS/EDT＝800-900ml/25-40ml/5-20ml/40-60ml/20-30ml；
Reaction formula is:
Figure FSB00000887598800093
Figure FSB00000887598800101
Bullion as the reduced form atropic west is soluble in water, and adding concentration is the ammoniacal liquor of 0.1～1.5mol/L, regulates pH to 7.5-10.0, and blowing air stirred 20～28 hours, filtration;
Reaction formula is:
Figure FSB00000887598800102
(13) purifying
Filtrate flow is through preparation HPLC C 18Post carries out purifying: follow the tracks of with analysis mode HPLC and collect needed effluent, after the sample peak merges, desalt, remove acetonitrile, freeze-drying, get white loose block finished product approximately.
4. method according to claim 3 is characterized in that, in the weight of resin as the reduced form atropic west, the add-on of cutting peptide reagent is 5～20ml/g.
5. method according to claim 3 is characterized in that, in the step (13), the moving phase of preparation HPLC is:
0.1-0.9M acetate buffer: acetonitrile 70-90: 30-10, volume ratio; Or 0.1-0.9M phosphate buffered saline buffer: acetonitrile 70-90: 30-10, volume ratio; Or 0.05%-0.1% trifluoracetic acid damping fluid: acetonitrile 70-90: 30-10, volume ratio.
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CN101696959B (en) * 2009-02-11 2012-10-17 海南中和药业有限公司 Acetic acid atosiban, and method for detecting content of preparation of acetic acid atosiban and relevant substances
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