CN104788546A - Preparation method of linear peptides containing 24 amino acid residues - Google Patents
Preparation method of linear peptides containing 24 amino acid residues Download PDFInfo
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Abstract
The invention relates to a preparation method of linear peptides containing 24 amino acid residues to mainly solve a problem of no existence of industrial methods for preparing the above compound at present. The method adopts a solid phase synthesis technology and a high performance liquid chromatography purifying technology, and mainly comprises the following steps: 1, coupling all protection amino acids one by one by adopting the Fmoc solid phase synthesis technology with Fmoc-Tyr(tbu)-Wang resin as initial resin to synthesize peptide chain resin with an all protected side chain; 2, cutting the peptide chain resin with an all protected side chain, cracking 24 peptides from the peptide chain resin, and removing side chain protection groups to obtain a crude antiviral polypeptide PY-24 and a crude antiviral polypeptide FY-24; and 3, purifying the above obtained crude products by adopting high performance liquid chromatography to prepare purified polypeptides with high purity of 98% or more. The method is used to low cost and large scale production of anti-HIV combination polypeptides PY-24 and FY-24.
Description
Technical field
The present invention relates to the preparation method of linear straight chain peptide, particularly a kind of preparation method containing the linear straight chain peptide of 24 amino-acid residues.
Background technology
The communicable disease that acquired immune deficiency syndrome (AIDS) is is feature with systemic immune system grievous injury, since it is found, the existence of the serious threat mankind, the healthy and socioeconomic development generation tremendous influence to population in the world.Along with research for many years, acquired immune deficiency syndrome (AIDS) makes great progress in treatment, such as the application of HAART (Highly active antiretroviral therapy, HAART, is also called therapeuticcocktail of anti-retrovirals).Drug cocktail therapy (treatment) treats acquired immune deficiency syndrome (AIDS) by multiple antiviral conbined usage, according to the ultimate principle of this method, membrane fusion inhibitor for HIV 1 adopts molecular biology method restructuring and expresses and design combination polypeptide and PY-24 and FY-24, these two polypeptide are for the gp41 of HIV, with the target spot of gp120, in the experiment in early stage, demonstrate them can have extraordinary inhibit activities to HIV 1.
PY-24 is a kind of linear straight chain peptide containing 24 amino-acid residues, and its molecular formula is C
110h
170n
36o
28, molecular weight: 2444.81, peptide sequence is H-Pro-His-Ser-Gly-Ala-His-Gly-Ile-Pro-Lys-Ile-Phe-Gly-Al a-Arg-Ala-Gln-Pro-Ala-Ala-Ala-Arg-Ala-Tyr-OH, structural formula following (cutting two sections):
FY-24 is a kind of linear straight chain peptide containing 24 amino-acid residues, and its molecular formula is C
115h
176n
42o
29, molecular weight: 2610.95, peptide sequence is H-Phe-Thr-Ala-His-Leu-His-Thr-His-His-Arg-Ile-Gly-Gly-Al a-Arg-Ala-Gln-Pro-Ala-Ala-Ala-Arg-Ala-Tyr-OH, structural formula following (cutting two sections):
。
Summary of the invention
The object of this invention is to provide a kind of high yield, low cost, reaction conditions is gentle, environmental pollution is little, be conducive to realizing the preparation method of the linear straight chain peptide containing 24 amino-acid residues of industrialization.Mainly solve the method also not having this compound of preparation of industrialization at present.
For achieving the above object, the present invention takes following technical scheme:
Contain a preparation method for the linear straight chain peptide of 24 amino-acid residues, comprise the steps:
1) with Fmoc-Tyr (tbu)-Wang Resin for initial resin, adopt Fmoc solid-phase synthesis each protected amino acid of coupling one by one, synthesis obtains the peptide chain resin of side chain full guard;
2) cut with the peptide chain resin of cutting reagent to side chain full guard, 24 peptides got off from cracking resin and removes side chain protected group, obtaining antiviral polypeptide PY-24 and FY-24 crude product;
3) the antiviral polypeptide PY-24 that obtains and FY-24 crude product are adopted high performance liquid chromatography, and choose suitable chromatographic column and moving phase separation and purification, thus prepare PY-24 and the FY-24 fine work that purity is greater than 98%.
Concrete steps are as follows:
1. get Fmoc-Tyr (tbu)-Wang Resin DMF to soak, make resin fully swelling, drain, add to take off and emit reagent, reaction 10-30 minute, drain, with DMF washing 5-10 time, drain, take DMF as solvent, add Fmoc-Ala-OH, with HBTU/HOBT, TBTU/HOBT, a kind of mixture in DIC/HOBT is as condensing agent, the one added again in NMM or DIEA regulates reaction soln PH environment as organic bases, reaction 0.5-2 hour, ninhydrin method detection reaction terminal, drain, with DMF washing 3-5 time, drain, obtain Fmoc-Ala-Tyr (tbu)-Wang Resin, ,
Fmoc-Tyr (tbu)-Wang Resin substitution value is: 0.2mmol/g ~ 0.8mmol/g, and the scheme more optimized is 0.35mmol/g; Fmoc-Ala-OH amount is 1-5 times of Fmoc-Tyr (tbu)-Wang Resin molar weight, and the scheme more optimized is 2 times of Fmoc-Tyr (tbu)-Wang Resin molar weight; The amount of condensation reagent (a kind of mixture in HBTU/HOBT, TBTU/HOBT, DIC/HOBT) be the 1-5 of resin molar weight doubly, 2 times of the scheme more optimized to be the amount of HBTU/HOBT be resin molar weight; The amount of organic bases (NMM or DIEA) be the 2-10 of Fmoc-Tyr (tbu)-Wang Resin molar weight doubly, 4 times of the scheme more optimized to be NMM be resin molar weight;
2. in the resin obtained in step (1), add reagent of raising one's hat, reaction 10-30 minute, drain, with DMF washing 5-10 time, drain, take DMF as solvent, add the amino acid with fluorenylmethyloxycarbonyl protection, with HBTU/HOBT, TBTU/HOBT, a kind of mixture in DIC/HOBT is as condensing agent, the one added again in NMM or DIEA regulates reaction soln PH environment as organic bases, reaction 0.5-2 hour, ninhydrin method detection reaction terminal, drain, with DMF washing 3-5 time, drain, and then add deprotecting regent, deprotection reaction, add the amino acid with fluorenylmethyloxycarbonyl protection again, so repeatedly, PY-24 is until connected last proline(Pro), FY-24 is until connected last phenylalanine, with DMF washing 3-5 time, deprotection reaction, DMF washing 5-10 time, washing is replaced 3 times again with methyl alcohol and methylene dichloride, finally add methyl alcohol shrinkage resin, drain, vacuum-drying, obtain peptide chain resin PY-24 and FY-24 of side chain full guard, PY-24 and Pro-His (trt)-Ser (tbu)-Gly-Ala-His (trt)-Gly-Ile-Pro-Lys (boc)-Ile-Phe-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-Wang Resin, FY-24 and Phe-Thr (tbu)-Ala-His (trt)-Leu-His (trt)-Thr (tbu)-His (trt)-His (trt)-Arg (pbf)-Ile-Gly-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-Wang Resin,
3. the proportioning of reagent of raising one's hat is PIP/DMF (v/v) solution of 10%-30%, and the scheme more optimized is PIP/DMF (v/v) solution of 20%;
4. the amino acid whose amount of Fmoc protection is 1-5 times of resin molar weight, and the scheme more optimized is 2 times of resin molar weight; The amount of condensation reagent (a kind of mixture in HBTU/HOBT, TBTU/HOBT, DIC/HOBT) be the 1-5 of resin molar weight doubly, 2 times of the scheme more optimized to be the amount of HBTU/HOBT be resin molar weight; The amount of organic bases (NMM or DIEA) be the 2-10 of resin molar weight doubly, 4 times of the scheme more optimized to be NMM be resin molar weight;
5. the said amino acid with fluorenylmethyloxycarbonyl protection of PY-24, comprise Fmoc-Arg (pbf)-OH successively, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gln (Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg (pbf)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Lys (boc)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Gly-OH, Fmoc-His (trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tbu)-OH, Fmoc-His (trt)-OH, Fmoc-Pro-OH,
6. the said amino acid with fluorenylmethyloxycarbonyl protection of FY-24, comprise Fmoc-Arg (pbf)-OH successively, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gln (Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg (pbf)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg (pbf)-OH, Fmoc-His (trt)-OH, Fmoc-His (trt)-OH, Fmoc-Thr (tbu)-OH, Fmoc-His (trt)-OH, Fmoc-Leu-OH, Fmoc-His (trt)-OH, Fmoc-Ala-OH, Fmoc-Thr (tbu)-OH, Fmoc-Phe-OH.
7. according to the present invention; described cutting in the peptide chain resin of the side chain full guard that peptide comprises the steps: above-mentioned steps 2 to obtain adds cutting reagent; the volume proportion of cutting reagent is trifluoroacetic acid: tri isopropyl silane: water=95%:3%:2%, and react 2.5 hours, suction filtration filters resin particle; and collect filtrate; then add crystallizing from ether precipitation, centrifugal collecting precipitation, then use washed with diethylether 3-6 time; vacuum-drying, obtains PY-24 and FY-24 crude product.
8. according to the present invention, described peptide purification technique comprises the steps: soluble in water for PY-24 and FY-24 polypeptide crude product, make the aqueous solution of 200mg/ml, with 0.45 μm of membrane filtration, required component is collected with reverse phase preparative HPLC, use the component collected by analysis mode HPLC analyzing and testing again, merge the component of purity more than 98%, amalgamation liquid is concentrating under reduced pressure under 35 ~ 40 DEG C of conditions, concentrated solution is put into low-temperature freeze-drying machine and carry out lyophilize, thus obtain PY-24 and FY-24 fine work.The preparative column that reverse preparation HPLC adopts is C18 type, and moving phase is one or more in water, ammonium acetate aqueous solution, acetonitrile, methyl alcohol.
in the present invention, some conventional abbreviations have following implication
Wang Resin: king's resin
HBTU:O-benzotriazole-N, N, N, N-tetramethyl-urea hexafluorophosphate
HATU:O-(7-azo benzotriazole-1-oxygen)-N, N, N, N-tetramethyl-urea hexafluorophosphate
TBTU:O-(benzotriazole-1-oxygen)-N, N, N, N-tetramethyl-urea hexafluoro borate
DIC: DIC
HOBT:1-hydroxybenzotriazole
HOAT:1-hydroxyl-7-azo benzotriazole
DIEA: diisopropylethylamine
NMM:N-methylmorpholine
Fmoc: fluorenylmethyloxycarbonyl
Pbf:2,2,4,6,7-pentamethyl--2H-cumarone-5 alkylsulfonyl
Trt: trityl
Tbu: the tertiary butyl
Boc: tertbutyloxycarbonyl
Pro: proline(Pro)
Thr: Threonine
Gly: glycine
Ala: L-Ala
Ser: Serine
Arg: arginine
Tyr: tyrosine
Gln: glutamine
Leu: leucine
Ile: Isoleucine
His: Histidine
Phe: phenylalanine
Lys: Methionin
PIP: piperidines
DMF:N, dinethylformamide
DCM: methylene dichloride.
Beneficial effect of the present invention: the industrialization that can realize these two antiviral compound polypeptide, provide a kind of high yield, low cost simultaneously, reaction conditions is gentle, environmental pollution is little, be conducive to realizing the preparation method of the linear straight chain peptide containing 24 amino-acid residues of industrialization, bring considerable economic benefit.
Embodiment
Hereinafter with reference to example, the present invention is described in further detail, but the invention is not restricted to this specific examples.
embodiment 1
1) Fmoc-Ala-Tyr (tbu)-Wang Resin is prepared
Take Fmoc-Tyr (tbu)-Wang Resin 23.1 grams of (0. 65mmol/g, 15mmol), 30 minutes are soaked with 800ml DMF, make resin fully swelling, drain, add Fmoc-Ala-OH (MW:311.3, 30mmol) 9.4g, TBTU (MW:321.1, 30mmol) 9.7g, HOBT (MW:135.1, 30mmol) 4.1g, DIEA (MW:129.24, 60mmol) 10ml, DMF500ml, react 1 hour, triketohydrindene hydrate detects resin water white transparency, drain, 3 times are washed with DMF, drain, obtain Fmoc-Ala-Tyr (tbu)-Wang Resin.
2) preparation Pro - His (TRT) - Ser (tbu) - Gly - Ala - His (TRT) - Gly - Ile - Pro - Lys (boc) - Ile - Phe - Gly - Ala - Arg (PBF) - Ala - Gln (TRT) - Pro - Ala - Ala - Ala - Arg (PBF) - Ala - Tyr (tbu) - Wang Resin (PY - 24) and Phe - Thr (tbu) - Ala - His (TRT) - Leu - His (TRT) - Thr (tbu) - ihs (TRT) - His (TRT) - Arg (PBF) - Ile - Gly Gly - Ala - Arg (PBF) - Ala - Gln (TRT) - Pro - Ala - Ala - Ala - Arg (PBF) - Ala - Tyr (tbu) - Wang Resin (FY - 24).
In Fmoc-Ala-Tyr (the tbu)-Wang Resin that step (1) obtains, add 800ml to raise one's hat reagent (PIP/DMF (v/v) solution of 20%), react 30 minutes, drain, 6 times are washed with DMF, drain, solvent is made with DMF, add the amino acid with Fmoc protection, TBTU/HOBT, DIEA, react 1 hour, detection reaction terminal, drain, with DMF washing 3-5 time, drain, and then add deprotecting regent, deprotection reaction, add the amino acid with Fmoc blocking group again, so repeatedly, PY-24 is until connected last proline(Pro), FY-24 is until connected last phenylalanine, with DMF washing 3-5 time, deprotection reaction, DMF washing 5-10 time, washing is replaced 3 times again with methyl alcohol and methylene dichloride, finally add methyl alcohol shrinkage resin, drain, vacuum-drying, obtain the peptide chain resin of side chain full guard, PY-24 and Pro-His (trt)-Ser (tbu)-Gly-Ala-His (trt)-Gly-Ile-Pro-Lys (boc)-Ile-Phe-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-Wang Resin, amount to 70g, FY-24 and Phe-Thr (tbu)-Ala-His (trt)-Leu-His (trt)-Thr (tbu)-His (trt)-His (trt)-Arg (pbf)-Ile-Gly-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-Wang Resin, amounts to 60g.
The amino acid whose amount that each step condensation reaction of PY-24 adds is respectively:
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Pro-OH(MW:337.4，30mmol)10.2g，
Fmoc-Gln(Trt)-OH(MW:610.7，30mmol)18.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-Phe-OH(MW:387.4，30mmol)11.7g，
Fmoc-Ile-OH(MW:353.4，30mmol)10.7g，
Fmoc-Lys(Boc)-OH(MW:468.55:，30mmol)14.1g，
Fmoc-Pro-OH(MW:337.4，30mmol)10.2g，
Fmoc-Ile-OH(MW:353.4，30mmol)10.7g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-Ser(tbu)-OH(MW:383.4，30mmol)11.6g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Pro-OH(MW:337.4，30mmol)10.2g；
The amino acid whose amount that each step condensation reaction of FY-24 adds is respectively:
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Pro-OH(MW:337.4，30mmol)10.2g，
Fmoc-Gln(Trt)-OH(MW:610.7，30mmol)18.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-Ile-OH(MW:353.4，30mmol)10.7g，
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Thr(tbu)-OH(MW:397.5，30mmol)12g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Leu-OH(MW:353.4，30mmol)10.7g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Thr(tbu)-OH(MW:397.5，30mmol)12g，
Fmoc-Phe-OH(MW:387.4，30mmol)11.7g。
The amount of the condensing agent that each step condensation reaction adopts is: TBTU (MW:321.1,30mmol) 9.7g, HOBT (MW:135.1,30mmol) 4.1g,
The amount of the organic bases that each step condensation reaction is added is: DIEA (MW:129.24,60mmol) 10ml.
3) PY-24 and FY-24 crude product is prepared
PY-24 and Pro-His (trt)-Ser (tbu)-Gly-Ala-His (trt)-Gly-Ile-Pro-Lys (boc)-Ile-Phe-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (the tbu)-Wang Resin got in step (2) amounts to 70g, be placed in the round-bottomed flask of 2L, add 700ml cutting reagent, proportioning is trifluoroacetic acid: tri isopropyl silane: water=95%:3%:2% (v/v), be placed in constant-temperature table 25 DEG C of oscillatory reactions 2.5 hours, suction filtration filters resin particle, collect filtrate, then 3500ml crystallizing from ether precipitation is added, centrifugal collecting precipitation, use washed with diethylether 3-6 time again, vacuum-drying, obtain PY-24 crude product 30g.
FY-24 and Phe-Thr (tbu)-Ala-His (trt)-Leu-His (trt)-Thr (tbu)-His (trt)-His (trt)-Arg (pbf)-Ile-Gly-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (the tbu)-Wang Resin got in step (2) amounts to the round-bottomed flask that 60g is placed in 2L, add 600ml cutting reagent, proportioning is trifluoroacetic acid: tri isopropyl silane: water=95%:3%:2% (v/v), be placed in constant-temperature table 25 DEG C of oscillatory reactions 2.5 hours, suction filtration filters resin particle, collect filtrate, then 3000ml crystallizing from ether precipitation is added, centrifugal collecting precipitation, use washed with diethylether 3-6 time again, vacuum-drying, obtain FY-24 crude product 29g.
4) high performance liquid chromatography separation and purification is passed through to PY-24 and FY-24 crude product, obtain the fine work that purity is greater than 98%.
The PY-24 crude product obtained in step (3) is total to 30g to be dissolved in 150ml water, with 0.45 μm of membrane filtration, required component is collected with reverse phase preparative HPLC, use the component collected by analysis mode HPLC analyzing and testing again, merge the component of purity more than 98%, amalgamation liquid is concentrating under reduced pressure under 35 ~ 40 DEG C of conditions, concentrated solution is put into low-temperature freeze-drying machine and carries out lyophilize 48 hours, obtain PY-24 fine work 7.5 grams, total recovery 20.5%
The FY-24 crude product obtained in step (3) is total to 29g to be dissolved in 145ml water, with 0.45 μm of membrane filtration, required component is collected with reverse phase preparative HPLC, use the component collected by analysis mode HPLC analyzing and testing again, merge the component of purity more than 98%, amalgamation liquid is concentrating under reduced pressure under 35 ~ 40 DEG C of conditions, concentrated solution is put into low-temperature freeze-drying machine and carries out lyophilize 48 hours, obtain FY-24 fine work 7.9 grams, total recovery 20.2%.
embodiment 2
1) Fmoc-Ala-Tyr (tbu)-Wang Resin is prepared
Take Fmoc-Tyr (tbu)-Wang Resin 42.9 grams of (0.35mmol/g, 15mmol), 30 minutes are soaked with 800ml DMF, make resin fully swelling, drain, add Fmoc-Ala-OH (MW:311.3, 30mmol) 9.4g, HBTU (MW:379.2, 30mmol) 11.4g, HOBT (MW:135.1, 30mmol) 4.1g, NMM (MW:102.1, 60mmol) 6.8ml, DMF500ml, react 0.5 hour, triketohydrindene hydrate detects resin water white transparency, drain, 3 times are washed with DMF, drain, obtain Fmoc-Ala-Tyr (tbu)-Wang Resin.
2) preparation Pro - His (TRT) - Ser (tbu) - Gly - Ala - His (TRT) - Gly - Ile - Pro - Lys (boc) - Ile - Phe - Gly - Ala - Arg (PBF) - Ala - Gln (TRT) - Pro - Ala - Ala - Ala - Arg (PBF) - Ala - Tyr (tbu) - Wang Resin (PY - 24) and Phe - Thr (tbu) - Ala - His (TRT) - Leu - His (TRT) - Thr (tbu) - ihs (TRT) - His (TRT) - Arg (PBF) - Ile - Gly Gly - Ala - Arg (PBF) - Ala - Gln (TRT) - Pro - Ala - Ala - Ala - Arg (PBF) - Ala - Tyr (tbu) - Wang Resin (FY - 24).
In Fmoc-Ala-Tyr (the tbu)-Wang Resin that step (1) obtains, add 800ml to raise one's hat reagent (PIP/DMF (v/v) solution of 20%), react 30 minutes, drain, 6 times are washed with DMF, drain, solvent is made with DMF, add the amino acid with Fmoc protection, HBTU/HOBT, NMM, react 0.5 hour, detection reaction terminal, drain, with DMF washing 3-5 time, drain, and then add deprotecting regent, deprotection reaction, add the amino acid with Fmoc blocking group again, so repeatedly, PY-24 is until connected last proline(Pro), FY-24 is until connected last phenylalanine, with DMF washing 3-5 time, deprotection reaction, DMF washing 5-10 time, washing is replaced 3 times again with methyl alcohol and methylene dichloride, finally add methyl alcohol shrinkage resin, drain, vacuum-drying, obtain the peptide chain resin of side chain full guard, PY-24 and Pro-His (trt)-Ser (tbu)-Gly-Ala-His (trt)-Gly-Ile-Pro-Lys (boc)-Ile-Phe-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-Wang Resin, amount to 96g, FY-24 and Phe-Thr (tbu)-Ala-His (trt)-Leu-His (trt)-Thr (tbu)-His (trt)-His (trt)-Arg (pbf)-Ile-Gly-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-Wang Resin, amounts to 92g.
The amino acid whose amount that each step condensation reaction of PY-24 adds is respectively:
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Pro-OH(MW:337.4，30mmol)10.2g，
Fmoc-Gln(Trt)-OH(MW:610.7，30mmol)18.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-Phe-OH(MW:387.4，30mmol)11.7g，
Fmoc-Ile-OH(MW:353.4，30mmol)10.7g，
Fmoc-Lys(Boc)-OH(MW:468.55:，30mmol)14.1g，
Fmoc-Pro-OH(MW:337.4，30mmol)10.2g，
Fmoc-Ile-OH(MW:353.4，30mmol)10.7g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-Ser(tbu)-OH(MW:383.4，30mmol)11.6g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Pro-OH(MW:337.4，30mmol)10.2g；
The amino acid whose amount that each step condensation reaction of FY-24 adds is respectively:
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Pro-OH(MW:337.4，30mmol)10.2g，
Fmoc-Gln(Trt)-OH(MW:610.7，30mmol)18.4g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-Gly-OH(MW:297.3，30mmol)9g，
Fmoc-Ile-OH(MW:353.4，30mmol)10.7g，
Fmoc-Arg(pbf)-OH(MW:648.8，30mmol)19.5g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Thr(tbu)-OH(MW:397.5，30mmol)12g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Leu-OH(MW:353.4，30mmol)10.7g，
Fmoc-His(Trt)-OH(MW:619.7，30mmol)18.6g，
Fmoc-Ala-OH(MW:311.3，30mmol)9.4g，
Fmoc-Thr(tbu)-OH(MW:397.5，30mmol)12g，
Fmoc-Phe-OH(MW:387.4，30mmol)11.7g。
The amount of the condensing agent that each step condensation reaction adopts is: HBTU (MW:379.2,30mmol) 11.4g, HOBT (MW:135.1,30mmol) 4.1g,
The amount of the organic bases that each step condensation reaction is added is: NMM (MW:102.1,60mmol) 6.8ml.
3) PY-24 and FY-24 crude product is prepared
PY-24 and Pro-His (trt)-Ser (tbu)-Gly-Ala-His (trt)-Gly-Ile-Pro-Lys (boc)-Ile-Phe-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (the tbu)-Wang Resin got in step (2) amounts to 96g, be placed in the round-bottomed flask of 2L, add 960ml cutting reagent, proportioning is trifluoroacetic acid: tri isopropyl silane: water=95%:3%:2% (v/v), be placed in constant-temperature table 25 DEG C of oscillatory reactions 2.5 hours, suction filtration filters resin particle, collect filtrate, then 4800ml crystallizing from ether precipitation is added, centrifugal collecting precipitation, use washed with diethylether 3-6 time again, vacuum-drying, obtain PY-24 crude product 35g.
FY-24 and Phe-Thr (tbu)-Ala-His (trt)-Leu-His (trt)-Thr (tbu)-His (trt)-His (trt)-Arg (pbf)-Ile-Gly-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (the tbu)-Wang Resin got in step (2) amounts to the round-bottomed flask that 92g is placed in 2L, add 920ml cutting reagent, proportioning is trifluoroacetic acid: tri isopropyl silane: water=95%:3%:2% (v/v), be placed in constant-temperature table 25 DEG C of oscillatory reactions 2.5 hours, suction filtration filters resin particle, collect filtrate, then 4600ml crystallizing from ether precipitation is added, centrifugal collecting precipitation, use washed with diethylether 3-6 time again, vacuum-drying, obtain FY-24 crude product 36g.
4) high performance liquid chromatography separation and purification is passed through to PY-24 and FY-24 crude product, obtain the fine work that purity is greater than 98%.
The PY-24 crude product obtained in step (3) is total to 35g to be dissolved in 175ml water, with 0.45 μm of membrane filtration, required component is collected with reverse phase preparative HPLC, use the component collected by analysis mode HPLC analyzing and testing again, merge the component of purity more than 98%, amalgamation liquid is concentrating under reduced pressure under 35 ~ 40 DEG C of conditions, concentrated solution is put into low-temperature freeze-drying machine and carries out lyophilize 48 hours, obtain PY-24 fine work 9 grams, total recovery 24.5%
The FY-24 crude product obtained in step (3) is total to 36g to be dissolved in 180ml water, with 0.45 μm of membrane filtration, required component is collected with reverse phase preparative HPLC, use the component collected by analysis mode HPLC analyzing and testing again, merge the component of purity more than 98%, amalgamation liquid is concentrating under reduced pressure under 35 ~ 40 DEG C of conditions, concentrated solution is put into low-temperature freeze-drying machine and carries out lyophilize 48 hours, obtain FY-24 fine work 10 grams, total recovery 25.5%.
embodiment 3
1) Fmoc-Ala-Tyr (tbu)-Wang Resin is prepared
Take Fmoc-Tyr (tbu)-Wang Resin 60 grams of (0.25mmol/g, 15mmol), 30 minutes are soaked with 800ml DMF, make resin fully swelling, drain, add Fmoc-Ala-OH (MW:311.3, 45mmol) 14.1g, DIC (MW:126, 45mmol) 7ml, HOBT (MW:135.1, 45mmol) 6.1g, DIEA (MW:129.24, 90mmol) 15ml, DMF500ml, react 2 hours, triketohydrindene hydrate detects resin water white transparency, drain, 3 times are washed with DMF, drain, obtain Fmoc-Ala-Tyr (tbu)-Wang Resin.
2) preparation Pro - His (TRT) - Ser (tbu) - Gly - Ala - His (TRT) - Gly - Ile - Pro - Lys (boc) - Ile - Phe - Gly - Ala - Arg (PBF) - Ala - Gln (TRT) - Pro - Ala - Ala - Ala - Arg (PBF) - Ala - Tyr (tbu) - Wang Resin (PY - 24) and Phe - Thr (tbu) - Ala - His (TRT) - Leu - His (TRT) - Thr (tbu) - ihs (TRT) - His (TRT) - Arg (PBF) - Ile - Gly Gly - Ala - Arg (PBF) - Ala - Gln (TRT) - Pro - Ala - Ala - Ala - Arg (PBF) - Ala - Tyr (tbu) - Wang Resin (FY - 24).
In Fmoc-Ala-Tyr (the tbu)-Wang Resin that step (1) obtains, add 800ml to raise one's hat reagent (PIP/DMF (v/v) solution of 20%), react 30 minutes, drain, 6 times are washed with DMF, drain, solvent is made with DMF, add the amino acid with Fmoc protection, DIC/HOBT, DIEA, react 2 hours, detection reaction terminal, drain, with DMF washing 3-5 time, drain, and then add deprotecting regent, deprotection reaction, add the amino acid with Fmoc blocking group again, so repeatedly, PY-24 is until connected last proline(Pro), FY-24 is until connected last phenylalanine, with DMF washing 3-5 time, deprotection reaction, DMF washing 5-10 time, washing is replaced 3 times again with methyl alcohol and methylene dichloride, finally add methyl alcohol shrinkage resin, drain, vacuum-drying, obtain the peptide chain resin of side chain full guard, PY-24 and Pro-His (trt)-Ser (tbu)-Gly-Ala-His (trt)-Gly-Ile-Pro-Lys (boc)-Ile-Phe-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-Wang Resin, amount to 105g, FY-24 and Phe-Thr (tbu)-Ala-His (trt)-Leu-His (trt)-Thr (tbu)-His (trt)-His (trt)-Arg (pbf)-Ile-Gly-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-Wang Resin, amounts to 100g.
The amino acid whose amount that each step condensation reaction of PY-24 adds is respectively:
Fmoc-Arg(pbf)-OH(MW:648.8，45mmol)29.2g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Pro-OH(MW:337.4，45mmol)15.2g，
Fmoc-Gln(Trt)-OH(MW:610.7，45mmol)27.5g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Arg(pbf)-OH(MW:648.8，45mmol)29.2g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Gly-OH(MW:297.3，45mmol)13.4g，
Fmoc-Phe-OH(MW:387.4，45mmol)17.5g，
Fmoc-Ile-OH(MW:353.4，45mmol)15.9g，
Fmoc-Lys(Boc)-OH(MW:468.55:，45mmol)21.1g，
Fmoc-Pro-OH(MW:337.4，45mmol)15.2g，
Fmoc-Ile-OH(MW:353.4，45mmol)15.9g，
Fmoc-Gly-OH(MW:297.3，45mmol)13.4g，
Fmoc-His(Trt)-OH(MW:619.7，45mmol)27.9g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Gly-OH(MW:297.3，45mmol)13.4g，
Fmoc-Ser(tbu)-OH(MW:383.4，45mmol)17.3g，
Fmoc-His(Trt)-OH(MW:619.7，45mmol)27.9g，
Fmoc-Pro-OH(MW:337.4，45mmol)15.2g；
The amino acid whose amount that each step condensation reaction of FY-24 adds is respectively:
Fmoc-Arg(pbf)-OH(MW:648.8，45mmol)29.2g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Pro-OH(MW:337.4，45mmol)15.2g，
Fmoc-Gln(Trt)-OH(MW:610.7，45mmol)27.5g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Arg(pbf)-OH(MW:648.8，45mmol)29.2g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Gly-OH(MW:297.3，45mmol)13.4g，
Fmoc-Gly-OH(MW:297.3，45mmol)13.4g，
Fmoc-Ile-OH(MW:353.4，45mmol)15.9g，
Fmoc-Arg(pbf)-OH(MW:648.8，45mmol)29.2g，
Fmoc-His(Trt)-OH(MW:619.7，45mmol)27.9g，
Fmoc-His(Trt)-OH(MW:619.7，45mmol)27.9g，
Fmoc-Thr(tbu)-OH(MW:397.5，45mmol)17.9g，
Fmoc-His(Trt)-OH(MW:619.7，45mmol)27.9g，
Fmoc-Leu-OH(MW:353.4，45mmol)15.9g，
Fmoc-His(Trt)-OH(MW:619.7，45mmol)27.9g，
Fmoc-Ala-OH(MW:311.3，45mmol)14.1g，
Fmoc-Thr(tbu)-OH(MW:397.5，45mmol)17.9g，
Fmoc-Phe-OH(MW:387.4，45mmol)17.5g。
The amount of the condensing agent that each step condensation reaction adopts is: DIC (MW:126,45mmol) 7ml, HOBT (MW:135.1,45mmol) 6.1g,
The amount of the organic bases that each step condensation reaction is added is: DIEA (MW:129.24,90mmol) 15ml.
3) PY-24 and FY-24 crude product is prepared
PY-24 and Pro-His (trt)-Ser (tbu)-Gly-Ala-His (trt)-Gly-Ile-Pro-Lys (boc)-Ile-Phe-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (the tbu)-Wang Resin got in step (2) amounts to 105g, be placed in the round-bottomed flask of 2L, add 1050ml cutting reagent, proportioning is trifluoroacetic acid: tri isopropyl silane: water=95%:3%:2% (v/v), be placed in constant-temperature table 25 DEG C of oscillatory reactions 2.5 hours, suction filtration filters resin particle, collect filtrate, then 5250ml crystallizing from ether precipitation is added, centrifugal collecting precipitation, use washed with diethylether 3-6 time again, vacuum-drying, obtain PY-24 crude product 32g.
FY-24 and Phe-Thr (tbu)-Ala-His (trt)-Leu-His (trt)-Thr (tbu)-His (trt)-His (trt)-Arg (pbf)-Ile-Gly-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (the tbu)-Wang Resin got in step (2) amounts to the round-bottomed flask that 100g is placed in 2L, add 1000ml cutting reagent, proportioning is trifluoroacetic acid: tri isopropyl silane: water=95%:3%:2% (v/v), be placed in constant-temperature table 25 DEG C of oscillatory reactions 2.5 hours, suction filtration filters resin particle, collect filtrate, then 5000ml crystallizing from ether precipitation is added, centrifugal collecting precipitation, use washed with diethylether 3-6 time again, vacuum-drying, obtain FY-24 crude product 31g.
4) high performance liquid chromatography separation and purification is passed through to PY-24 and FY-24 crude product, obtain the fine work that purity is greater than 98%.
The PY-24 crude product obtained in step (3) is total to 32g to be dissolved in 160ml water, with 0.45 μm of membrane filtration, required component is collected with reverse phase preparative HPLC, use the component collected by analysis mode HPLC analyzing and testing again, merge the component of purity more than 98%, amalgamation liquid is concentrating under reduced pressure under 35 ~ 40 DEG C of conditions, concentrated solution is put into low-temperature freeze-drying machine and carries out lyophilize 48 hours, obtain PY-24 fine work 6.9 grams, total recovery 18.9%
The FY-24 crude product obtained in step (3) is total to 31g to be dissolved in 160ml water, with 0.45 μm of membrane filtration, required component is collected with reverse phase preparative HPLC, use the component collected by analysis mode HPLC analyzing and testing again, merge the component of purity more than 98%, amalgamation liquid is concentrating under reduced pressure under 35 ~ 40 DEG C of conditions, concentrated solution is put into low-temperature freeze-drying machine and carries out lyophilize 48 hours, obtain FY-24 fine work 7 grams, total recovery 17.8%.
Claims (8)
1. a preparation method for the linear straight chain peptide containing 24 amino-acid residues, is characterized in that comprising the following steps:
1) with Fmoc-Tyr (tbu)-king's resin for initial resin, adopt Fmoc solid-phase synthesis each protected amino acid of coupling one by one, synthesis obtains the peptide chain resin of side chain full guard;
2) cut with the peptide chain resin of cutting reagent to side chain full guard, 24 peptides got off from cracking resin and removes side chain protected group, obtaining antiviral polypeptide PY-24 and FY-24 crude product;
3) the antiviral polypeptide PY-24 that obtains and FY-24 crude product are adopted high performance liquid chromatography, and choose suitable chromatographic column and moving phase separation and purification, thus prepare PY-24 and the FY-24 fine work that purity is greater than 98%.
2. method according to claim 1, is characterized in that, step 1) particular content comprises the steps:
1) Fmoc-Tyr (tbu)-king's resin N is got, dinethylformamide soaks, make resin fully swelling, drain, add to take off and emit reagent, reaction 10-30 minute, drain, with N, dinethylformamide washing 5-10 time, drain, with N, dinethylformamide is solvent, add Fmoc-Ala-OH, with O-benzotriazole-N, N, N, N-tetramethyl-urea hexafluorophosphate/I-hydroxybenzotriazole, O-(benzotriazole-1-oxygen)-N, N, N, N-tetramethyl-urea hexafluoro borate/I-hydroxybenzotriazole, a kind of mixture in DIC/I-hydroxybenzotriazole is as condensing agent, the one added again in N-methylmorpholine or diisopropylethylamine regulates reaction soln PH environment as organic bases, reaction 0.5-2 hour, ninhydrin method detection reaction terminal, drain, with N, dinethylformamide washing 3-5 time, drain, obtain Fmoc-Ala-Tyr (tbu)-king's resin,
2) in Fmoc-Ala-Tyr (the tbu)-king's resin obtained in step (1), add reagent of raising one's hat, reaction 10-30 minute, drain, with N, dinethylformamide washing 5-10 time, drain, with N, dinethylformamide is solvent, add the amino acid with fluorenylmethyloxycarbonyl protection, with O-benzotriazole-N, N, N, N-tetramethyl-urea hexafluorophosphate/I-hydroxybenzotriazole, O-(benzotriazole-1-oxygen)-N, N, N, N-tetramethyl-urea hexafluoro borate/I-hydroxybenzotriazole, a kind of mixture in DIC/I-hydroxybenzotriazole is as condensing agent, the one added again in N-methylmorpholine or diisopropylethylamine regulates reaction soln PH environment as organic bases, reaction 0.5-2 hour, ninhydrin method detection reaction terminal, drain, with N, dinethylformamide washing 3-5 time, drain, and then add deprotecting regent, deprotection reaction, add the amino acid with fluorenylmethyloxycarbonyl protection again, so repeatedly, PY-24 is until connected last proline(Pro), FY-24 is until connected last phenylalanine, with N, dinethylformamide washing 3-5 time, deprotection reaction, N, dinethylformamide washing 5-10 time, washing is replaced 3 times again with methyl alcohol and methylene dichloride, finally add methyl alcohol shrinkage resin, drain, vacuum-drying, obtain peptide chain resin PY-24 and FY-24 of side chain full guard, PY-24 and Pro-His (trt)-Ser (tbu)-Gly-Ala-His (trt)-Gly-Ile-Pro-Lys (boc)-Ile-Phe-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-king's resin, FY-24 and Phe-Thr (tbu)-Ala-His (trt)-Leu-His (trt)-Thr (tbu)-His (trt)-His (trt)-Arg (pbf)-Ile-Gly-Gly-Ala-Arg (pbf)-Ala-Gln (Trt)-Pro-Ala-Ala-Ala-Arg (pbf)-Ala-Tyr (tbu)-king's resin,
There is involved by PY-24 the amino acid of fluorenylmethyloxycarbonyl protection, comprise Fmoc-Arg (pbf)-OH successively, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gln (Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg (pbf)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Lys (boc)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Gly-OH, Fmoc-His (trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tbu)-OH, Fmoc-His (trt)-OH, Fmoc-Pro-OH,
There is involved by FY-24 the amino acid of fluorenylmethyloxycarbonyl protection, comprise Fmoc-Arg (pbf)-OH successively, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gln (Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg (pbf)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg (pbf)-OH, Fmoc-His (trt)-OH, Fmoc-His (trt)-OH, Fmoc-Thr (tbu)-OH, Fmoc-His (trt)-OH, Fmoc-Leu-OH, Fmoc-His (trt)-OH, Fmoc-Ala-OH, Fmoc-Thr (tbu)-OH, Fmoc-Phe-OH.
3. method according to claim 2, is characterized in that, the substitution value of Fmoc-Tyr (tbu)-king's resin is between 0.2mmol/g-0.8mmol/g, and Fmoc-Ala-OH amount is 1-5 times of Fmoc-Tyr (tbu)-king's resin molar weight; Reagent of raising one's hat is the piperidines/N of volumn concentration 10-30%; dinethylformamide mixing solutions; the amount of each step protected amino acid be the 1-5 of Fmoc-Tyr (tbu)-king's resin molar weight doubly; the amount of condensation reagent is 1-5 times of Fmoc-Tyr (tbu)-king's resin molar weight; the amount of organic bases be the 2-10 of Fmoc-Tyr (tbu)-king's resin molar weight doubly, what the detection method of each step reaction end adopted is triketohydrindene hydrate detection method.
4. method according to claim 3, is characterized in that, the substitution value of Fmoc-Tyr (tbu)-king's resin is 2 times that 0.35mmol/g, Fmoc-Ala-OH measure as Fmoc-Tyr (tbu)-king's resin molar weight; Reagent of raising one's hat is the piperidines/N of volumn concentration 20%; dinethylformamide mixing solutions; the amount of each step protected amino acid be 2 times of Fmoc-Tyr (tbu)-king's resin molar weight; condensation reagent is O-(benzotriazole-1-oxygen)-N; N; N; N-tetramethyl-urea hexafluoro borate/I-hydroxybenzotriazole; its consumption is 2 times of Fmoc-Tyr (tbu)-king's resin molar weight; organic bases is N-methylmorpholine, and its consumption is 4 times of Fmoc-Tyr (tbu)-king's resin molar weight.
5. method according to claim 1; it is characterized in that; described step 2) comprise the steps: to add cutting reagent in the peptide chain resin of side chain full guard, reaction 1-3 hour, suction filtration filters resin particle; and collect filtrate; then add crystallizing from ether precipitation, centrifugal collecting precipitation, then use washed with diethylether 3-6 time; vacuum-drying, obtains PY-24 and FY-24 crude product.
6. method according to claim 5, is characterized in that the volume proportion of described cutting reagent is trifluoroacetic acid: tri isopropyl silane: water=95%:3%:2%, reacts 2.5 hours.
7. method according to claim 1, it is characterized in that, described step 3) particular content is: by soluble in water for PY-24 and FY-24 polypeptide crude product, make the aqueous solution of 200mg/ml, with 0.45 μm of membrane filtration, required component is collected with reverse phase preparative HPLC, use the component collected by analysis mode HPLC analyzing and testing again, merge the component of purity more than 98%, amalgamation liquid is concentrating under reduced pressure under 35 ~ 40 DEG C of conditions, concentrated solution is put into low-temperature freeze-drying machine and carry out lyophilize, thus obtain PY-24 and FY-24 fine work.
8. method according to claim 7, it is characterized in that the preparative column that reverse preparation HPLC adopts is C18 type, moving phase is one or more in water, ammonium acetate aqueous solution, acetonitrile, methyl alcohol.
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Application publication date: 20150722 |