CN102268065A - Amino-protecting serine oligopeptide as well as preparation method and application thereof - Google Patents
Amino-protecting serine oligopeptide as well as preparation method and application thereof Download PDFInfo
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- CN102268065A CN102268065A CN201110170807A CN201110170807A CN102268065A CN 102268065 A CN102268065 A CN 102268065A CN 201110170807 A CN201110170807 A CN 201110170807A CN 201110170807 A CN201110170807 A CN 201110170807A CN 102268065 A CN102268065 A CN 102268065A
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Abstract
The invention belongs to the technical field of amino acid modified compounds, in particular relates to amino-protecting serine oligopeptide as well as a preparation method and application thereof. The structure of amino-protecting serine oligopeptide is shown in a formula I, wherein X is tBu or Trt, and n is 1-4. According to the amino-protecting serine oligopeptide, a novel polypeptide segment is provided for synthesis of polypeptide medicaments, thus impurities are reduced when the polypeptide is synthesized, and the yield and purity are improved.
Description
Technical field
The invention belongs to amino acid modified compound technical, particularly amido protecting Serine oligopeptides and its production and use.
Technical background
Polypeptide drug is a biologically active substance, and they participate in the many important physical processes of human body mostly, such as hematopoiesis, inhibition activity of tumor cells, immunity etc.
The research and development of polypeptide drug is the hot fields of biological medicine industry always; in the preparation process of polypeptide; often run into the multiple aminoacid sequence; cause coupling incomplete; thereby reduced the preparation yield significantly; preparation cost is increased substantially simultaneously, and patent of the present invention overcomes the problems referred to above by the amino acid oligopeptides of preparation protection, has good application prospects.
Only there are Serine and protection Serine in the prior art, yet there are no the relevant report of amido protecting Serine oligopeptides.
Summary of the invention
First technical problem to be solved by this invention provides the new amido protecting Serine oligopeptides of a class, and structure is suc as formula shown in the I:
X is tBu or Trt, and n is 1～4.
Preferably, n is 1～3.
Fmoc is a 9-fluorenylmethyloxycarbonyl, and tBu is that the tertiary butyl, Trt are triphenyl methane.
Second technical problem to be solved by this invention provides the preparation method of the oligopeptides of amido protecting Serine shown in the formula I: with Fmoc-[Ser (X)]
y-2-Cl-Trt-resin is a raw material, obtains Fmoc-[Ser (X) by solid phase coupling synthesis method and the coupling of Fmoc-protection Serine fragment]
N+1-2-Cl-Trt-resin, after the cracking promptly; X is tBu or Trt, and n is 1～4, y=1～4.
Fmoc-protection Serine fragment is at least a among Fmoc-Ser (X)-OH, Fmoc-Ser (X)-Ser (X)-OH, Fmoc-Ser (X)-Ser (X)-Ser (X)-OH, Fmoc-Ser (X)-Ser (X)-Ser (X)-Ser (X)-OH, and X is tBu or Trt.
Fmoc-[Ser (X)]
y-2-Cl-Trt-resin substitution value is 0.2～1.2mmol/g resin, preferably 0.4～0.6mmol/g resin.
Fmoc-protection Serine fragment consumption is Fmoc-[Ser (X)]
x1.2～6 times of the amino total mole number of-2-Cl-Trt-resin, preferred 2.5～3.5 times.
Fmoc-[Ser (X)]
ySlough Fmoc protection during the coupling of-2-Cl-Trt-resin earlier, the reagent of sloughing the Fmoc protection is 10～30% (V/V) piperidines (PIP)/N, dinethylformamide (DMF) solution, preferably 20%.It is every gram resin 5～15ml that feeds intake that going of using protected reagent dosage, is preferably every gram resin 10ml that feeds intake.The protective reaction time is 10～60 minutes, is preferably 15～25 minutes.
Linked reaction need be added condensation reagent and activating reagent, condensation reagent is selected from N, N-DIC (DIC), N, N-dicyclohexylcarbodiimide (DCC), phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus (PyBOP), 2-(7-azepine-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-urea phosphofluoric acid ester (HATU), benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate (HBTU) or O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid ester (TBTU); N preferably, the N-DIC.
The mole dosage of condensation reagent is 1.2～6 times of amino total mole number in the aminoresin, is preferably 2.5～3.5 times.
Activating reagent is selected from I-hydroxybenzotriazole (HOBt), N-hydroxyl-7-azepine benzotriazole (HOAt), preferably I-hydroxybenzotriazole.
The activating reagent consumption is 1.2～6 times of amino total mole number in the aminoresin, preferably 2.5～3.5 times.
The linked reaction time is 60～300 minutes, preferably 100～140 minutes.
Add lytic reagent during cracking, lytic reagent is selected from hexafluoroisopropanol/DCM (methylene dichloride) solution or trifluoroethanol/acetic acid/DCM solution, preferred hexafluoroisopropanol/DCM (methylene dichloride) solution.The concentration of hexafluoroisopropanol is 20～60% (V/V) in hexafluoroisopropanol/DCM solution; The concentration of trifluoroethanol is 10～50% (V/V) in trifluoroethanol/acetic acid/DCM solution, and the concentration of acetic acid is 10～50% (V/V), and surplus is DCM.
The Serine of amido protecting shown in formula I oligopeptides can be used in the preparation polypeptide, and synthetic a kind of new polypeptide fragment, minimizing impurity generation when making polypeptide synthetic, the raising productive rate and the purity of providing of polypeptide drug is provided.
Embodiment
The present invention will be helped to understand by following embodiment, but content of the present invention can not be limited:
Embodiment 1Fmoc-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-OH's is synthetic
Get 3.0mol Ser (tBu) and 3.0mol HOBt, with an amount of DMF dissolving; Other gets 3.0mol DIC (N, N-DIC), stirs slowly to be added to down in the protection amino acid DMF solution, and stirring reaction is 30 minutes in room temperature environment, the protection amino acid solution after obtaining activating.
Get Fmoc-Ser (tBu)-2-Cl-Trt-resin 1Kg (substitution value is 1.0mmol/g); , adopt 6L PIP/DMF solution to go to protect 25 minutes, wherein the volumetric concentration of PIP is 20%.Filter the back resin respectively with MDF, DCM washing 3 times, add above-mentioned protection amino acid solution, stirring at room reaction 3 hours after reaction is finished, is filtered the back resin respectively with MDF, DCM washing 3 times, obtains Fmoc-Ser (tBu)-Ser (tBu)-OH.
Repeat above-mentioned reaction, insert other 3 Ser (tBu), make Fmoc-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-2-Cl-Trt-resin.
Get Fmoc-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-2-Cl-Trt-resin, add 20L hexafluoroisopropanol/DCM solution, the volumetric concentration of hexafluoroisopropanol is 30%.Stirring reaction 2 hours filters and collects filtrate, the evaporated under reduced pressure solvent, and drying under reduced pressure gets Fmoc-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-OH 742g, and yield is 91.3%, and purity is 95.7%, MS m/z:814 (M+1).
The application of embodiment 2Fmoc-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-OH in polypeptide is synthetic
Get Fmoc-Lys (Boc)-2-Cl-Trt-resin, after Fmoc goes protection,, make 5 peptide resins with Fmoc-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-OH coupling.
Coupling method is specially:
1, the activation of Fmoc-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-OH:
Get 0.3mol Fmoc-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-OH and 0.3mol HOBt, with an amount of DMF dissolving; Other gets 0.3mol DIC, stirs down slowly to be added in the aforementioned DMF solution, and stirring reaction is 30 minutes in room temperature environment, the protection amino acid solution after obtaining activating.
2, the Fmoc that takes off of Fmoc-Lys (Boc)-2-Cl-Trt-resin protects:
Get Fmoc-Lys (Boc)-2-Cl-Trt-resin (substitution value is 0.5mmol/g) 200g, adopt 1L 20% PIP/DMF solution to go to protect 25 minutes, filter the back and wash the resin that obtains Fmoc after 5 times with DMF, standby.
3, coupling:
Protection amino acid solution after the resin that removes Fmoc adds activation, linked reaction 60～300 minutes, the back resin washs 5 times with DMF after finishing.
Go to protect 25 minutes with 2L PIP (20%)/DMF solution; use DMF, DCM, methanol wash 3 times respectively after finishing reaction; drying under reduced pressure gets 5 peptide resins: H-Ser (tBu)-Ser (tBu)-Ser (tBu)-Ser (tBu)-Lys (Boc)-2-Cl-Trt-resin 233g, yield 94.0%.
4, acidolysis:
5 peptide resins are added to lytic reagent [TFA/ water/EDT=95: in 5: 5 (V/V) (10ml/ restrains resin), stir, stirring at room reaction 3 hours, reaction mixture uses sand core funnel to filter, and collects filtrate, and resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure behind the merging filtrate, add the anhydrous diethyl ether precipitation, wash precipitation 3 times with anhydrous diethyl ether again, drain to such an extent that white powder is 5 peptide crude product H-Ser-Ser-Ser-Ser-Lys-OH.
5, purifying:
5 peptide crude products are dissolved in the 30% acetonitrile water, with 0.45 μ m membrane filtration, adopt the liquid chromatography purifying, chromatogram is leant on and is anti-phase C18 post, purifying moving phase is 5% acetic acid/water-5% acetic acid mixed solvent, detect wavelength 230nm, collect and merging main peak solution, at concentrating under reduced pressure below 40 ℃, with the concentrated solution lyophilize, get H-Ser-Ser-Ser-Ser-His-OH pentapeptide product 30.9g, purity is 99.1%, and total recovery is 62.4%.MS?m/z：495(100％?M+1)。
Claims (10)
1. amido protecting Serine oligopeptides, structure is suc as formula shown in the I:
X is tBu or Trt, and n is 1～4.
2. amido protecting Serine oligopeptides according to claim 1 is characterized in that: n is 1～3.
3. the preparation method of claim 1 or 2 described amido protecting Serine oligopeptides: with Fmoc-[Ser (X)]
y-2-Cl-Trt-resin is a raw material, obtains Fmoc-[Ser (X) by solid phase coupling synthesis method and the coupling of Fmoc-protection Serine fragment]
N+1-2-Cl-Trt-resin, after the cracking promptly; X is tBu or Trt, and n is 1～4, y=1～4.
4. preparation method according to claim 3 is characterized in that: Fmoc-protection Serine fragment is at least a among Fmoc-Ser (X)-OH, Fmoc-Ser (X)-Ser (X)-OH, Fmoc-Ser (X)-Ser (X)-Ser (X)-OH, Fmoc-Ser (X)-Ser (X)-Ser (X)-Ser (X)-OH.
5. according to claim 3 or 4 described preparation methods, it is characterized in that: Fmoc-[Ser (X)]
y-2-Cl-Trt-resin substitution value is 0.2～1.2mmol/g resin.
6. preparation method according to claim 5 is characterized in that: Fmoc-[Ser (X)]
y-2-Cl-Trt-resin substitution value is 0.4～0.6mmol/g resin.
7. according to claim 3 or 4 described preparation methods, it is characterized in that: Fmoc-protection Serine fragment consumption is Fmoc-[Ser (X)]
x1.2～6 times of-2-Cl-Trt-resin total mole number.
8. according to claim 3 or 4 described preparation methods, it is characterized in that: Fmoc-[Ser (X)]
ySlough Fmoc protection during the coupling of-2-Cl-Trt-resin earlier, the reagent of sloughing the Fmoc protection is piperidines/N, and dinethylformamide solution, consumption are every gram resin 5～15ml that feeds intake.
9. according to claim 3 or 4 described preparation methods, it is characterized in that: linked reaction need be added condensation reagent and activating reagent, condensation reagent is selected from N, N-DIC, N, the N-dicyclohexylcarbodiimide, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus, 2-(7-azepine-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-urea phosphofluoric acid ester, benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate or O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid ester;
Activating reagent is selected from I-hydroxybenzotriazole or N-hydroxyl-7-azepine benzotriazole.
10. claim 1 or 2 described amido protecting Serine oligopeptides are in the purposes of preparation in the polypeptide.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1450906A (en) * | 1999-07-07 | 2003-10-22 | 特莱默里斯公司 | Methods and compositions for peptide synthesis |
| CN101974073A (en) * | 2010-11-05 | 2011-02-16 | 中国人民解放军第三军医大学第三附属医院 | Anti-inflammatory hexapeptide |
-
2011
- 2011-06-23 CN CN201110170807A patent/CN102268065A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1450906A (en) * | 1999-07-07 | 2003-10-22 | 特莱默里斯公司 | Methods and compositions for peptide synthesis |
| CN101974073A (en) * | 2010-11-05 | 2011-02-16 | 中国人民解放军第三军医大学第三附属医院 | Anti-inflammatory hexapeptide |
Non-Patent Citations (6)
| Title |
|---|
| 《Tetrahedron Letters》 19970811 Carlo Unverzagt Building blocks for glycoproteins: Synthesis of the ribonuclease B fragment 21-25 containing an undecasaccharide N-glycan 5627-5630 1-10 第38卷, 第32期 * |
| CARLO UNVERZAGT: "Building blocks for glycoproteins: Synthesis of the ribonuclease B fragment 21–25 containing an undecasaccharide N-glycan", 《TETRAHEDRON LETTERS》 * |
| JOHN H JONES ET AL.: "Abbreviations and symbols in peptide science: a revised guide and commentary", 《JOURNAL OF PEPTIDE SCIENCE》 * |
| KLEOMENIS BARLOS ET AL.: "Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide", 《INT.J.PEPTIDE PROTEIN RES.》 * |
| KLEOMENIS BARLOS ET AL.: "Fmoc/Trt-amino acids: comparison to Fmoc/tBu-amino acids in peptide synthesis", 《J.PEPTIDE RES.》 * |
| 孔凡琳: "多肽固相合成氨基酸树脂替代度的研究", 《硅谷》 * |
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Application publication date: 20111207 |