CN1858060A - Process for preparing solid phase polypeptide synthetic eptifibatide - Google Patents
Process for preparing solid phase polypeptide synthetic eptifibatide Download PDFInfo
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Abstract
The preparation process of solid phase polypeptide synthesized eptifibatide includes the following steps: (1) connecting amino acids one by one with Rink Amide resin, Rink Amide MBHA resin or Rink Amide AM resin as initial material, and Fmoc protected amino aids as monomer, with the last peptide chain being S-benzyl mercapto propionic acid Map(SBzl); (2) adding peptide cutting agent (TFA/HBr/HAc/TIS/EDT) to cut peptide; (3) precipitating and collecting coarse reductant eptifibatide product in ether solvent; (4) dissolving coarse reductant eptifibatide product in water, regulating pH value with ammonia water to 7.5-10.0, introducing air for oxidation, and collecting coarse eptifibatide product; and (5) separating and purifying the coarse product in C18 column to obtain the target product. The present invention has high yield, and is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of preparation method of Integrilin, be specifically related to the preparation method of solid phase polypeptide synthetic eptifibatide.
Background technology
Integrilin, English Eptifibatide by name, structural formula is:
Molecular formula is C
35H
49N
11O
9S
2
Integrilin is a kind of specificity GP IIb/IIIa receptor antagonist that can quiet notes clinically.By stop fiber former with GP IIb/IIIa receptors bind, thereby anticoagulant and prevent thrombosis, this product is as the adjuvant drug of heparin or acetylsalicylic acid clinically.
According to luxuriant and rich with fragrance Bart is artificial synthetic ring-type seven peptides, it is platelet glycoprotein GP IIb/IIIa receptor antagonist, the platelet aggregation reaction that causes by various activator capable of blocking, it is the strongest known specificity platelet aggregation inhibitor, be mainly used in and prevent the myocardial oxygen delivery arterial occlusion, heart attack, the sudden death that unstable angina pectoris, non-q wave myocardial infarction, coronary artery interventional therapy cause.
This kind cooperated make it in the U.S. to go on the market trade(brand)name Integrelin in 1998 by COR Therapeutics company and Schering-Plough company by the exploitation of U.S. COR Therapeutics company.
Chinese patent application 02145292.X discloses a kind of preparation method according to luxuriant and rich with fragrance Bart; with Fmoc-Rink AmideAM resin for opening the beginning raw material; the 5th protection propylhomoserin is S-triphenyl sulfydryl propionyl-N; N-two tertbutyloxycarbonyls-homoarginine; the raw material sources difficulty; be not easy preparation, apply and have any problem.Exist the defective of using severe toxicity, corrosive reagents.
Summary of the invention
The objective of the invention is to disclose a kind of preparation method of solid phase polypeptide synthetic eptifibatide, 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, the amino acid of protecting with Fmoc is monomer, connects amino acid one by one, and last peptide chain uses S-benzyl thiohydracrylic acid (Map (SBzl));
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 as 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, precipitation is also collected reduced form Integrilin crude product (MW=833.96);
(4) reduced form Integrilin crude product is soluble in water, regulate PH to 7.5-10.0 with ammoniacal liquor, oxidized form Integrilin crude product is collected in the blowing air oxidation;
(5) crude product of collecting is passed through C
18Column separating purification obtains target product.Total recovery can reach more than 23%.
According to the present invention; with Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin is starting raw material; amino acid with the Fmoc protection is monomer; connect amino acid one by one, last peptide chain uses the method for S-benzyl thiohydracrylic acid (Map (SBzl)) to comprise the steps:
Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin
---→ Fmoc-Cys-resin---→ Fmoc-Pro-Cys-resin
---→ Fmoc-Asp (OtBu)-Trp-Pro-Cys-resin
---→ Fmoc-Gly-Asp (OtBu)-Trp-Pro-Cys-resin
---→ Fmoc-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys-resin
---→ Map (SBzl)-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys-resin.
More specifically comprise the steps:
(1) pre-treatment of resin: with Rink Amide resin, Rink Amide mbha resin or Rink AmideAM resin, make the abundant swelling of resin with the DMF immersion, nitrogen dries up, the DMF solution that adds hexahydropyridine, 10～35 ℃ were reacted 20～40 minutes, nitrogen blows elimination and removes hexahydropyridine, the DMF washing, and 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-Cys (Trt)-resin:
The pretreated resin of step (1) is mixed with Fmoc-Cys (Trt)-OH (MW:585.7), TBTU (MW:321), HOBT (MW:135.1), NMM (MW=101.2) and DMF, mixture was reacted under 20～35 ℃ condition 0.5～1.5 hour, nitrogen dries up, the DMF washing, nitrogen dries up;
NMM: DMF=1: 5～15, volume ratio;
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;
The DMF solution that adds hexahydropyridine then, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, and with the DMF washing, nitrogen dries up, and obtains Fmoc-Cys (Trt)-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-Pro-Cys (Trt)-resin:
In the Fmoc-Cys of step (2) (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Pro-OH (MW:337.4), TBTU (MW:321), HOBT (MW:135.1), NMM (MW=101.2) and DMF and mix, 20～35 ℃ were reacted 0.5～2 hour, and nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Pro-Cys (Trt)-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:
(4) preparation of Fmoc-Trp-Pro-Cys (Trt)-resin:
In the Fmoc-Pro-Cys of step (3) (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Trp-OH (MW:426.5), TBTU (MW:321), HOBT (MW:135.1), NMM (MW=101.2) and DMF and mix, 20～35 ℃ were reacted 0.5～2 hour, and nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Trp-Pro-Cys (Trt)-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Trp-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:
(5) preparation of Fmoc-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin
In the Fmoc-Trp-Pro-Cys of step (4) (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Asp (OtBu)-OH (MW:411.5), TBTU MW:321), HOBT (MW:135.1), NMM (MW=101.2) and DMF mix, with 20～35 ℃ of reactions of mixture 0.5～2 hour, nitrogen dried up, DMF washs, nitrogen dries up; Obtain Fmoc-Trp-Pro-Cys (Trt)-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Asp (OtBu)-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:
(6) preparation of Fmoc-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin
In the Fmoc-Asp of step (5) (OtBu)-Trp-Pro-Cys (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Gly-OH (MW:297.3), TBTU, HOBT, NMM and DMF and mix, with 20～35 ℃ of reactions of mixture 0.5～2 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
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;
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:
(7) preparation of Fmoc-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin
In the Fmoc-Gly-Asp of step (6) (OtBu)-Trp-Pro-Cys (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Fmoc-Har (Pbf)-OH (MW:662.8), TBTU, HOBT, NMM and DMF and mix, with 20～35 ℃ of reactions of mixture 0.5～2 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin;
NMM: DMF=1: 5～15, volume ratio;
In the reaction system:
The mole number of Fmoc-Har (Pbf)-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:
(8) preparation of Map (SBzl)-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin
In the Fmoc-Har of step (7) (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, the DMF washing, nitrogen dries up.Add Map (SBzl)-OH (MW:196), TBTU, HOBT, NMM and DMF, with 20～35 ℃ of reactions of mixture 0.5～2 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 (SBzl) 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 reduced form Integrilin resin;
Reaction expression is:
(8) cut peptide
Will cut peptide reagent add the Integrilin resin of protection, 20～35 ℃ of reactions 2～4 hours are filtered, drain, the filtrate precipitation that adds diethyl ether, the filtration collecting precipitation gets reduced form Integrilin crude product (MW=833.96), 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:
(9) oxidation (formation of disulfide linkage)
Reduced form Integrilin crude product 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 oxidation in 15～30 hours is filtered;
Reaction expression is:
(10) purifying
Filtrate flow is through C
18Post carries out purifying, moving phase: 0.1MNH
4Ac: acetonitrile (85: 15); Flow velocity is: 200-400ml/min; Follow the tracks of the needed effluent liquid of collection with HPLC.After the sample peak merges, adopt conventional method to desalt, remove acetonitrile, freeze-drying, get white loose block finished product (MW:831.96) approximately; About 23% (in the 61.9mmol of Rink Amide-resin) of total recovery.
S-benzyl thiohydracrylic acid (preparation of Map (SBzl):
With thiohydracrylic acid 300 gram and 1000 ml distilled waters, adds in 3000 milliliters the three-necked bottle, add strong aqua to more than the pH8, be cooled to 10 ℃ with ice-water bath, fierce stirring.Drip 300 milliliters of Benzyl Chlorides in 2 hours, keep about pH9, continue stirring at room 2 hours, filter, use distilled water wash, take out solid then, place interior the grinding of beaker to wash after-filtration, use 95% washing with alcohol again with distilled water, must about 300 gram S-benzyl thiohydracrylic acids (Map (SBzl)) after the drying.
By above-mentioned disclosed technical scheme as seen, method of the present invention, adopting Rink Amide mbha resin is starting raw material, the amino acid of protecting with Fmoc is monomer, connects amino acid whose method one by one, connects peptide yield height.Select Fmoc-Har (Pbf)-OH for use, raw material sources are convenient, and last peptide chain uses S-benzyl thiohydracrylic acid (Map (SBzl)), 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 crude product, avoids using poisonous reagents such as hydrogen fluoride, and three-waste pollution is few, and improves yield, adopts C
18Post carries out separation and purification, avoids using TFA (trifluoracetic acid) to carry out purifying, reduces the three wastes, and 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: 23%.This shows that method of the present invention is convenient to industrializing implementation, have bigger industrialization prospect.
Embodiment:
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 | Fmoc-Cys(Trt)-OH | Chengdu, Sichuan three high biochemical stock company |
| 2 | Fmoc-Pro-OH | Chengdu, Sichuan three high biochemical stock company |
| 3 | Fmoc-Trp-OH | Chengdu, Sichuan three high biochemical stock company |
| 4 | Fmoc-Asp(OtBu)-OH | Chengdu, Sichuan three high biochemical stock company |
| 6 | Fmoc-HAr(pbf)-OH | Chengdu, Sichuan three high biochemical stock company |
| 7 | Map (SBzl) (S-benzyl thiohydracrylic acid) | Self-control |
| 8 | Rink Amide mbha resin | Tianjin with become scientific ﹠ technical corporation |
| 9 | Rink Amide mbha resin | Tianjin with become scientific ﹠ technical corporation |
| 10 | HOBt (1-hydroxy benzo triazole) | Chengdu, Sichuan three high biochemical stock company |
| 11 | TBTU | Chengdu, Sichuan three high biochemical stock company |
| 12 | TFA (trifluoroacetic acid) | Merck company |
| 13 | TIS (tri isopropyl silane) | Merck company |
| 14 | EDT (dithioglycol) | Merck company |
| 15 | NMM (N-methylmorpholine) | Sail company of Shanghai section |
| 16 | DMF (dimethyl formamide) | Merck company |
| 17 | PIP (hexahydropyridine) | Merck company |
Embodiment 1
The peptide chain preparation:
Take by weighing Rink Amide mbha resin 72 gram (150 orders, 0.86mmol/g, 61.9mmol), in the special-purpose reactor of packing into, open the synthesizer general supply, open workstation, call the peptide program that connects of writing in advance, soak with 800mlDMF, make the abundant swelling of resin, nitrogen dries up.The DMF solution that adds 800 milliliter of 20% hexahydropyridine, 25 ℃ of joltings 35 minutes.Nitrogen blows the elimination hexahydropyridine, and with DMF, washing three times, nitrogen dries up respectively.
The preparation of Fmoc-Cys (Trt)-resin:
Add Fmoc-Cys (Trt)-OH (MW:585.7, the 2-6 of resin mole number is doubly) 108.9g, TBTU (MW:321, the 2-6 of resin mole number is doubly) 59.7g, HOBT (MW:135.1, the 2-6 of resin mole number are doubly) 25.1g, NMM 41.4ml (MW=101.2), 400mlDMF was with 25 ℃ of joltings of mixture 1 hour.Nitrogen dries up, DMF washing three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter of 20% hexahydropyridine, 25 ℃ of joltings 30 minutes.Nitrogen dries up, and with DMF washing five times, nitrogen dries up.
The preparation of Fmoc-Pro-Cys (Trt)-resin:
Fmoc-Pro-OH (MW:337.4, the 2-6 of resin mole number is doubly) 62.8g, TBTU (MW:321, the 2-6 of resin mole number is doubly) 59.7g, HOBT (MW:135.1, the 2-6 of resin mole number are doubly) 25.1g, NMM 41.4ml (MW=101.2), 400mlDMF was with 25 ℃ of joltings of mixture 1 hour.Nitrogen dries up, DMF washing three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter of 20% hexahydropyridine, 25 ℃ of joltings 30 minutes.Nitrogen dries up, and with DMF washing five times, nitrogen dries up.
The preparation of Fmoc-Trp-Pro-Cys (Trt)-resin:
Add Fmoc-Trp-OH (MW:426.5, the 2-6 of resin mole number is doubly) 79.3g, TBTU (MW:321, the 2-6 of resin mole number is doubly) 59.7g, HOBT (MW:135.1, the 2-6 of resin mole number are doubly) 25.1g, NMM 41.4ml (MW=101.2), 400ml DMF was with 25 ℃ of joltings of mixture 1 hour.Nitrogen dries up, DMF washing three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter of 20% hexahydropyridine, 25 ℃ of joltings 30 minutes.Nitrogen dries up, and with DMF washing five times, nitrogen dries up.
The preparation of Fmoc-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin:
Add Fmoc-Asp (OtBu)-OH (MW:411.5, the 2-6 of resin mole number are doubly) 76.5g TBT (MW:321, the 2-6 of resin mole number are doubly)
59.7g, HOBT (MW:135.1, the 2-6 of resin mole number are doubly) 25.1g, NMM 41.4ml (MW=101.2), 400mlDMF was with 25 ℃ of joltings of mixture 1 hour.Nitrogen dries up, DMF washing three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter of 20% hexahydropyridine, 25 ℃ of joltings 30 minutes.Nitrogen dries up, and with DMF washing five times, nitrogen dries up.
The preparation of Fmoc-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin:
Add Fmoc-Gly-OH (MW:297.3, the 2-6 of resin mole number are doubly) 55.3g, TBTU (MW:321, the 2-6 of resin mole number are doubly)
59.7g, HOBT (MW:135.1, the 2-6 of resin mole number are doubly) 25.1g, NMM 41.4ml (MW=101.2), 400mlDMF was with 25 ℃ of joltings of mixture 1 hour.Nitrogen dries up, DMF washing three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter of 20% hexahydropyridine, 25 ℃ of joltings 30 minutes.Nitrogen dries up, and with DMF washing five times, nitrogen dries up.
The preparation of Fmoc-Har (pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin:
Add Fmoc-Har (Pbf)-OH (662.8, the 2-6 of resin mole number doubly) 123.3g, TBTU (MW:321, the 2-6 of resin mole number are doubly)
59.7g, HOBT (MW:135.1, the 2-6 of resin mole number are doubly) 25.1g, NMM 41.4ml (MW=101.2), 400mlDMF was with 25 ℃ of joltings of mixture 1 hour.Nitrogen dries up, DMF washing three times, and nitrogen dries up.
The DMF solution that adds 500 milliliter of 20% hexahydropyridine, 25 ℃ of joltings 30 minutes.Nitrogen dries up, and with DMF washing five times, nitrogen dries up.
The preparation of Map (SBzl)-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin:
Add Map (SBzl)-OH (MW:196, the 2-6 of resin mole number is doubly) 36.5g, TBTU (MW:321, the 2-6 of resin mole number is doubly) 59.7g, HOBT (MW:135.1, the 2-6 of resin mole number is doubly) 25.1g, NMM 41.4ml (MW=101.2), 400mlDMF was with 25 ℃ of joltings of mixture 1 hour.Nitrogen dries up, DMF washing three times, and nitrogen dries up.
Again with anhydrous methanol washing three times.After draining, put into vacuum nitrogen blowing dryer nitrogen and dry up, weigh, must protect the about 97g of Integrilin resin.
Cut peptide:
The Integrilin resin transfer of getting the 97g protection is to cutting in the peptide bottle, and cooling adds while stirring and cuts peptide reagent down: (TFA/HBr/HAc/TIS/EDT=850ml/34ml/l1ml/51ml/25ml), 25 ℃ were stirred 3 hours.Filter, drain, filtrate adds the anhydrous diethyl ether precipitation, filter collecting precipitation must about 37g reduced form Integrilin crude product (MW=833.96,44.4mmol), yield is 95.4%.
Oxidation (formation of disulfide linkage):
37g reduced form Integrilin crude product is dissolved in the 40L purified water, under agitation slowly adds 1mol/L ammoniacal liquor, regulate PH to 8, blowing air stirred 24 hours, filtered.
Purifying:
Filtrate is through the C18 column purification, moving phase: 0.1MNH
4Ac: acetonitrile (85: 15); Flow velocity is: 300ml/min; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph.Desalt, remove acetonitrile, freeze-drying after the sample peak merges, approximately 11.8g white loose block finished product (MW:831.96,14.2mmol); About 23% (in the 61.9mmol of Rink Amide-resin) of total recovery.
Claims (7)
1. the preparation method of a solid phase polypeptide synthetic eptifibatide 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, the amino acid of protecting with Fmoc is monomer, connects amino acid one by one, and last peptide chain uses S-benzyl thiohydracrylic acid (Map (SBzl));
(2) adding is cut peptide reagent (TFA/HBr/HAc/TIS/EDT) and is cut peptide then;
(3) with the ether be solvent, precipitation is also collected reduced form Integrilin crude product;
(4) reduced form Integrilin crude product is soluble in water, regulate PH to the oxidation of 7.5-10.0 blowing air with ammoniacal liquor, collect oxidized form Integrilin crude product;
(5) crude product of collecting is passed through C
18Column separating purification obtains target product.
2. method according to claim 1; it is characterized in that; with Rink Amide resin, Rink AmideMBHA resin or Rink Amide AM resin is starting raw material; amino acid with the Fmoc protection is monomer; connect amino acid one by one, last peptide chain uses the method for S-benzyl thiohydracrylic acid (Map (SBzl)) to comprise the steps:
Rink Amide resin, Rink Amide mbha resin or Rink Amide AM resin
→ Fmoc-Cys-resin → Fmoc-Pro-Cys-resin
→ Fmoc-Asp (OtBu)-Trp-Pro-Cys-resin
→ Fmoc-Gly-Asp (OtBu)-Trp-Pro-Cys-resin
→ Fmoc-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys-resin
→ Map (SBzl)-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys-resin.
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 AmideAM resin, make the abundant swelling of resin with the DMF immersion, nitrogen dries up, the DMF solution that adds hexahydropyridine, 10～35 reaction 20～40 minutes, nitrogen blows elimination and removes hexahydropyridine, the DMF washing, and nitrogen dries up.
(2) preparation of Fmoc-Cys (Trt)-resin:
The resin of step (1) is mixed with Fmoc-Cys (Trt)-OH, TBTU, HOBT, NMM and DMF, reacted 0.5～1.5 hour, nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Cys (Trt)-resin;
Reaction expression is:
In the Fmoc-Cys of step (2) (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, and with the DMF washing, nitrogen dries up.Add Fmoc-Pro-OH, TBTU, HOBT, NMM and DMF and mix, reacted 0.5～2 hour, nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Pro-Cys (Trt)-resin.
Reaction expression is:
(4) preparation of Fmoc-Trp-Pro-Cys (Trt)-resin:
In the Fmoc-Pro-Cys of step (3) (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, and with the DMF washing, nitrogen dries up.Add Fmoc-Trp-OH, TBTU, HOBT, NMM and DMF and mix, reacted 0.5～2 hour, nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Trp-Pro-Cys (Trt)-resin.
Reaction expression is:
(5) preparation of Fmoc-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin
In the Fmoc-Trp-Pro-Cys of step (4) (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ were reacted 20～40 minutes, and nitrogen dries up, and with the DMF washing, nitrogen dries up.Add Fmoc-Asp (OtBu)-OH, TBTU, HOBT, NMM and DMF and mix, reacted 0.5～2 hour, nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin.
Reaction expression is:
(6) preparation of Fmoc-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin
In the Fmoc-Asp of step (5) (OtBu)-Trp-Pro-Cys (Trt)-resin, add the DMF solution of hexahydropyridine, 20～35 ℃ of reactions 20～40 minutes, nitrogen dries up, with the DMF washing, nitrogen dries up.Add Fmoc-Gly-OH, TBTU, HOBT, NMM and DMF and mix, reacted 0.5～2 hour, nitrogen dries up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin.
Reaction expression is:
(7) preparation of Fmoc-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin
In the Fmoc-Gly-Asp of step (6) (OtBu)-Trp-Pro-Cys (Trt)-resin, add the DMF solution of hexahydropyridine, reacted 20～40 minutes, nitrogen dries up, and with the DMF washing, nitrogen dries up.Add Fmoc-Har (Pbf)-OH, TBTU, HOBT, NMM and DMF and mix, with 20～35 ℃ of reactions of mixture 0.5～2 hour, nitrogen dried up, the DMF washing, and nitrogen dries up; Obtain Fmoc-Har (pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin.
Reaction expression is:
(8) preparation of Map (SBzl)-Har (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin
In the Fmoc-Har of step (7) (Pbf)-Gly-Asp (OtBu)-Trp-Pro-Cys (Trt)-resin, add the DMF solution of hexahydropyridine, reacted 20～40 minutes, nitrogen dries up, and with the DMF washing, nitrogen dries up.Add Map (SBzl)-OH, TBTU, HOBT, NMM and DMF, reacted 0.5～2 hour, nitrogen dries up, the DMF washing, and nitrogen dries up.Methanol wash, nitrogen dries up, and obtains the reduced form Integrilin resin of band protecting group.
Reaction expression is:
(8) cut peptide
With cutting the reduced form Integrilin resin that peptide reagent adds protection, react 2～4 hours, filter, drain, the filtrate precipitation that adds diethyl ether, the filtration collecting precipitation gets reduced form Integrilin crude product;
Saidly cut the mixture that peptide reagent is TFA/HBr/HAc/TIS/EDT;
Reaction expression is:
(9) oxidation
Reduced form Integrilin crude product is soluble in water, add ammoniacal liquor, regulate pH to 7.5-10.0, blowing air oxidation in 15～30 hours is followed the tracks of reaction process with HPLC;
Reaction expression is:
(10) purifying
Filtrate flow is through C
18Post carries out purifying, and moving phase is 0.1MNH
4Ac and acetonitrile; Flow velocity is: 200-800ml/min; Collect needed effluent liquid, adopt conventional method to desalt, remove acetonitrile, freeze-drying, get finished product.
4. method according to claim 3 is characterized in that, in the step (10), moving phase is 0.1MNH
4Ac: acetonitrile (75-85: 25-15), volume ratio.
5. method according to claim 3 is characterized in that, said ratio of cutting peptide reagent is (TFA/HBr/HAc/TIS/EDT=850ml/34ml/11ml/51ml/25ml).
6. method according to claim 3 is characterized in that, is starting raw material with Rink Amide mbha resin or RinkAmide AM resin, and the amino acid of protecting with Fmoc is monomer, and the temperature of reaction when connecting amino acid one by one is 20～35 ℃.
7. method according to claim 3 is characterized in that, cutting the peptide temperature is 20～35 ℃.
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