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;
(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
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 is doubly) 76.5g TBT (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.
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 is doubly) 55.3g, 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 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.
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 is doubly) 123.3g, 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 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.
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)-Cly-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/11ml/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.