CN104098656A - Polypeptide synthesis method for sinapultide - Google Patents
Polypeptide synthesis method for sinapultide Download PDFInfo
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- CN104098656A CN104098656A CN201410332391.7A CN201410332391A CN104098656A CN 104098656 A CN104098656 A CN 104098656A CN 201410332391 A CN201410332391 A CN 201410332391A CN 104098656 A CN104098656 A CN 104098656A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention discloses a polypeptide synthesis method for sinapultide. The method comprises the steps as follows: 2-chloro-triphenyl chlorine resin is used as a starting raw material and sequentially connected with amino acids containing protecting groups with a solid-phase synthesis method to obtain protecting 21 peptide resin, Fmoc-protecting groups are removed sequentially, a crude product is obtained after TFA (trifluoroacetic acid) peptide cutoff, the crude product is separated and purified by a C18 column, and a high-quality sinapultide product is prepared after freeze-drying. The method is stable in technology, convenient in raw and auxiliary material source, short in preparation cycle, high in yield, stable in quality, low in preparation cost, high in connecting peptide yield and little in thee-waste pollution due to avoidance of usage of highly toxic reagents such as hydrogen fluoride and the like. The connecting peptide yield in each step is higher than 98.5%; the yield after peptide cutoff is 98.3%, the total yield is 47.9%, and the HPLC (high performance liquid chromatography) measured product purity is 99.6%.
Description
Technical field
The present invention relates to the preparation method of sinapultide, relate in particular to the preparation method of the synthetic sinapultide of solid-phase polypeptide.
Background technology
Sinapultide, English name: Surfaxin, Sinapultide, molecular formula: C
126h
238n
26o
22; CAS registration number: 138531-07-4; Molecular weight: 2469.5; Carbon, hydrogen, nitrogen ratio: C:61.28%, H:9.71%, N:14.75%.Aminoacid sequence:
H-Lys-Leu-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Lys-OH；
Structural formula:
Sinapultide is 21 peptides of a synthetic, is used for the treatment of hyaline membrane disease of prematurity.Discovery Laboratories IncWei Yuanyan company, was gone on the market by U.S. FDA approval in March, 2012.
Chinese Patent Application No. is 201210345662.3, and the application for a patent for invention that name is called " a kind of chemical synthesis process of sinapultide " discloses a kind of method of synthetic sinapultide.The method specific implementation that this invention provides is: by synthetic 4 polypeptide small segments, as intermediate, utilize the mode of salvage to obtain sinapultide resin through 3 step linked reactions, then carry out cracking purifying and obtain sinapultide sterling.The need of the method consume 4 resins, and cost compare is high.
Summary of the invention
The preparation method who the object of this invention is to provide the synthetic sinapultide of a kind of solid-phase polypeptide, only adopts resin 1 time in preparation method of the present invention, given full play to the advantage of solid phase synthesis, the high efficiency sinapultide of having prepared.
Synthetic route is as follows:
The chloro-resin of the chloro-triphenyl of 2-
↓ 1. Fmoc-Lys (Boc)-OH; 2. DIPEA; 3. methyl alcohol
Fmoc-Lys (Boc)-resin
↓①PIP；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Lys (Boc)-resin
↓①PIP；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Lys (Boc)-resin
↓①PIP；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP；②HATU/HOAt，Fmoc-Leu?-OH
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP；②HATU/HOAt，Fmoc-Lys(Boc)-OH
Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP；②HATU/HOAt，Fmoc-?Leu-OH
Fmoc-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Lue-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP；②HATU/HOAt，Fmoc-Lys(Boc)-OH
Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Lys(Boc)-OH
Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Leu-OH
Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓①PIP%；②HATU/HOAt，Fmoc-Lys(Boc)-OH
Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
↓ 1. PIP; 2. TFA/TIS/ mercaptoethanol; 3. phosphate solution/acetonitrile; 4. lyophilize
Lys-Leu-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Lys-OH
Technical scheme of the present invention is as follows:
The preparation method of the synthetic sinapultide of solid-phase polypeptide, comprise the steps: with the chloro-triphenyl chlorine of 2-resin starting raw material, according to the method for solid phase synthesis, connect successively amino acid Fmoc-Lys (the Boc)-OH with blocking group, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH carries out condensation reaction, obtain protection 21 peptide resins, slough successively Fmoc-blocking group therebetween, synchronously take off side chain protected group, cut peptide, obtain sinapultide crude product, after purifying, make sinapultide.
Preferably, described according to the method for solid phase synthesis, obtain protection 21 peptide resins, comprise the steps:
One, preparation Fmoc-Lys (Boc)-resin
1) get the chloro-triphenyl chlorine of 2-resin and soak with DMF, make the abundant swelling of resin, add DIPEA, Fmoc-Lys (Boc)-OH, reaction, then add methyl alcohol, reaction;
2) drain, with DMF, methyl alcohol and DMF washing, drain respectively;
Two, preparation Fmoc-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Three, preparation Fmoc-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Four, preparation Fmoc-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Five, preparation Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Six, preparation Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Lys (Boc)-OH, HATU, HOAt, with connecing peptide reagent, dissolve reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain respectively;
Seven, preparation Fmoc-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Eight, preparation Fmoc-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Nine, preparation Fmoc-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Ten, preparation Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (tBu)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
11, preparation Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains;
2) get Fmoc-Lys (Boc)-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
12, preparation Fmoc-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
13, preparation Fmoc-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
14, preparation Fmoc-Leu-Lue-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
15, preparation Fmoc-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
16, preparation Fmoc-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains;
2) get Fmoc-Lys (Boc)-OH, HATU, HOAt, with connecing peptide reagent, dissolve reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
17, preparation Fmoc-Lue-Lys (Boc) Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
18, preparation Fmoc-Lue-Lue-Lys (Boc) Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
19, preparation Fmoc-Lue-Lue-Lue-Lys (Boc) Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
20, preparation Fmoc-Lue-Lue-Lue-Lue-Lys (Boc) Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
21, preparation Fmoc-Lys (Boc)-Lue-Lue-Lue-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains;
2) get Fmoc-Lys (Boc)-OH, HATU, HOAt, with connecing peptide reagent, dissolve reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
4) add the reagent of raising one's hat, reaction, drains, and by DMF, methyl alcohol, DMF and methanol wash, drains;
The described peptide reagent that connects is: DIPEA: DMF=1: 10, and volume ratio;
The described reagent of raising one's hat is: PIP: DMF=24.5: 75.5, and volume ratio;
The described reactive polypeptide temperature that connects is: 0-5 ℃; Connecing the peptide time is: 55 minutes;
The described temperature of reaction of raising one's hat is: 21-23 ℃; Raise one's hat 18 minutes time.
Preferably, the chloro-triphenyl chlorine of described 2-resin substitution value is: 0.45-0.55mmol/g.
Preferably, described in to slough the used reagent of raising one's hat of Fmoc-blocking group be PIP: DMF=24.5: 75.5, volume ratio.
Preferably, described in to carry out the used peptide reagent that connects of condensation reaction be DIPEA: DMF=1: 10, volume ratio.
Preferably, described in, cutting the used peptide reagent of cutting of peptide is: TFA/TIS=932ml/68ml.
Preferably, described in, cutting peptide comprises the steps:
By described protection 21 peptide resins: Fmoc-Lys (Boc)-Lue-Lue-Lue-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin adds in advance and is chilled to-10 to-7 ℃ cut in peptide reagent, reaction, collects and cuts the crude product after peptide;
The said peptide reagent of cutting is: TFA/TIS=932ml/68ml;
The said reactive polypeptide temperature of cutting is: 13-16 ℃;
Saidly cut the reactive polypeptide time and be: 65 minutes.
Preferably, described purifying specific implementation is: described 21 peptide crude products, through C18 column separating purification, desalination, after freeze-drying, are made to sinapultide.
Preferably, described purifying specific implementation is: sinapultide crude product is dissolved in acetic acid, filters, filtrate is through C18 column purification, and flow velocity is 800-900ml/min; Detection wavelength is: 230nm; With liquid chromatograph, follow the tracks of to collect needed effluent liquid, desalination after sample peak merges, desolventizes, freeze-drying, obtains finished product;
Described moving phase is: 0.122Mol/L phosphoric acid triethylamine/acetonitrile=6.9/3.1.
beneficial effect
Product structure of the present invention is comprised of 21 amino acid, 16 Leu wherein, 5 Lys.Therefore to polypeptide, synthesize and bring certain difficulty.The present invention adopts the technique of unique combination to prepare, and has obtained beyond thought effect.It is starting raw material that the chloro-trityl resin of 2-is take in the present invention, adopt Fmoc-Lys (Boc)-OH with first amino acid of resin condensation, then, one by one with Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH carries out condensation reaction, condensing agent is HATU/HOAt, obtain Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin, with the special peptide reagent (TFA/TIS=93.2/6.8) of cutting, polypeptide is cut from resin, by special moving phase (0.122Mol/L phosphoric acid triethylamine/acetonitrile=6.9/3.1), carry out separation and purification, obtain the sinapultide that purity is greater than 99.6%.Method of the present invention, process stabilizing, raw and auxiliary material convenient sources, with short production cycle, yield is high, steady quality, production cost is low, connects peptide yield high, avoids using the poisonous reagents such as hydrogen fluoride, and three-waste pollution is few.Every step connects peptide yield all more than 98.5%; After cutting peptide, yield is: 98.3%, and total recovery is: 47.9%, HPLC measures product purity and is: 99.6%.
Embodiment
Embodiment 1
The raw material adopting in embodiment and aforementioned process is listed as follows:
| No | The name of an article | Production firm |
| 1 | The chloro-triphenyl chlorine of 2-resin | Tianjin He Cheng Science and Technology Ltd. |
| 2 | Fmoc-Lys(Boc)-OH | Suzhou heavenly steed Pharmaceutical Group |
| 3 | Fmoc-Leu-OH | Suzhou heavenly steed Pharmaceutical Group |
| 4 | 1-hydroxyl-7-azo benzotriazole (HOAt) | Suzhou heavenly steed Pharmaceutical Group |
| 5 | 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester (HATU) | Suzhou heavenly steed Pharmaceutical Group |
| 6 | DIPEA (DIPEA) | Chemical Reagent Co., Ltd., Sinopharm Group |
| 7 | Dimethyl formamide (DMF) | Chemical Reagent Co., Ltd., Sinopharm Group |
| 8 | Methyl alcohol | Chemical Reagent Co., Ltd., Sinopharm Group |
| 9 | Hexahydropyridine (PIP) | Chemical Reagent Co., Ltd., Sinopharm Group |
| 11 | Tri isopropyl silane (TIS) | Chemical Reagent Co., Ltd., Sinopharm Group |
| 12 | Trifluoracetic acid (TFA) | Chemical Reagent Co., Ltd., Sinopharm Group |
In embodiment:
Connecing peptide reagent is: DIPEA: DMF=1: 10, and volume ratio;
The reagent of raising one's hat is: PIP: DMF=24.5: 75.5, and volume ratio;
One, preparation Fmoc-Lys (Boc)-resin
1) (0.5mmol/g resin, 50mmol) 100g soak 30 minutes with 1000ml DMF, make the abundant swelling of resin, add DIPEA 20ml, Fmoc-Lys (Boc)-OH 23.5g, 25 ℃ of reactions 65 minutes to get the chloro-triphenyl chlorine of 2-resin.Add methyl alcohol 50ml, 25 ℃ are reacted 60 minutes again.
2) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Two, preparation Fmoc-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Three, preparation Fmoc-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Four, preparation Fmoc-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Five, preparation Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Six, preparation Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 70.5g Fmoc-Lys (Boc)-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Seven, preparation Fmoc-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 27-29 ℃ of reaction 15 minutes, drains, and with DMF, methyl alcohol and DMF washing three times, drains respectively.
2) get 74.2g Fmoc-Leu-OH, 79.5g HATU, 28.6g HOAt, connect peptide reagent dissolve with 760ml, add reaction vessel, 19-21 ℃ is reacted 45 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Eight, preparation Fmoc-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Nine, preparation Fmoc-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
Ten, preparation Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-D-Tyr (tBu)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
11, preparation Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 70.5g Fmoc-Lys (Boc)-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
12, preparation Fmoc-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
13, preparation Fmoc-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
14, preparation Fmoc-Leu-Lue-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
15, preparation Fmoc-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
16, preparation Fmoc-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 70.5g Fmoc-Lys (Boc)-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
17, preparation Fmoc-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
18, preparation Fmoc-Lue-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
19, preparation Fmoc-Lue-Lue-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
20, preparation Fmoc-Lue-Lue-Lue-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 53.0g Fmoc-Leu-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
21, preparation Fmoc-Lys (Boc)-Lue-Lue-Lue-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, drains.
2) get 70.5g Fmoc-Lys (Boc)-OH, 57.0g HATU, 20.5g HOAt, connect peptide reagent dissolve with 520ml, add reaction vessel, 0-5 ℃ is reacted 55 minutes.
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain.
4) add the 1000ml reagent of raising one's hat, 21-23 ℃ of reaction 18 minutes, drains, and respectively washs three times respectively with DMF, methyl alcohol and DMF, and methanol wash six times, drains.
22, prepare sinapultide crude product
Protection 21 peptide resins are transferred in 2000ml reactor, add the reagent (TFA/TIS=932ml/68ml) for preparing in advance and be chilled in advance approximately-10 to-7 ℃, 13-16 ℃ is stirred 65 minutes.Decompression desolventizes to small volume, adds the cold anhydrous diethyl ether of 1000ml, and precipitation is filtered collecting precipitation, after washing fully with anhydrous diethyl ether, and vacuum-drying.Obtain sinapultide crude product 84.5g(68.8mmol), yield 98.3%.
23, separation and purification
Sinapultide crude product is dissolved in 8.1% acetic acid, filters, filtrate is through C18 column purification, moving phase: 0.122Mol/L phosphoric acid triethylamine/acetonitrile=6.9/3.1; Flow velocity is 870ml/min; The detection wavelength of preparation HPLC is: 230nm; With analysis mode HPLC, follow the tracks of and collect needed effluent liquid, after sample peak merges, removal of solvent under reduced pressure.Upper prop is removed phosphoric acid salt again, and freeze-drying approximately obtains 41.2g white block finished product (MW:2469.5,33.56mmol), and total recovery 47.9%(is in the 50mmol of resin), HPLC measures purity and is: 99.6%.
Claims (9)
1. the polypeptide synthesis method of a sinapultide, it is characterized in that, comprise the steps: with the chloro-triphenyl chlorine of 2-resin starting raw material, according to the method for solid phase synthesis, connect successively amino acid Fmoc-Lys (the Boc)-OH with blocking group, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH carries out condensation reaction, obtain protection 21 peptide resins, slough successively Fmoc-blocking group therebetween, synchronously take off side chain protected group, cut peptide, obtain sinapultide crude product, after purifying, make sinapultide.
2. method according to claim 1, is characterized in that, described according to the method for solid phase synthesis, obtains protection 21 peptide resins, comprises the steps:
One, preparation Fmoc-Lys (Boc)-resin
1) get the chloro-triphenyl chlorine of 2-resin and soak with DMF, make the abundant swelling of resin, add DIPEA, Fmoc-Lys (Boc)-OH, reaction, then add methyl alcohol, reaction;
2) drain, with DMF, methyl alcohol and DMF washing, drain respectively;
Two, preparation Fmoc-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Three, preparation Fmoc-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Four, preparation Fmoc-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Five, preparation Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Six, preparation Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Lys (Boc)-OH, HATU, HOAt, with connecing peptide reagent, dissolve reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain respectively;
Seven, preparation Fmoc-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Eight, preparation Fmoc-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Nine, preparation Fmoc-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF, wash three times respectively, drain;
Ten, preparation Fmoc-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (tBu)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
11, preparation Fmoc-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains;
2) get Fmoc-Lys (Boc)-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
12, preparation Fmoc-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
13, preparation Fmoc-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
14, preparation Fmoc-Leu-Lue-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
15, preparation Fmoc-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
16, preparation Fmoc-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains;
2) get Fmoc-Lys (Boc)-OH, HATU, HOAt, with connecing peptide reagent, dissolve reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
17, preparation Fmoc-Lue-Lys (Boc) Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
18, preparation Fmoc-Lue-Lue-Lys (Boc) Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
19, preparation Fmoc-Lue-Lue-Lue-Lys (Boc) Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
20, preparation Fmoc-Lue-Lue-Lue-Lue-Lys (Boc) Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains respectively;
2) get Fmoc-Leu-OH, HATU, HOAt, with connecing peptide reagent, dissolve, add reaction vessel, reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
21, preparation Fmoc-Lys (Boc)-Lue-Lue-Lue-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin
1) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains;
2) get Fmoc-Lys (Boc)-OH, HATU, HOAt, with connecing peptide reagent, dissolve reaction;
3) drain, with DMF, methyl alcohol and DMF washing, drain;
4) add the reagent of raising one's hat, reaction, drains, and with DMF, methyl alcohol and DMF washing, drains;
The described peptide reagent that connects is: DIPEA: DMF=1: 10, and volume ratio;
The described reagent of raising one's hat is: PIP: DMF=24.5: 75.5, and volume ratio;
The described reactive polypeptide temperature that connects is: 0-5 ℃; Connecing the peptide time is: 55 minutes;
The described temperature of reaction of raising one's hat is: 21-23 ℃; Raise one's hat 18 minutes time.
3. method according to claim 1, is characterized in that, the chloro-triphenyl chlorine of described 2-resin substitution value is: 0.45-0.55mmol/g.
4. method according to claim 1, is characterized in that, described in to slough the used reagent of raising one's hat of Fmoc-blocking group be PIP: DMF=24.5: 75.5, volume ratio.
5. method according to claim 1, is characterized in that, described in to carry out the used peptide reagent that connects of condensation reaction be DIPEA: DMF=1: 10, volume ratio.
6. method according to claim 1, is characterized in that, described in cut the used peptide reagent of cutting of peptide and be: TFA/TIS=932ml/68ml.
7. method according to claim 1, is characterized in that, described in cut peptide and comprise the steps:
By described protection 21 peptide resins: Fmoc-Lys (Boc)-Lue-Lue-Lue-Lue-Lys (Boc)-Leu-Lue-Leu-Lue-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-Leu-Leu-Leu-Leu-Lys (Boc)-resin adds in advance and is chilled to-10 to-7 ℃ cut in peptide reagent, reaction, collects and cuts the crude product after peptide;
The said peptide reagent of cutting is: TFA/TIS=932ml/68ml;
The said reactive polypeptide temperature of cutting is: 13-16 ℃;
Saidly cut the reactive polypeptide time and be: 65 minutes.
8. method according to claim 1, is characterized in that, described purifying specific implementation is: described 21 peptide crude products, through C18 column separating purification, desalination, after freeze-drying, are made to sinapultide.
9. method according to claim 1, is characterized in that, described purifying specific implementation is: sinapultide crude product is dissolved in acetic acid, filters, filtrate is through C18 column purification, and flow velocity is 800-900ml/min; Detection wavelength is: 230nm; With liquid chromatograph, follow the tracks of to collect needed effluent liquid, desalination after sample peak merges, desolventizes, freeze-drying, obtains finished product; Described moving phase is: 0.122Mol/L phosphoric acid triethylamine/acetonitrile=6.9/3.1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104817631A (en) * | 2015-05-26 | 2015-08-05 | 成都圣诺生物科技股份有限公司 | Method for synthesizing sinapultide |
| CN106821982A (en) * | 2017-01-12 | 2017-06-13 | 东南大学 | A kind of sinapultide microbubble agents and preparation method thereof |
| WO2017107241A1 (en) * | 2015-12-22 | 2017-06-29 | 何润泽 | Method for preparing polypeptide of kpv peptide and analogue thereof |
| CN110938115A (en) * | 2018-09-25 | 2020-03-31 | 深圳翰宇药业股份有限公司 | Whole liquid phase synthesis method of sinapultide |
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| CN102850440A (en) * | 2012-09-18 | 2013-01-02 | 深圳翰宇药业股份有限公司 | Chemical synthesis method of sinapultide |
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| CN102850440A (en) * | 2012-09-18 | 2013-01-02 | 深圳翰宇药业股份有限公司 | Chemical synthesis method of sinapultide |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104817631A (en) * | 2015-05-26 | 2015-08-05 | 成都圣诺生物科技股份有限公司 | Method for synthesizing sinapultide |
| CN104817631B (en) * | 2015-05-26 | 2018-04-13 | 成都圣诺生物科技股份有限公司 | A kind of method for synthesizing sinapultide |
| WO2017107241A1 (en) * | 2015-12-22 | 2017-06-29 | 何润泽 | Method for preparing polypeptide of kpv peptide and analogue thereof |
| CN106821982A (en) * | 2017-01-12 | 2017-06-13 | 东南大学 | A kind of sinapultide microbubble agents and preparation method thereof |
| CN110938115A (en) * | 2018-09-25 | 2020-03-31 | 深圳翰宇药业股份有限公司 | Whole liquid phase synthesis method of sinapultide |
| WO2020062782A1 (en) * | 2018-09-25 | 2020-04-02 | 深圳翰宇药业股份有限公司 | Complete liquid-phase synthesis method of sinapultide |
| CN110938115B (en) * | 2018-09-25 | 2021-07-06 | 深圳翰宇药业股份有限公司 | Whole liquid phase synthesis method of sinapultide |
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