CN1923849B - Preparation method of synthesizing octriotide from solid phase polypeptide - Google Patents
Preparation method of synthesizing octriotide from solid phase polypeptide Download PDFInfo
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- CN1923849B CN1923849B CN2005100292212A CN200510029221A CN1923849B CN 1923849 B CN1923849 B CN 1923849B CN 2005100292212 A CN2005100292212 A CN 2005100292212A CN 200510029221 A CN200510029221 A CN 200510029221A CN 1923849 B CN1923849 B CN 1923849B
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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
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Abstract
The invention discloses an aoqu-peptide preparing method of solid-phase polypeptide, which comprises the following steps: adopting 2-chloride-trityl resin, 4-methyl trityl resin or 4-methoxyl trityl resin as raw material; connecting amino acid with protective group according to solid-phase synthetic method; obtaining protected octapeptide resin; removing Fmoc-protective group sequently; stripping side-chain protective group; cutting peptide to obtain reduced aoqu-peptide; oxidizing through air under pH 7-11 condition; separating and purifying rought product through C18 (C8) column to produce exquisite.
Description
Technical field
The present invention relates to a kind of preparation method of Sostatin, be specifically related to the preparation method of synthesizing octriotide from solid phase polypeptide.
Background technology
Sostatin, Chinese name: Sostatin LAR; English name: Octreotide Acetate
Structural formula:
Molecular formula: C
49H
66N
10O
10S
2XC
2H
4O
2
Sostatin is Somatostatin 8 peptides of synthetic, is one of hormone medicine of the tool effect of the clinical application nineties.Identical with the pharmacological action of spontaneous growth chalone, but the transformation period prolonged 30 times, therefore have using value.Sostatin can suppress secretion and other multiple endocrine function of multiple gastrointestinal hormone, be widely used at present that cirso-is hemorrhage at the bottom of the various pancreatitis of clinical treatment, pancreas fistula, intestinal fistula, the liver cirrhosis esophagus-stomach, digestive tract ulcer is hemorrhage, seriously diarrhoea and acromegaly, and be expected to be used for the treatment of Digestive tract endocrine tumors.
Sostatin is a kind of choice drug for the treatment of upper gastrointestinal hemorrhage, acute pancreatitis safely and effectively.But at present this product does not still have home made article, whole dependence on import, and import product (trade(brand)name: Sandostatin, rather kind, the manufacturer is a Switzerland mountain pass scholar company) retail price at home is 801 yuan/box (5), specification is 2ml:0.1mg.
At home, the pathogenesis of cirrhosis rate surpasses 1%, and the patient of the profuse bleeding that annual generation rupture of esophageal varices causes adds the massive bleeding from upper digestive tract patient that peptide ulceration causes, sum surpasses 3,000,000, so the market of Sostatin is huge.We will be significantly less than the import product by Sandostatin (Sostatin LAR) cost of development, will reduce patient's treatment cost greatly.
The preparation method of reported in literature all exists certain defective at present, 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 use hydrogen fluoride is cut peptide, trifluoracetic acid carries out purifying, contaminate environment, and yield is lower.Therefore present method has limited the scale operation and the use of Sostatin.
Summary of the invention
The technical issues that need to address of the present invention are the preparation methods that disclose a kind of synthesizing octriotide from solid phase polypeptide, to overcome the above-mentioned defective that prior art exists.
Method of the present invention comprises the steps:
With 2-chloro-trityl resin (2-Chlorotrityl Chloride Resin); trityl resin is made resin (TritylChloride Resin); 4-methyl trityl resin (4-Methyltrityl Chloride Resin) or 4-methoxyl group trityl resin (4-Methoxytrityl Chloride Resin) are starting raw material; method according to solid phase synthesis connects the amino acid with blocking group successively; obtain protection octapeptide resin; slough the Fmoc-blocking group therebetween successively; with TBTU or HBTU and HOBT is that condensing agent connects reactive polypeptide; behind the reduced form octapeptide resin that must protect; take off the side chain protected group synchronously and cut peptide; obtain the reduced form Sostatin; and under the condition of pH7-11, use atmospheric oxidation; obtain the Sostatin crude product; again through C18 (or C8) column separating purification; after the lyophilize, make the Sostatin elaboration.
According to the present invention, connect amino acid successively with blocking group, obtain protection octapeptide resin, the method for sloughing the Fmoc-blocking group therebetween successively comprises the steps:
(1) preparation Fmoc-Thr (tBu)-resin:
With 2-chloro-trityl resin, trityl resin, 4-methyl trityl resin or 4-methoxyl group trityl resin, soaked 10～60 minutes with DMF, make the abundant swelling of resin, add DIPEA or DMPA and Fmoc-Thr (tBu)-ol10～50 ℃ reaction 1～24 hour;
Add 10～50 ℃ of reactions of methyl alcohol 1～20 hour again, nitrogen dries up, and resin washs with DMF, obtains Fmoc-Thr (tBu)-resin;
Said resin replacement amount is: 0.3-1.5mmol/g; Particle diameter is the 100-400 order;
The mole number of Fmoc-Thr (tBu)-ol is 2～5 times of resin;
The mole number of DIPEA or DMPA is 2～20 times of resin;
The add-on of reagent of raising one's hat is that benchmark is 5～20ml/g with the weight of resin, down with;
The mole number of methyl alcohol is 2～20 times of resin;
(2) preparation Fmoc-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-Thr of step (1) (tBu)-resin, add the reagent of raising one's hat, 10～50 ℃ were reacted 10～60 minutes, and dried up, use DMF and washing with alcohol respectively, dry up, add with the mixture that meets peptide reagent dissolved Fmoc-Cys (Trt)-OH, TBTU/HBTU and HOBT, 10～50 ℃ were reacted 1～5 hour, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Cys (Trt)-Thr (tBu)-resin;
The said reagent of raising one's hat is: PIP: DMF=1: 2～5, and volume ratio, down together;
The add-on of reagent of raising one's hat is that benchmark is 5～20ml/g with the weight of resin, down with;
In the mixture:
The mole number of Fmoc-Cys (Trt)-OH is 2～5 times of resin;
The mole number of TBTU/HBTU and HOBT is 2～5 times of resin;
The said peptide reagent that connects is: NMM: DMF=1: 5～15, and volume ratio, down together;
Connect in the peptide reagent, the mole number of Fmoc-Cys (Trt)-OH is 2～5 times of resin;
" TBTU/HBTU and HOBT " refers to: the mixture of TBTU and HOBT, or the mixture of HBTU and HOBT, down together;
(3) preparation Fmoc-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin
In the Fmoc-Cys of step (2) (Trt)-Thr (tBu)-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-Thr (tBu)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-Thr (tBu)-OH is 2～5 times of resin.
All the other operations and processing condition are the same;
(4) preparation Fmoc-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin
In the Fmoc-Cys of step (2) (Trt)-Thr (tBu)-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-Lys (Boc)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-Lys (Boc)-OH is 2～5 times of resin.
All the other operations and processing condition are the same;
(5) preparation Fmoc-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-D-Trp-Lys of step (3) (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-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-Trp-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dries up, and obtains Fmoc-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-D-Trp-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
(6) preparation Fmoc-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-D-Trp-Lys of step (5) (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-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-Thr (tBu)-OH, TBTU/HBTU and HOBT, reaction dries up, and uses DMF and washing with alcohol respectively, dry up, obtain Fmoc-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-Phe-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
(7) preparation Fmoc-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-Phe-D-Trp-Lys of step (6) (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-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-Cys (Trt)-OH, TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-Lys (Boc)-OH is 2～5 times of resin.
All the other operations and processing condition are the same;
(8) preparation Boc-D-Phe-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu) resin:
In the Fmoc-Cys of step (7) (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-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 Boc-D-Phe-OH, TBTU/HBTU and HOBT, reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Boc-D-Phe-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu) resin;
In the mixture:
The mole number of Boc-D-Phe-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
(9) preparation D-Phe-Cys (SH)-Phe-D-Trp-Lys-Thr-Cys (SH)-Thr-ol:
In step (8) Boc-D-Phe-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu) resin, add and be chilled to-10 ℃～20 ℃ the peptide reagent of cutting in advance, 10 ℃～50 ℃ were reacted 1～5 hour, remove solvent under reduced pressure, add ether sedimentation, the collecting precipitation thing is washed with ether, P
2O
5Vacuum-drying, the reduced form Sostatin behind the peptide is cut in acquisition;
Said component of cutting peptide reagent is: TFA/EDT/H
2O/TIS=90-95/2-5/2-5/1-5, volume ratio;
(10) preparation
In the D-Phe-Cys of step (9) (SH)-Phe-D-Trp-Lys-Thr-Cys (SH)-Thr-ol, regulate pH to 7-12 with sodium hydroxide or ammoniacal liquor, under 10 ℃～50 ℃, bubbling air or dioxygen oxidation reaction 8～30 hours obtain the Sostatin crude product.
According to the present invention, crude product comprises the steps: through the method for C18 (or C8) column separating purification separation and purification
The Sostatin 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, and total recovery is 30-40% (in the mmol of resin).
The weight concentration of acetate is 0.5～10%, and the weight concentration of Sostatin crude product is 1～50% in the acetate.
Sostatin 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 the raising yield adopts C18 (or C8) post to carry out separation and purification, avoids using TFA (trifluoracetic acid) to carry out purifying, reduce the three wastes, per step connects the peptide yield all more than 98%; Total recovery is 30-40%.
Embodiment
Embodiment 1
The raw material that is adopted in embodiment and the aforementioned process is listed as follows:
One, synthetic peptide chain
(1) preparation Fmoc-Thr (tBu)-resin:
(100-200 order, 1.0mmol/g 50mmol), soaked 30 minutes with DMF, made the abundant swelling of resin, added 44ml DIPEA, and 39.7g Fmoc-Thr (tBu)-ol reacted 3 hours, added 50ml methyl alcohol again and continued reaction 3 hours to get 2-chloro-trityl resin 50 grams.Nitrogen dries up, and resin is given a baby a bath on the third day after its birth inferior with DMF.Obtain Fmoc-Thr (tBu)-resin;
(2) preparation of Fmoc-Cys (Trt)-Thr-resin:
Add 800 ml volumes concentration and be the DMF solution of 20% hexahydropyridine, 25 ℃ of reactions 30 minutes.Nitrogen blows the elimination hexahydropyridine, respectively washs three times with DMF, anhydrous methanol, DMF respectively, and nitrogen dries up.
Add Fmoc-Cys (Trt)-OH (MW:585.7,200mmol) 117.1g, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW=101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter 20% hexahydropyridine, 25 ℃ were reacted 30 minutes.Nitrogen dries up, and respectively washs three times with DMF, anhydrous methanol, DMF respectively, and nitrogen dries up.
(3) preparation of Fmoc-Thr (tBu)-Cys (Trt)-Thr-resin
In the above-mentioned reacted resin of raising one's hat, add 79.5g (397.5,200mmol) Fmoc-Thr (tBu)-OH, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW=101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter 20% hexahydropyridine, 25 ℃ were reacted 30 minutes.Nitrogen dries up, and respectively washs three times with DMF, anhydrous methanol, DMF respectively, and nitrogen dries up.
(4) preparation of Fmoc-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr-resin
In the above-mentioned reacted resin of raising one's hat, add 93.7g (468.5,200mmol) Fmoc-Lys (Boc)-OH, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW=101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter 20% hexahydropyridine, 25 ℃ were reacted 30 minutes.Nitrogen dries up, and respectively washs three times with DMF, anhydrous methanol, DMF respectively, and nitrogen dries up.
(5) preparation of Fmoc-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr-resin
In the above-mentioned reacted resin of raising one's hat, add 85.3g (426.5,200mmol) Fmoc-D-Trp-OH, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW=101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter 20% hexahydropyridine, 25 ℃ were reacted 30 minutes.Nitrogen dries up, and respectively washs three times with DMF, anhydrous methanol, DMF respectively, and nitrogen dries up.
(6) preparation of Fmoc-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr-fat
In the above-mentioned reacted resin of raising one's hat, add 77.5g (FW:387.4,200mmol) Fmoc-Phe-OH, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW=101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter 20% hexahydropyridine, 25 ℃ were reacted 30 minutes.Nitrogen dries up, and respectively washs three times with DMF, anhydrous methanol, DMF respectively, and nitrogen dries up.
(7) preparation of Fmoc-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr-resin
In the above-mentioned reacted resin of raising one's hat, add 117.1g (585.7,200mmol) Fmoc-Cys (Trt)-OH, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW=101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter 20% hexahydropyridine, 25 ℃ were reacted 30 minutes.Nitrogen dries up, and respectively washs three times with DMF, anhydrous methanol DMF respectively, and nitrogen dries up.
(8) preparation of Boc-D-Phe-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr-resin
In the above-mentioned reacted resin of raising one's hat, add 53g (FW:265.3,200mmol) Boc-D-Phe-OH, TBTU (MW:321,200mmol) 64.2g, HOBT (MW:153,200mmol) 30.6g, NMM 44.4ml (MW=101.2), 400mlDMF was with 25 ℃ of reactions of mixture 1 hour.Nitrogen dries up, and DMF, anhydrous methanol, DMF respectively wash three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter 20% hexahydropyridine, 25 ℃ were reacted 30 minutes.Nitrogen dries up, and respectively washs three times with DMF, anhydrous methanol DMF respectively, and nitrogen dries up.
Again with anhydrous methanol washing three times.After draining, it is dry to put into vacuum drier, weighs, and (MW=931 43mmol), connects the peptide total recovery and is about 86% to get the about 90g of octapeptide resin.
Two, cut peptide
Get 90.2g octapeptide resin transfer in eggplant-shape bottle, cooling adds down cuts peptide reagent: (TFA/ water/dithioglycol/thioanisole=650ml/17ml/17ml/34ml), 25 ℃ were stirred 2 hours.Filter, drain, filtrate adds the anhydrous diethyl ether precipitation, and filtering collecting precipitation must about 30g reduced form Sostatin crude product.
Three, oxidation (formation of disulfide linkage)
30g reduced form Sostatin crude product is dissolved in 30L water, under agitation slowly drips 2M NH
4OH to pH7.8, about 24 hours of room temperature blowing air oxidation transfers to pH5.5 with Glacial acetic acid, adds activated carbon and stirs 30 minutes, filters.
Four, purifying
Filtrate is in batches through the C18 column purification, moving phase: 0.1MNH
4Ac: acetonitrile (7.5: 2.5); Flow velocity is: 380ml/min; The detection wavelength is: 280nm; The sample peak merges the back freeze-drying, approximately 15.6g white loose block finished product (MW:1019,15.3mmol); Yield 30.6% (in the mmol of Fmoc-Thr-resin).Follow the tracks of the needed effluent liquid of collection with liquid chromatograph.
Embodiment 2～3
Adopt method and the processing condition identical with embodiment 1, wherein:
Make starting raw material with 4-methyl trityl resin or 4-methoxyl group trityl resin respectively, after the same method connects Fmoc-Thr-ol, add the reagent of raising one's hat again, 25 ℃ were reacted 0.5 hour, add with the mixture that meets peptide reagent dissolved Fmoc-amino acid, TBTU/HBTU and HOBT, 25 ℃ were reacted 2 hours, again through cutting reactions such as peptide, oxidation, obtain 16.3g and 14.9g white loose block finished product, yield is respectively 32.0% and 29.2% (in the mmol of Fmoc-Thr-resin).
Claims (3)
1. the preparation method of a synthesizing octriotide from solid phase polypeptide, it is characterized in that, comprise the steps: with 2-chloro-trityl resin, 4-methyl trityl resin or 4-methoxyl group trityl resin are starting raw material, method according to solid phase synthesis connects the amino acid with blocking group successively, obtain protection octapeptide resin, slough the Fmoc-blocking group therebetween successively, with TBTU or HBTU and HOBT is that condensing agent connects reactive polypeptide, behind the reduced form octapeptide resin that must protect, take off the side chain protected group synchronously and cut peptide, obtain the reduced form Sostatin, and under the condition of pH7-11, use atmospheric oxidation, obtain the Sostatin crude product, again through C18 or C8 column separating purification, after the lyophilize, make the Sostatin elaboration;
Connect the amino acid with blocking group successively, obtain protection octapeptide resin, the method for sloughing the Fmoc-blocking group therebetween successively comprises the steps:
(1) preparation Fmoc-Thr (tBu)-resin:
With 2-chloro-trityl resin, 4-methyl trityl resin or 4-methoxyl group trityl resin, the 100-400 order, 0.3-1.5mmol/g soaked 5～60 minutes with DMF, add DIPEA or DMPA and Fmoc-Thr (tBu)-ol, 10～50 ℃ were reacted 1～24 hour;
Add 10～50 ℃ of reactions of methyl alcohol 1～24 hour again, nitrogen dries up, and resin washs with DMF, obtains Fmoc-Thr (tBu)-resin;
The mole number of Fmoc-Thr (tBu)-ol is 2～5 times of resin;
The mole number of DIPEA or DMPA is 2～20 times of resin;
The mole number of methyl alcohol is 2～20 times of resin;
(2) preparation Fmoc-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-Thr of step (1) (tBu)-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-Cys (Trt)-OH, TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Cys (Trt)-Thr (tBu)-resin;
The said reagent of raising one's hat is: PIP: DMF=1: 2～5, and volume ratio, down together;
The add-on of reagent of raising one's hat is that benchmark is 5～20ml/g with the weight of resin, down with;
In the mixture:
The mole number of Fmoc-Cys (Trt)-OH is 2～5 times of resin;
The mole number of TBTU/HBTU and HOBT is 2～5 times of resin;
The said peptide reagent that connects is: NMM: DMF=1: 5～15, and volume ratio, down together;
Connect in the peptide reagent, the mole number of Fmoc-Cys (Trt)-OH is 2～5 times of resin;
(3) preparation Fmoc-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin
In the Fmoc-Cys of step (2) (Trt)-Thr (tBu)-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-Thr (tBu)-OH, TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-Thr (tBu)-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
(4) preparation Fmoc-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-Thr of step (3) (tBu)-Cys (Trt)-Thr (tBu)-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-Lys (Boc)-OH, TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-Lys (Boc)-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
(5) preparation Fmoc-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-Lys of step (4) (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-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-D-Trp-OH, TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-D-Trp-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
(6) preparation Fmoc-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-D-Trp-Lys of step (5) (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-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-Phe-OH, TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-Phe-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
(7) preparation Fmoc-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-Phe-D-Trp-Lys of step (6) (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-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-Cys (Trt)-OH, TBTU/HBTU and HOBT reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Fmoc-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin;
In the mixture:
The mole number of Fmoc-Cys (Trt)-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
(8) preparation Boc-D-Phe-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-resin:
In the Fmoc-Cys of step (7) (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu)-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 Boc-D-Phe-OH, TBTU/HBTU and HOBT, reaction, dry up, use DMF and washing with alcohol respectively, dry up, obtain Boc-D-Phe-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu) resin;
In the mixture:
The mole number of Boc-D-Phe-OH is 2～5 times of resin;
All the other operations and processing condition are the same;
Take off the side chain protected group synchronously and cut peptide, the method that obtains the reduced form Sostatin comprises the steps:
In step (8) Boc-D-Phe-Cys (Trt)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Trt)-Thr (tBu) resin, add and be chilled to-10 ℃～20 ℃ the peptide reagent of cutting in advance, 10 ℃～50 ℃ were reacted 1～5 hour, remove solvent under reduced pressure, add ether sedimentation, the collecting precipitation thing is washed with ether, P
2O
5Vacuum-drying, the reduced form Sostatin behind the peptide is cut in acquisition;
Said component of cutting peptide reagent is: TFA/EDT/H
2O/TIS=90-95/2-5/2-5/1-5, volume ratio;
Described oxidising process comprises the steps:
Above-mentioned reduced form Sostatin is regulated pH to 7-12 with sodium hydroxide or ammoniacal liquor, and under 10 ℃～50 ℃, bubbling air or dioxygen oxidation reaction 8～30 hours obtain the Sostatin crude product;
Crude product comprises the steps: through the method for C18 or C8 column separating purification
The Sostatin 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; The weight concentration of acetate is 0.5～10%.
2. method according to claim 1 is characterized in that, adds the reagent of raising one's hat, and 10～50 ℃ were reacted 10～60 minutes.
3. method according to claim 1 is characterized in that, adds with the mixture that meets peptide reagent dissolved Fmoc-Cys (Trt)-OH, TBTU/HBTU and HOBT, and 10～50 ℃ were reacted 1～5 hour.
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| CN101357938B (en) * | 2007-07-31 | 2013-07-03 | 周逸明 | Method for synthesizing Exenatide from solid phase polypeptide |
| CN102229645B (en) * | 2011-06-03 | 2013-08-14 | 程云 | Method for preparing immune seven peptides |
| CN102336807B (en) * | 2011-07-01 | 2016-03-16 | 上海苏豪逸明制药有限公司 | A kind of industrialized process for preparing of RDG |
| CN102850438B (en) * | 2012-09-19 | 2014-11-05 | 上海昂博生物技术有限公司 | Solid phase preparation method of crude octrotide |
| CN103351426B (en) * | 2013-08-02 | 2018-11-02 | 上海苏豪逸明制药有限公司 | A kind of polypeptide synthesis method of octreotide acetate |
| CN107778351B (en) * | 2016-08-25 | 2021-04-13 | 成都圣诺生物制药有限公司 | Method for synthesizing octreotide by all-solid-phase method |
| CN111378009A (en) * | 2018-12-27 | 2020-07-07 | 江苏金斯瑞生物科技有限公司 | Preparation method of octreotide |
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Non-Patent Citations (4)
| Title |
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| Frank J.Dekker et al.Replacement of the intervening amino acid sequence of a syk-binding diphosphopeptide by a nonpeptide spacer with preservation of high affinity.ChemBioChem.2002,(02-03),238-242. * |
| 上海苏豪逸明制药有限公司.固相多肽合成醋酸奥曲肽.国科网科技频道首页.2004, * |
| 刘作家.醋酸奥曲肽的合成研究.中国优秀博硕士学位论文全文数据库（硕士）医药卫生科技辑（季刊） 2004年第04期.2004,(2004年第04期),E079-9. |
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