CN101314613A - Solid phase synthesis method for atosiban - Google Patents

Abstract

The invention relates to a synthetic process of atosiban and solves the technical problem with poor solubility and long reaction time in the air liquid phase oxidation of the prior art. The synthetic process comprises the steps as follows: aminomethyl resin is adopted as a carrier; amino acids are combined one by one according to the Fmoc/tbu solid-phase peptide synthetic method; S-S bonds are formed by adopting the iodine oxidization method on the resin; peptides are cut from the resin with the mixed solution of trifluoroacetic acid, tri-isopropyl silicane, water and p-cresol to obtain a coarse product with S-S bonds; and the product is subjected to the HPLC purification and then frozen to obtain the atosiban. The synthetic process is simple and applicable to the large-scale synthesis of atosiban.

Description

A kind of solid phase synthesis process of Atosiban

Technical field:

The present invention relates to a kind of solid phase synthesis process of Atosiban.

Background technology:

Atosiban is the Uteracon competitive antagonist of acceptor on intrauterine and decidua, the fetal membrane, is a kind of breakthrough product in the obstetric drugs, in Austria, Denmark and Sweden's listing, is used to postpone not term birth.Estimating soon also will be in Holland, Germany and Britain's listing, China has the listing of import at present but does not also domesticize at present. but Atosiban dosage correlation ground suppresses uterine contraction, and the prostaglandin(PG) secretion of Uteracon mediation is reduced, reach the purpose of preventing miscarriage, in China, year natality is 2,000 ten thousand, by present clinical statistics numeral, the pregnant woman of the about 5-15% treatment of need preventing miscarriage is by average 10%, number of patients is about 2,000,000, so this kind has bigger market outlook.

The structure of Atosiban is as follows:

c[Mpr-D-Tyr(OEt)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH ₂

Be the ring-type polypeptide, illustrious name is: Atosiban, and according to the feature of its sequence, synthetic route has by mbha resin, and Boc method solid phase is closed polypeptide, forms the product of sulphur sulfide linkage Cheng Huan again by iodine oxidation in the solution, sees patent EP0710243.

Summary of the invention:

The process for solid phase synthesis that the purpose of this invention is to provide a kind of Atosiban.The technical issues that need to address of the present invention are: select a kind of method for oxidation, it is satisfied: (1) has stability in polypeptide is synthetic, (2) carrier reaction easily, (3) can avoid causing the long or incomplete defective of oxidation of oxidization time because of polypeptide is poorly soluble in the atmospheric oxidation, (4) can shorten oxidization time and raise the efficiency greatly.

Some abbreviations commonly used have following implication among the present invention;

Fmoc: fluorenylmethyloxycarbonyl

Fmoc-AA: the amino acid of fluorenylmethyloxycarbonyl protection

TBu: the tertiary butyl

HoBt:1-hydroxybenzene a pair of horses going side by side triazole

HBTU, benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester

DIC:N, N-di-isopropyl carbodiimide

DIEA:N, the N-diisopropylethylamine

MPa: thiohydracrylic acid

D-Tyr (Et): the tyrosine chain of D-type is protected with ethyl

Ile: Isoleucine

Thr: Threonine

Asn: asparagine

Cys: halfcystine

Pro: proline(Pro)

Orn: ornithine

Gly: glycine

Trt: trityl

Et: ethyl

Boc: tertbutyloxycarbonyl

DMF:N, dinethylformamide

Piperidine: hexahydropyridine

Fmoc-Rink Amide Am resin: with the Rink aminomethyl resin of Fmoc protection

TFA: trifluoracetic acid

TIS: tri isopropyl silane

MeOH: methyl alcohol

CH ₂CL ₂: methylene dichloride

Kniser test detection reagent is: triketohydrindene hydrate

Technical scheme of the present invention is: a kind of solid phase synthesis process of Atosiban, at first use the aminomethyl resin to make carrier, according to Fmoc/tbu solid-phase polypeptide synthetic method amino acid is connected one by one, method with iodine oxidation on the resin forms the sulphur sulfide linkage again, use trifluoracetic acid at last again: tri isopropyl silane: water: the mixing solutions of p-cresol downcuts peptide from resin, just can directly obtain forming the crude product of sulphur sulfide linkage, through the freezing Atosiban that obtains of preparation HPLC purifying.

In connecing the amino acid process, (volume ratio is 1: 1: 1: 2) with Fmoc-AA/HBTU/HOBT/DIEA, the excessive 2-5 of Fmoc-AA condensation course doubly, each step condensation is all passed through Caesar's reagent (Kaiser test) and is detected, be positive as color and then repeat to connect amino acid, use 20% piperidines/DMF to take off Fmoc then, formed the sulphur sulfide linkage with the methanol solution of iodine in nine amino acid whose resin reaction 30-90 minutes with connecting again, every gram resin is with 6-10ml volume ratio trifluoracetic acid: tri isopropyl silane: water: p-cresol=88: 2: 5: 5 mixed solution downcuts peptide from resin.The crude product productive rate that obtains is about 92.3%, and HPLC is 78.2%.It should be noted that the iodine oxidation has formed the sulphur sulfide linkage on the resin, so can not be with the cutting liquid that contains two mercaptan when selecting cutting liquid cutting resin.As can be seen with the synthetic Atosiban of this method, productive rate and purity are all more satisfactory from last.In addition, this method also has certain meaning to the sulphur sulfide linkage cyclisation peptide of synthetic other amino acids.

The invention has the beneficial effects as follows: we select the method for iodine oxidation on the resin to form the sulphur sulfide linkage, on the one hand, its reaction selection condition is stable, can apply in the method for solid phase Fmoc/tbu and go, avoided in the air oxidation process causing the crude product loss shortcoming such as the long and oxidation of oxidization time is incomplete on the other hand because of poorly soluble.

Embodiment:

Embodiment 1:1, the nonapeptide resin of anamorphic zone protection

Peptide sequence (c[mpa-D-Tyr (Et)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH ₂)

The calculating of the charging capacity of each amino acid charging capacity and some reagent in the sequence

K=2.5 * m resin * Loading=2.5 * 1g * 0.52mmol/g=1.3mmol

Activator: m (HoBt)=M (HoBt) * K=0.135g/mmol * 1.3mmol=0.176g

Condensing agent: V (DIC)=M (DIC) * K/0.81=0.126g/mmol * 1.3mmol/0.81g/ml=0.2ml

Alkali reagent V (collidine)=2 * M (collidine) * K/0.80g/ml=2 * 0.124g/mmol * 1.3mmol/0.80g/ml=0.40ml

m(Gly)＝M(Gly)×K＝0.2973g/mmol×1.3mmol＝0.386g

m(orn)＝M(orn)×K＝0.4545g/mmol×1.3mmol＝0.591g

m(pro)＝M(pro)×K＝0.3374g//mmol×1.3mmol＝0.439g

m(cys)＝M(cys)×K＝0.5857g/mmol×1.3mmol＝0.761g

m(Asn)＝M(Asn)×K＝0.5967g/mmol×1.3mmol＝0.776g

m(Thr)＝M(Thr)×K＝0.3974g/mmol×1.3mmol＝0.517g

m(Ile)＝M(Ile)×K＝0.3534g/mmol×1.3mmol＝0.459g

m(D-Tyr)＝M(D-Tyr)×K＝0.4315×1.3mmol＝0.561g

m(Mpa)＝M(Mpa)×K＝0.3480×1.3mmol＝0.452g

1. take off the Fmoc blocking group

Getting 5g Fmoc-RinkAmide Am resin puts into and connects the peptide bottle, the substitution value of this resin is 0.52mmol/g, add 30～40ml 20%piperidine/DMF solution vibration 5min, drain, add 30～40ml 20%piperidine/DMF solution vibration 15min again, drain then, wash 3 times with DMF, MeOH washes 3 times, CH ₂CL ₂Wash 3 times, drain, get 10-20 resin and make Kaiser test and detect to show blueness and be positive,, then repeat the above step of raising one's hat if be to be negative.

2., the condensation of Fmoc-Gly-OH

Take by weighing Fmoc-Gly-OH 1.93g, HoBt 0.88g connects in the peptide bottle above adding.Add an amount of DMF, 2mlDIEA, 2.87g HBTU puts into vibrator reaction 1 hour after the sealing, and reaction solution is drained in the washing after reaction finishes, and washes resin 3 times with DMF, and MeOH washes 3 times, CH ₂CL ₂Wash 3 times, drain, get 10-20 resin and make the apparent feminine gender of Kaiser test, pass through up to the apparent feminine gender of the Kaiser test that reacts completely if the journey positive then repeats top condensation reaction operation.

Meet Fmoc-orn (Boc)-OH more successively, Fmoc-Cys (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Thr (tBn)-OH, Fmoc-Ile-OH, Fmoc-D-Tyr (OEt)-(OH), the same Fmoc-Gly-OH of Mpa (Trt) method

Embodiment 2,

Take by weighing 0.67g iodine (this iodine amount is excessive 5 times) and be dissolved in 10ml methyl alcohol, with embodiment 1 resin reaction 30min 1., temperature is controlled at 0 ℃, and reaction finishes with using the ether dry adsorbent behind the DMF repetitive scrubbing resin.

The resin that obtains is joined the volume ratio trifluoracetic acid of 10ml: tri isopropyl silane: water: p-cresol=88: 2: 5: in 5 the mixed solution, room temperature vibration 1.5 hours, filter, wash resin 2 times with trifluoracetic acid, filtrate concentrates, add anhydrous diethyl ether and get white group, with the anhydrous diethyl ether washing, get Atosiban crude product 0.48g repeatedly.The last separation and purification lyophilize of ESI:994.87 HPLC:53.2% gets the finished product Atosiban.

Embodiment 3,

Take by weighing 0.67g iodine (this iodine amount is excessive 5 times) and be dissolved in 10ml methyl alcohol, with embodiment 1 resin reaction 75min 1., temperature is controlled at 0 ℃, and reaction finishes with using the ether dry adsorbent behind the DMF repetitive scrubbing resin.Aftertreatment gets Atosiban crude product 0.45g with example 2.The last separation and purification lyophilize of ESI:994.45 HPLC:78.2% gets the finished product Atosiban.

Embodiment 4,

Take by weighing 0.67g iodine (this iodine amount is excessive 5 times) and be dissolved in 10ml methyl alcohol, with embodiment 1 resin reaction 90min 1., temperature is controlled at 0 ℃, and reaction finishes with using the ether dry adsorbent behind the DMF repetitive scrubbing resin.Aftertreatment gets Atosiban crude product 0.39g with example 2.The last separation and purification lyophilize of ESI:995.03 HPLC:63.2% gets the finished product Atosiban.

Claims (5)

1, a kind of solid phase synthesis process of Atosiban, it is characterized in that may further comprise the steps: at first use the aminomethyl resin to make carrier, according to the fluorenylmethyloxycarbonyl/tertiary butyl solid-phase polypeptide synthetic method amino acid is connected one by one, method with iodine oxidation on the resin forms the sulphur sulfide linkage again, use trifluoracetic acid at last again: tri isopropyl silane: water: the mixing solutions of p-cresol downcuts peptide from resin, just can directly obtain forming the crude product of sulphur sulfide linkage, through the freezing Atosiban that obtains of high-efficient liquid phase chromatogram purification.

2, the solid phase synthesis process of a kind of Atosiban according to claim 1; it is characterized in that: in connecing the amino acid process; with volume ratio fluorenylmethyloxycarbonyl protection amino acid/benzotriazole-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester/1-hydroxybenzene a pair of horses going side by side triazole/N, N-diisopropylethylamine=1: 1: 1: 2, the excessive 2-5 of amino acid condensation process of fluorenylmethyloxycarbonyl protection is doubly.

3, the solid phase synthesis process of a kind of Atosiban according to claim 1 is characterized in that: Caesar's detection is all passed through in each step condensation in connecing the amino acid process, and color is positive and then repeats to connect amino acid.

4, the solid phase synthesis process of a kind of Atosiban according to claim 1 is characterized in that: described iodine is oxidized to: iodine is dissolved in the methanol solution and connected nine amino acid whose resin reaction 30-90 minutes, and temperature is controlled at 0 ℃.

5, the solid phase synthesis process of a kind of Atosiban according to claim 1, it is characterized in that: described peptide is downcut from resin, every gram resin is with 6-10ml volume ratio trifluoracetic acid: tri isopropyl silane: water: p-cresol=88: 2: 5: 5 mixed solution.