CN102603869B - Synthetic method of hexapeptide - Google Patents
Synthetic method of hexapeptide Download PDFInfo
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- CN102603869B CN102603869B CN2012100830840A CN201210083084A CN102603869B CN 102603869 B CN102603869 B CN 102603869B CN 2012100830840 A CN2012100830840 A CN 2012100830840A CN 201210083084 A CN201210083084 A CN 201210083084A CN 102603869 B CN102603869 B CN 102603869B
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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 relates to a synthetic method of hexapeptide, which comprises the following steps: swelling resins; preparing amino resins; synthetizing protection peptide resins; synthesizing H2N-Arg(pbf)-HN-Trt-resin; synthetizing NH2-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH-resin peptide resin; synthetizing Fmoc-Glu(tbu)-COOSu; synthetizing Fmoc-Glu(tbu)-Glu(tbu)-OH; synthesizing Fmoc-Glu(tbu)-Glu(tbu)-OOSu; synthetizing Fmoc-Glu(tbu)-Glu(tbu)-Met-COOH; synthesizing Fmoc-Glu(tbu)-Glu(tbu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH-resin peptide; synthesizing H2N-Glu(tbu)-Glu(tbu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH-resin peptide; synthesizing Ac-HN-Glu(tbu)-Glu(tbu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH-resin peptide and preparing hexapeptide.
Description
Technical field
The invention belongs to the makeup technical field, be specifically related to the small peptide in the polypeptide class.
Background technology
Argireline is a kind of six amino acid whose active polypeptide that contain, and aminoacid sequence is Ac-Glu-Glu-Met-Gln-Arg-Arg-NH
2, have the biological activity of skin wrinkle, be widely used in the various senior cosmetics.
The at present classical solid phase method production of main employing of the preparation method of Argireline.Be about to protected amino acid and extended one by one to the N end by the C end, prepare purity by condensation, deprotection, again condensation, cutting resin, centrifugal, freeze-drying and high performance liquid phase and be higher than 90% Argireline product.Needed resin market value is comparatively expensive in the method, and its exchange activity is little, and in synthetic the condensing agent that uses and alkali all obviously excessive, caused increasing of Argireline raw materials cost.As cosmetic material, the market value of Argireline is 1000 yuan/g at present, causes the makeup finished product that contains Argireline expensive, is difficult to accept for the general population, and therefore, the processing method research that reduces the Argireline production cost is significant.
Summary of the invention
Technical problem to be solved by this invention is to overcome the shortcoming of above-mentioned Argireline solid phase synthesis process, and the synthetic method of the Argireline that a kind of method is simple, product cost is low is provided in conjunction with solid phase and liquid-phase synthesis process.
Solving the problems of the technologies described above the technical scheme that adopts is comprised of following step:
1, resin swelling
The Rink-Amide-AM-Resin resin is added in the flask, and every gram Rink-Amide-AM-Resin resin adds the 10mL methylene dichloride, and stirring at room is soaked and made its abundant swelling in 30 minutes.
2, preparation aminoresin
In the resin of step 1 swelling, pass into ammonia, sealing, stirring at room was soaked 30～60 minutes, and 300 order cores remove by filter solvent, clean resin with dimethyl formamide, get aminoresin.
3, synthetic protection peptide resin
In the aminoresin of step 2, add N; dinethylformamide; stirring at room; add Fmoc-Arg (pbf)-OH; O-benzotriazole-N; N; N ', N '-tetramethyl-urea Tetrafluoroboric acid; benzotriazole; diisopropylethylamine, aminoresin and Fmoc-Arg (pbf)-OH; O-benzotriazole-N; N; N ', N '-tetramethyl-urea Tetrafluoroboric acid; benzotriazole; the mol ratio of diisopropylethylamine is 1: 3: 3: 3: 6, and stirring at room reaction 3～5 hours; use N; dinethylformamide cleans resin, gets the protection peptide resin, and sequence is Fmoc-Arg (pbf)-HN-Trt-resin.
4, synthetic H
2N-Arg (pbf)-HN-Trt-resin
Adding 10mL massfraction is 20% piperidines DMF solution in every gram protection peptide resin, and room temperature reaction 20～40 minutes removes by filter solvent with core, and DMF cleans resin, gets H
2N-Arg (pbf)-HN-Trt-resin.
5, synthetic NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin peptide resin
Repeating step 3,4 carries out coupling to the protection arginine first, again the protection glutamine is carried out coupling, removes the Fmoc protection, and core removes by filter solvent, the three peptide prod NH that must link to each other with resin
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
6, synthetic Fmoc-Glu (tbu)-COOSu
Fmoc-Glu (tbu)-COOH is placed reaction flask, add 10～15m L ethyl acetate by every gram Fmoc-Glu (tbu)-COOH, stirring at room, add nitrogen maloyl imines, the mol ratio of Fmoc-Glu (tbu)-COOH and nitrogen maloyl imines is 1: 1～1.5, stirring and dissolving adds dicyclohexylcarbodiimide.The mol ratio of Fmoc-Glu (tbu)-COOH and dicyclohexylcarbodiimide is 1: 1～2, room temperature reaction 20～24 hours, and filtration under diminished pressure is removed solid insoluble, and filtrate decompression is concentrated into dried, gets Fmoc-Glu (tbu)-COOSu.
7, synthetic Fmoc-Glu (tbu)-Glu (tbu)-OH
The product of step 6 gained is placed reaction flask, and by every gram Fmoc-Glu (tbu)-COOSu adding 10～15mL dioxane, stirring at room is dissolved fully to Fmoc-Glu (tbu)-COOSu.
γ-tert-butyl ester L-glutamic acid and sodium bicarbonate are dissolved in the distilled water, gained solution is added drop-wise in the dioxane solution of Fmoc-Glu (tbu)-COOSu, the mol ratio of γ-tert-butyl ester L-glutamic acid and Fmoc-Glu (tbu)-COOSu is 1.5: 1, the mol ratio of sodium bicarbonate and Fmoc-Glu (tbu)-COOSu is 2: 1, stirring at room reaction 18～20 hours, be that to transfer pH be 2～3 to 5% hydrochloric acid soln with massfraction, add ethyl acetate extraction, tell organic phase, organic phase is water successively, the saturated common salt washing, organic phase is through anhydrous sodium sulfate drying, filter, filtration under diminished pressure, filtrate decompression inspissation Fmoc-Glu (tbu)-Glu (the tbu)-COOH that contracts to get.
8, synthetic Fmoc-Glu (tbu)-Glu (tbu)-OOSu
Adopt the described method of step 6 to react Fmoc-Glu (tbu)-Glu (tbu)-COOH, get Fmoc-Glu (tbu)-Glu (tbu)-COOSu.
9, synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH
Adopt the described method of step 7 to react step 8Fmoc-Glu (tbu)-Glu (tbu)-COOSu, methionine(Met), get Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH.
10, synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
The NH that step 5 is synthetic
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the mol ratio of diisopropylethylamine is 1: 2～5: 2～5: 2～5: 6～10, room temperature reaction 3～5 hours, filtration under diminished pressure, use N, dinethylformamide is washed resin 1～3 time, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
11, synthetic H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
With the method for step 4, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin is removed the Fmoc blocking group, gets H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
12, synthetic Ac-HN-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
At H
2Add methylene dichloride, diacetyl oxide, triethylamine in N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide, by every gram H
2Add 20mL methylene dichloride, H in N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
2The mass ratio of N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and diacetyl oxide, triethylamine is 1: 0.5: 1.Room temperature reaction 3～5 hours filters, and washes resin 3 times with DMF, gets Ac-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
13, preparation Argireline
Preparation resin cutting liquid: be 5: 2.5: 5 by volume with phenol, 1，2-ethandithiol, thioanisole, trifluoroacetic acid, water: join beaker at 4: 83.5 and to mix.
Cutting resin: in step 12 resin dai, add cutting liquid, add 10～15mL cutting liquid by every gram Ac-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide, stirring at room reaction 3 hours, filter, filtrate is poured in the freezing anhydrous diethyl ether, stir, centrifugal, isolate supernatant liquor, the room temperature decompression drying, the Argireline crude product, get purity greater than 95% Argireline through reverse-phase chromatography purifying, concentrated, freeze-drying.
In synthetic Fmoc-Glu of the present invention (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 10, the NH that step 5 is synthetic
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the preferred molar ratio of diisopropylethylamine is 1: 2～4: 2～4: 2～4: 6～10, room temperature reaction 3～5 hours, filtration under diminished pressure, use N, dinethylformamide is washed resin 1～3 time, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf0-NH-resin resin peptide.
In synthetic Fmoc-Glu of the present invention (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 10, the NH that step 5 is synthetic
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the optimum mole ratio of diisopropylethylamine is 1: 3: 3: 3: 8, room temperature reaction 3～5 hours, filtration under diminished pressure, use N, dinethylformamide is washed resin 1～3 time, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
The present invention adopts conventional liquid phase synthesizing method to prepare three amino acid whose protection tripeptides of the N end of Argireline; three peptide resins that adopt the solid phase method preparation to be connected with resin; adopt the solid phase method condensation to obtain the Argireline resin as a fragment peptide and tripeptides three amino acid whose protection tripeptides of the N of Argireline end, through resin cutting, centrifugal, high performance liquid phase prepares, freeze-drying gets pure Argireline.The present invention compares with solid phase method, the steric effect impact when having overcome one by one amino acid condensation, and production cost can reduce by 20%～30%.The present invention has the advantages such as synthetic method is simple, product cost is low, can be used for synthetic Argireline.
Description of drawings
Fig. 1 is the liquid chromatogram that adopts the synthetic Argireline of the embodiment of the invention 1.
Fig. 2 is the mass spectrum that adopts the synthetic Argireline of the embodiment of the invention 1.
Embodiment
The present invention is described in more detail below in conjunction with drawings and Examples, but the invention is not restricted to these embodiment.
Embodiment 1
Prepare Argireline as an example of raw materials used Rink-Amide-AM-Resin resin 10g example, used other raw material and preparation method are as follows:
1, resin swelling
Rink-Amide-AM-Resin resin 10g is added in the 250ml flask, add the 100ml methylene dichloride, stirring at room is soaked and was made its abundant swelling in 30 minutes.
2, preparation aminoresin
In the resin of step 1 swelling, pass into ammonia, sealing, stirring at room was soaked 30 minutes, and 300 order cores remove by filter solvent, and clean resin with dimethyl formamide, get aminoresin, and resins exchange activity is 17mmol.
3, synthetic protection peptide resin
In aminoresin, add 100ml N, dinethylformamide, and O-benzotriazole-N of the Fmoc-Arg (pbf) of 51mmol-OH, 51mmol, N, N ', the benzotriazole of N '-tetramethyl-urea Tetrafluoroboric acid, 51mmol and the diisopropylethylamine of 102mmol, aminoresin and Fmoc-Arg (pbf)-OH, O-benzotriazole-N, N, N ', the mol ratio of N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine is 1: 3: 3: 3: 6, and stirring at room reaction 3～5 hours.Triketohydrindene hydrate detects resin particle should show bright look, shows to react completely.Core removes by filter solvent, and cleans resin with DMF, gets the protection peptide resin, and sequence is Fmoc-Arg (pbf)-HN-Trt-resin.
4, synthetic H
2N-Arg (pbf)-HN-Trt-resin
Adding 100ml massfraction is 20% piperidines DMF solution in the protection peptide resin that is synthesized, stirring at room reaction 20～40 minutes, and triketohydrindene hydrate detects resin particle should show blueness, shows to react to finish.Core removes by filter solvent, and cleans resin with DMF, gets H
2N-Arg (pbf)-HN-Trt-resin.
5, synthetic NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin peptide resin
Repeating step 3,4 carries out coupling to Fmoc-Arg (pbf)-OH protected amino acid first, again Fmoc-Gln (Trt)-OH amino acid is carried out coupling, the three peptide prod NH that must link to each other with resin
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
6, synthetic Fmoc-Glu (tbu)-COOSu
Fmoc-Glu (tbu)-COOH 12.8g is placed the 250ml reaction flask, add the 150ml ethyl acetate, stirring at room, add nitrogen maloyl imines 4.15g, the mol ratio of Fmoc-Glu (tbu)-COOH and nitrogen maloyl imines is 1: 1.2, stirring and dissolving, add dicyclohexylcarbodiimide 7.43g, the mol ratio of Fmoc-Glu (tbu)-COOH and dicyclohexylcarbodiimide is 1: 1.2, room temperature reaction 20 hours, filtration under diminished pressure is removed solid insoluble, and filtrate decompression is concentrated into dried, gets Fmoc-Glu (tbu)-COOSu.
7, synthetic Fmoc-Glu (tbu)-Glu (tbu)-OH
The product 5.23g that gets step 6 gained places reaction flask, adds in the 60ml dioxane stirring at room, fully dissolving.Getting γ-tert-butyl ester L-glutamic acid 2.64g and sodium bicarbonate 2.18g is dissolved in the 60ml distilled water, gained solution is added drop-wise in the dioxane solution of Fmoc-Glu (tbu)-COOSu, the mol ratio of γ-tert-butyl ester L-glutamic acid and Fmoc-Glu (tbu)-COOSu is 1.3: 1, the mol ratio of sodium bicarbonate and Fmoc-Glu (tbu)-COOSu is 2: 1, stirring at room reaction 18～20 hours, be that to transfer pH be 2～3 to 5% hydrochloric acid soln with massfraction, add ethyl acetate extraction, tell organic phase, organic phase is water successively, the saturated common salt washing, organic phase is through anhydrous sodium sulfate drying, filter, filtration under diminished pressure, filtrate decompression inspissation Fmoc-Glu (tbu)-Glu (the tbu)-COOH4.8g that contracts to get, yield 78.7%.
8, synthetic Fmoc-Glu (tbu)-Glu (tbu)-OOSu
With Fmoc-Glu (tbu)-Glu (tbu)-COOH 4.8g, adopt described method of the 6th step to react, get Fmoc-Glu (tbu)-Glu (tbu)-COOSu 6.0g.
9, synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH
Get 8 Fmoc-Glu (tbu) that synthesizes-Glu (tbu)-COOSu 6.0g and methionine(Met) and adopt the described method of step 7 to react, get Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH.
10, synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
The NH that step 5 is synthesized
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH 37.8g, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid 16.4g, benzotriazole 6.9g, diisopropylethylamine 17.6g, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the mol ratio of diisopropylethylamine is 1: 3: 3: 3: 8, room temperature reaction 3～5 hours, filtration under diminished pressure, use N, dinethylformamide is washed resin 1～3 time, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
11, synthetic H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
With the method for step 4, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin is removed the Fmoc blocking group, gets H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
12, synthetic Ac-HN-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
The Ac-HN-Glu (tbu) that step 11 is synthetic-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide 10g adds the 200mL methylene dichloride, adds 5.0g diacetyl oxide, 10.0g triethylamine, H
2The mass ratio of N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and diacetyl oxide, triethylamine is 1: 0.5: 1.Room temperature reaction 4 hours filters, and washes resin 3 times with DMF, gets Ac-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
13, preparation Argireline
Preparation resin cutting liquid: with phenol 10mL, 1,2-dithioglycol 5mL, thioanisole 10mL, trifluoroacetic acid 8mL, water 167mL join in the beaker and mix, the volume ratio of phenol and 1，2-ethandithiol, thioanisole, trifluoroacetic acid, water is 5: 2.5: 5: 4: 83.5, for subsequent use.
Cutting resin: get the synthetic Ac-Glu (tbu) of step 12-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide 10g, add 120ml resin cutting liquid, stirring at room reaction 3 hours is filtered, and filtrate is poured in the freezing anhydrous diethyl ether, stir, centrifugal, isolate supernatant liquor, the room temperature decompression drying gets Argireline crude product 9.0g, efficient liquid phase chromatographic analysis, purity is greater than 50%.
Take acetonitrile and the water that contains 0.1%TFA as moving phase, oppositely prepare purifying with high performance liquid chromatography.Collect the principal constituent cut, decompression is concentrated into 10% ± 5% of original volume, and freeze-drying gets Argireline, and purity is greater than 95%.
Embodiment 2
Prepare Argireline as an example of raw materials used Rink-Amide-AM-Resin resin 10g example, used other raw material and preparation method are as follows:
The resin swelling step 1 of present embodiment, preparation aminoresin step 2, synthetic protection peptide resin step 3, synthetic H
2N-Arg (pbf)-HN-Trt-resin step 4, synthetic NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin peptide resin step 5 is identical with embodiment 1.
In synthetic Fmoc-Glu (tbu)-COOSu step 6, Fmoc-Glu (tbu)-COOH 12.8g is placed the 250ml reaction flask, add the 128ml ethyl acetate, stirring at room, add nitrogen maloyl imines 3.45g, the mol ratio of Fmoc-Glu (tbu)-COOH and nitrogen maloyl imines is 1: 1, stirring and dissolving, add dicyclohexylcarbodiimide 6.18g, the mol ratio of Fmoc-Glu (tbu)-COOH and dicyclohexylcarbodiimide is 1: 1, room temperature reaction 20 hours, and filtration under diminished pressure is removed solid insoluble, filtrate decompression is concentrated into dried, gets Fmoc-Glu (tbu)-COOSu.
Synthetic Fmoc-Glu (tbu)-Glu (tbu)-OH step 7, synthetic Fmoc-Glu (tbu)-Glu (tbu)-OOSu step 8, synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH step 9 is identical with embodiment 1.
In synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 10, the NH that step 5 is synthesized
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH25.2g, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid 10.9g, benzotriazole 4.6g, diisopropylethylamine 11.7g, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the mol ratio of diisopropylethylamine is 1: 2: 2: 2: 6, other step in this step is identical with embodiment 1, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
Synthetic H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 11, synthetic Ac-HN-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 12 is identical with embodiment 1.In preparation Argireline step 13, get the synthetic Ac-Glu (tbu) of step 12-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide 10g, add 100ml resin cutting liquid, stirring at room reaction 3 hours is filtered, filtrate is poured in the freezing anhydrous diethyl ether, stir, centrifugal, isolate supernatant liquor, room temperature decompression drying, other step in this step is identical with embodiment 1, gets the Argireline crude product.
Other step is identical with embodiment 1, is prepared into Argireline.
Embodiment 3
Prepare Argireline as an example of raw materials used Rink-Amide-AM-Resin resin 10g example, used other raw material and preparation method are as follows:
The resin swelling step 1 of present embodiment, preparation aminoresin step 2, synthetic protection peptide resin step 3, synthetic H
2N-Arg (pbf)-HN-Trt-resin step 4, synthetic NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin peptide resin step 5 is identical with embodiment 1.
In synthetic Fmoc-Glu (tbu)-COOSu step 6, Fmoc-Glu (tbu)-COOH 12.8g is placed the 250ml reaction flask, add the 128ml ethyl acetate, stirring at room, add nitrogen maloyl imines 4.15g, the mol ratio of Fmoc-Glu (tbu)-COOH and nitrogen maloyl imines is 1: 1.2, stirring and dissolving, add dicyclohexylcarbodiimide 7.43g, the mol ratio of Fmoc-Glu (tbu)-COOH and dicyclohexylcarbodiimide is 1: 1.2, room temperature reaction 20 hours, and filtration under diminished pressure is removed solid insoluble, filtrate decompression is concentrated into dried, gets Fmoc-Glu (tbu)-COOSu.
Synthetic Fmoc-Glu (tbu)-Glu (tbu)-OH step 7, synthetic Fmoc-Glu (tbu)-Glu (tbu)-OOSu step 8, synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH step 9 is identical with embodiment 1.
In synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 10, the NH that step 5 is synthesized
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH 63g, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid 27.3g, benzotriazole 11.5g, diisopropylethylamine 21.9g, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the mol ratio of diisopropylethylamine is 1: 5: 5: 5: 10, other step in this step is identical with embodiment 1, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
Synthetic H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 11, synthetic Ac-HN-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 12 is identical with embodiment 1.In preparation Argireline step 13, get the synthetic Ac-Glu (tbu) of step 12-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide 10g, add 100ml resin cutting liquid, stirring at room reaction 3 hours is filtered, filtrate is poured in the freezing anhydrous diethyl ether, stir, centrifugal, isolate supernatant liquor, room temperature decompression drying, other step in this step is identical with embodiment 1, gets the Argireline crude product.
Other step is identical with embodiment 1, is prepared into Argireline.
Embodiment 4
Prepare Argireline as an example of raw materials used Rink-Amide-AM-Resin resin 10g example, used other raw material and preparation method are as follows:
The resin swelling step 1 of present embodiment, preparation aminoresin step 2, synthetic protection peptide resin step 3, synthetic H
2N-Arg (pbf)-HN-Trt-resin step 4, synthetic NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin peptide resin step 5 is identical with embodiment 1.
In synthetic Fmoc-Glu (tbu)-COOSu step 6, Fmoc-Glu (tbu)-COOH 12.8g is placed the 250ml reaction flask, add the 192ml ethyl acetate, stirring at room, add nitrogen maloyl imines 5.19g, the mol ratio of Fmoc-Glu (tbu)-COOH and nitrogen maloyl imines is 11.5, stirring and dissolving, add dicyclohexylcarbodiimide 12.39g, the mol ratio of Fmoc-Glu (tbu)-COOH and dicyclohexylcarbodiimide is 1: 2, room temperature reaction 20～24 hours, and filtration under diminished pressure is removed solid insoluble, filtrate decompression is concentrated into dried, gets Fmoc-Glu (tbu)-COOSu.
Synthetic Fmoc-Glu (tbu)-Glu (tbu)-OH step 7, synthetic Fmoc-Glu (tbu)-Glu (tbu)-OOSu step 8, synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH step 9 is identical with embodiment 1.
In synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 10, the NH that step 5 is synthesized
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH 50.4g, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid 21.8g, benzotriazole 9.18g, diisopropylethylamine 21.9g, Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the mol ratio of diisopropylethylamine is 1: 4: 4: 4: 10, other step in this step is identical with embodiment 1, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
Synthetic H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 11, synthetic Ac-HN-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step 12 is identical with embodiment 1.In preparation Argireline step 13, get the synthetic Ac-Glu (tbu) of step 12-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide 10g, add 100ml resin cutting liquid, stirring at room reaction 3 hours is filtered, filtrate is poured in the freezing anhydrous diethyl ether, stir, centrifugal, isolate supernatant liquor, room temperature decompression drying, other step in this step is identical with embodiment 1, gets the Argireline crude product.
Other step is identical with embodiment 1, is prepared into Argireline.
In order to verify beneficial effect of the present invention, the contriver adopts the embodiment of the invention 1 synthetic Argireline to adopt hplc determination purity, and liquid chromatogram is seen Fig. 1, and as seen from Figure 1, the purity of prepared Argireline is greater than 95%.
Prepared Argireline is measured its structure with mass spectrograph, and test result is seen Fig. 2, and as seen from Figure 2, prepared Argireline molecular weight and molecular ion peak are consistent with Argireline molecular weight and molecular ion peak.
Claims (3)
1. the synthetic method of an Argireline is comprised of following step:
(1) resin swelling
The Rink-Amide-AM-Resin resin is added in the flask, and every gram Rink-Amide-AM-Resin resin adds the 10mL methylene dichloride, and stirring at room is soaked and made its abundant swelling in 30 minutes;
(2) preparation aminoresin
In the resin of step (1) swelling, pass into ammonia, sealing, stirring at room was soaked 30～60 minutes, and 300 order cores remove by filter solvent, clean resin with dimethyl formamide, get aminoresin;
(3) synthetic protection peptide resin
In the aminoresin of step (2), add N, dinethylformamide, stirring at room, add Fmoc-Arg (pbf)-OH, O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine, aminoresin and Fmoc-Arg (pbf)-OH, O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the mol ratio of diisopropylethylamine is 1:3:3:3:6, stirring at room reaction 3～5 hours, use N, dinethylformamide cleans resin, gets the protection peptide resin, and sequence is Fmoc-Arg (pbf)-HN-resin;
(4) synthetic H
2N-Arg (pbf)-HN-resin
Adding 10mL massfraction is 20% piperidines DMF solution in every gram protection peptide resin, and room temperature reaction 20～40 minutes removes by filter solvent with core, and DMF cleans resin, gets H
2N-Arg (pbf)-HN-resin;
(5) synthetic NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin peptide resin
Coupling is carried out to the protection arginine first in repeating step (3), (4), again the protection glutamine is carried out coupling, removes the Fmoc protection, and core removes by filter solvent, the three peptide prod NH that must link to each other with resin
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide;
(6) synthetic Fmoc-Glu (tbu)-COOSu
Fmoc-Glu (tbu)-COOH is placed reaction flask, add 10～15m L ethyl acetate by every gram Fmoc-Glu (tbu)-COOH, stirring at room, add nitrogen maloyl imines, the mol ratio of Fmoc-Glu (tbu)-COOH and nitrogen maloyl imines is 1:1～1.5, stirring and dissolving, add dicyclohexylcarbodiimide, the mol ratio of Fmoc-Glu (tbu)-COOH and dicyclohexylcarbodiimide is 1:1～2, room temperature reaction 20～24 hours, filtration under diminished pressure is removed solid insoluble, and filtrate decompression is concentrated into dried, gets Fmoc-Glu (tbu)-COOSu;
(7) synthetic Fmoc-Glu (tbu)-Glu (tbu)-OH
The product of step (6) gained is placed reaction flask, and by every gram Fmoc-Glu (tbu)-COOSu adding 10～15mL dioxane, stirring at room is dissolved fully to Fmoc-Glu (tbu)-COOSu;
γ-tert-butyl ester L-glutamic acid and sodium bicarbonate are dissolved in the distilled water, gained solution is added drop-wise in the dioxane solution of Fmoc-Glu (tbu)-COOSu, the mol ratio of γ-tert-butyl ester L-glutamic acid and Fmoc-Glu (tbu)-COOSu is 1.5:1, the mol ratio of sodium bicarbonate and Fmoc-Glu (tbu)-COOSu is 2:1, stirring at room reaction 18～20 hours, be that to transfer pH be 2～3 to 5% hydrochloric acid soln with massfraction, add ethyl acetate extraction, tell organic phase, organic phase is water successively, the saturated common salt washing, organic phase is through anhydrous sodium sulfate drying, filter, filtration under diminished pressure, filtrate decompression inspissation Fmoc-Glu (tbu)-Glu (the tbu)-COOH that contracts to get;
(8) synthetic Fmoc-Glu (tbu)-Glu (tbu)-COOSu
Adopt the described method of step (6) to react Fmoc-Glu (tbu)-Glu (tbu)-COOH, get Fmoc-Glu (tbu)-Glu (tbu)-
COOSu；
(9) synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH
Adopt the described method of step (7) to react step (8) Fmoc-Glu (tbu)-Glu (tbu)-COOSu, methionine(Met), get Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH;
(10) synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
The NH that step (5) is synthetic
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine, NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N', the mol ratio of N'-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine is 1:2～5:2～5:2～5:6～10, room temperature reaction 3～5 hours, filtration under diminished pressure, use N, dinethylformamide is washed resin 1～3 time, get Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-
Arg (pbf)-NH-resin resin peptide;
(11) synthetic H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
With the method for step (4), Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin is removed the Fmoc blocking group, gets H
2N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide;
(12) synthetic Ac-HN-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
At H
2Add methylene dichloride, diacetyl oxide, triethylamine in N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide, by every gram H
2Add 20mL methylene dichloride, H in N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide
2The mass ratio of N-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and diacetyl oxide, triethylamine is 1:0.5:1, room temperature reaction 3～5 hours, filter, use N, dinethylformamide is washed resin 3 times, gets Ac-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide;
(13) preparation Argireline
Preparation resin cutting liquid: phenol, 1，2-ethandithiol, thioanisole, trifluoroacetic acid, water are mixed for 5:2.5:5:4:83.5 joins in the beaker by volume;
Cutting resin: in step (12) resin peptide, add cutting liquid, by every gram Ac-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-
Arg (pbf)-NH-resin resin peptide adds 10～15mL cutting liquid, stirring at room reaction 3 hours, filter, filtrate is poured in the freezing anhydrous diethyl ether, stir, centrifugal, isolate supernatant liquor, room temperature decompression drying, the Argireline crude product, get purity greater than 95% Argireline through reverse-phase chromatography purifying, concentrated, freeze-drying.
2. according to the synthetic method of Argireline claimed in claim 1, it is characterized in that: synthetic Fmoc-Glu (tbu)-Glu (tbu)-
In Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step (10), the NH that step (5) is synthetic
2-Gln (Trt)-Arg (pbf)-
Arg (pbf)-NH-resin resin peptide is dissolved in the DMF, adds successively Fmoc-Glu (tbu)-Glu (tbu)-Met-
COOH, O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine, NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the mol ratio of diisopropylethylamine is 1:2～4:2～4:2～4:6～10, room temperature reaction 3～5 hours, filtration under diminished pressure, use N, dinethylformamide is washed resin 1～3 time, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
3. according to the synthetic method of Argireline claimed in claim 1, it is characterized in that: in synthetic Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide step (10), the NH that step (5) is synthetic
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide is dissolved in N, in the dinethylformamide, add successively Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, diisopropylethylamine, NH
2-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide and Fmoc-Glu (tbu)-Glu (tbu)-Met-COOH, O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid, benzotriazole, the mol ratio of diisopropylethylamine is 1:3:3:3:8, room temperature reaction 3～5 hours, filtration under diminished pressure, use N, dinethylformamide is washed resin 1～3 time, gets Fmoc-Glu (tbu)-Glu (tbu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH-resin resin peptide.
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| CN103694316B (en) * | 2013-12-31 | 2015-09-09 | 杭州华津药业股份有限公司 | A kind of preparation method of A Ji Rayleigh |
| CN106632608A (en) * | 2017-01-04 | 2017-05-10 | 陕西慧康生物科技有限责任公司 | Purifying method for arigireline |
| CN106632609B (en) * | 2017-01-05 | 2020-05-19 | 陕西慧康生物科技有限责任公司 | Preparation method of hexapeptide and product thereof |
| CN106749529A (en) * | 2017-01-05 | 2017-05-31 | 陕西慧康生物科技有限责任公司 | A kind of preparation method of Argireline and products thereof |
| CN110426476A (en) * | 2019-08-15 | 2019-11-08 | 珠海伊斯佳科技股份有限公司 | The measuring method of Argireline content in a kind of cosmetics |
| CN110684077B (en) * | 2019-10-18 | 2021-03-26 | 陕西慧康生物科技有限责任公司 | Large-scale synthesis method of achirelin |
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