CN101314612B - Method for solid phase polypeptide synthesis of hexarelin - Google Patents
Method for solid phase polypeptide synthesis of hexarelin Download PDFInfo
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- CN101314612B CN101314612B CN2008100433464A CN200810043346A CN101314612B CN 101314612 B CN101314612 B CN 101314612B CN 2008100433464 A CN2008100433464 A CN 2008100433464A CN 200810043346 A CN200810043346 A CN 200810043346A CN 101314612 B CN101314612 B CN 101314612B
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- fluorenylmethyloxycarbonyl
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 26
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 21
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 19
- RVWNMGKSNGWLOL-GIIHNPQRSA-N (2s)-6-amino-2-[[(2r)-2-[[(2s)-2-[[(2s)-2-[[(2r)-2-[[(2s)-2-amino-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(2-methyl-1h-indol-3-yl)propanoyl]amino]propanoyl]amino]-3-(1h-indol-3-yl)propanoyl]amino]-3-phenylpropanoyl]amino]hexanamide Chemical compound C([C@H](N)C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CN=CN1 RVWNMGKSNGWLOL-GIIHNPQRSA-N 0.000 title claims abstract description 18
- 108010070965 hexarelin Proteins 0.000 title claims abstract description 18
- 239000007790 solid phase Substances 0.000 title claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 title claims description 11
- 238000003786 synthesis reaction Methods 0.000 title claims description 11
- 239000011347 resin Substances 0.000 claims abstract description 86
- 229920005989 resin Polymers 0.000 claims abstract description 86
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 49
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims abstract description 43
- 238000006243 chemical reaction Methods 0.000 claims abstract description 38
- 238000005520 cutting process Methods 0.000 claims abstract description 6
- 238000000746 purification Methods 0.000 claims abstract description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims abstract description 5
- 150000001413 amino acids Chemical class 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims abstract description 5
- 238000004108 freeze drying Methods 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 claims abstract description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 54
- 238000005406 washing Methods 0.000 claims description 50
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 239000003153 chemical reaction reagent Substances 0.000 claims description 33
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 24
- 239000004576 sand Substances 0.000 claims description 20
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 18
- 239000012043 crude product Substances 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 7
- -1 hexafluorophosphate Chemical compound 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- IWDCLRJOBJJRNH-UHFFFAOYSA-N p-cresol Chemical compound CC1=CC=C(O)C=C1 IWDCLRJOBJJRNH-UHFFFAOYSA-N 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 5
- 238000010025 steaming Methods 0.000 claims description 5
- 230000000903 blocking effect Effects 0.000 claims description 4
- 235000002597 Solanum melongena Nutrition 0.000 claims description 3
- 244000061458 Solanum melongena Species 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 230000003252 repetitive effect Effects 0.000 claims description 3
- 238000005201 scrubbing Methods 0.000 claims description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims 9
- 239000002253 acid Substances 0.000 claims 7
- 239000012964 benzotriazole Substances 0.000 claims 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 5
- 150000002148 esters Chemical class 0.000 claims 5
- 229910052739 hydrogen Inorganic materials 0.000 claims 5
- 239000001257 hydrogen Substances 0.000 claims 5
- 229920001721 polyimide Polymers 0.000 claims 5
- 239000009719 polyimide resin Substances 0.000 claims 5
- YXFVVABEGXRONW-UHFFFAOYSA-N toluene Substances CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims 5
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims 2
- 239000006035 Tryptophane Substances 0.000 claims 2
- 229960002885 histidine Drugs 0.000 claims 2
- 150000003003 phosphines Chemical class 0.000 claims 2
- 229960004799 tryptophan Drugs 0.000 claims 2
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 claims 1
- 239000003463 adsorbent Substances 0.000 claims 1
- 229940024606 amino acid Drugs 0.000 claims 1
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 1
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 abstract description 17
- 238000002360 preparation method Methods 0.000 abstract description 15
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 abstract description 9
- 239000007821 HATU Substances 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000010189 synthetic method Methods 0.000 abstract description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 abstract description 2
- 125000006239 protecting group Chemical group 0.000 abstract 3
- 239000000126 substance Substances 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- HOPRXXXSABQWAV-UHFFFAOYSA-N anhydrous collidine Natural products CC1=CC=NC(C)=C1C HOPRXXXSABQWAV-UHFFFAOYSA-N 0.000 description 8
- UTBIMNXEDGNJFE-UHFFFAOYSA-N collidine Natural products CC1=CC=C(C)C(C)=N1 UTBIMNXEDGNJFE-UHFFFAOYSA-N 0.000 description 8
- GFYHSKONPJXCDE-UHFFFAOYSA-N sym-collidine Natural products CC1=CN=C(C)C(C)=C1 GFYHSKONPJXCDE-UHFFFAOYSA-N 0.000 description 8
- 206010019280 Heart failures Diseases 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 230000002107 myocardial effect Effects 0.000 description 3
- SJVFAHZPLIXNDH-JOCHJYFZSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-JOCHJYFZSA-N 0.000 description 2
- XXMYDXUIZKNHDT-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(N=C1)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXMYDXUIZKNHDT-QNGWXLTQSA-N 0.000 description 2
- ADOHASQZJSJZBT-SANMLTNESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[1-[(2-methylpropan-2-yl)oxycarbonyl]indol-3-yl]propanoic acid Chemical compound C12=CC=CC=C2N(C(=O)OC(C)(C)C)C=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ADOHASQZJSJZBT-SANMLTNESA-N 0.000 description 2
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 2
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 2
- 239000012317 TBTU Substances 0.000 description 2
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229910000040 hydrogen fluoride Inorganic materials 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 210000004165 myocardium Anatomy 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 102000005862 Angiotensin II Human genes 0.000 description 1
- 101800000733 Angiotensin-2 Proteins 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- CZGUSIXMZVURDU-JZXHSEFVSA-N Ile(5)-angiotensin II Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=[NH2+])NC(=O)[C@@H]([NH3+])CC([O-])=O)C(C)C)C1=CC=C(O)C=C1 CZGUSIXMZVURDU-JZXHSEFVSA-N 0.000 description 1
- 102100022831 Somatoliberin Human genes 0.000 description 1
- 101710142969 Somatoliberin Proteins 0.000 description 1
- 229950006323 angiotensin ii Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
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- 230000000955 neuroendocrine Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
-
- 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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- Peptides Or Proteins (AREA)
Abstract
The invention relates to a preparation method of hexarelin, and mainly solves the technical problems with long synthetic period and applications of hypertoxic substances in the prior synthetic method. The method for synthesizing the hexarelin by adopting the solid phase polypeptide synthetic method comprises the steps as follows: Fmoc-Linker-AM-Resin or Fmoc-Linker-MBHA-Resin is adopted as the initial raw material; amino acids with Fmoc protective groups are sequentially connected according to the solid phase polypeptide synthetic method to obtain a protected six peptide resin, while the Fmoc protective groups are sequentially removed and a peptide synthetic reaction is carried out in the presence of one of DIC/HOBt, DIC/HOAt, BOP/HOBt, BOP/HOAt, HBTU/HOBt, HBTU/HOAt, HATU/HOBt, HATU/HOAt or HCTU/HOBt as a condensing agent; side chain protective groups removal and peptide cutting are carried out synchronously to obtain a coarse product; the coarse product is subjected to separation and purification to obtain the hexarelin which is further subjected to lyophilization to obtain hexarelin acetate or trifluoroacetate product. The method is applicable to the large-scale hexarelin production.
Description
Technical field:
The present invention relates to the sea sand Rayleigh preparation method of (comprising acetate, trifluoroacetate), relate in particular to the preparation method of solid phase polypeptide synthesis of hexarelin.
Background technology:
The sea sand Rayleigh, Chinese name: sea sand Rayleigh, English name: Hexarelin
Structural formula: His-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH
2
Molecular formula and molecular weight: C47H58N12O6; 887.0
Discovered in recent years that apoptosis caused that it is one of important mechanisms that takes place in heart failure that the myocardial cell loses, a kind of important neuroendocrine factor when Angiotensin II (Angiotension II) is in heart failure the generation can be induced apoptosis of cardiac muscle.Great mass of data shows that hexarelin has obvious provide protection to cardiovascular systems.By studies show that Hexarelin can obviously suppress AngII inductive apoptosis of cardiac muscle.Myocardial cell's hypertrophy is one of important pathogenic process in heart failure, and the myocardial cell that hexarelin has obvious anti-Ang II to bring out is loose and to the effect of SHR myocardial hypertrophy.
Usually synthetic GHRH series polypeptide such as GHRH-2, GHRH-6 etc. adopt the Boc method, and per step takes off Boc and all need use TFA, uses MBHA or AM Resin, with the hydrogen fluoride cutting of strong stimulation, huge poison, is that the cycle is long by the condensation of liquid phase method segment in addition.With respect to above two kinds, the scale operation and the use of sea sand Rayleigh have been limited.And along with the amino acid whose commercialized degree of Fmoc is more and more higher, the decline of cost, the Fmoc solid phase method is than faster, and is convenient, in a large number the method for large-scale production.
Summary of the invention:
The method that the purpose of this invention is to provide the synthetic sea sand Rayleigh of a kind of phase polypeptide mainly solves the synthesis cycle length that has synthetic method now and exist, the technical problem of using violent in toxicity in synthetic.
Technical scheme of the present invention is: the method for the synthetic sea sand Rayleigh of a kind of phase polypeptide comprises the steps:
With Fmoc-Linker-AM-Resin or Fmoc-Linker-MBHA-Resin is starting raw material; method according to solid phase synthesis connects the amino acid with Fmoc protection successively; obtain six peptide resins of protection; slough the Fmoc blocking group therebetween successively; connect reactive polypeptide with wherein a kind of of DIC/HOBt or DIC/HOAt or BOP/HOBt or BOP/HOAt or HBTU/HOBt or HBTU/HOAt or HATU/HOBt or HATU/HOAt or HCTU/HOBt for condensing agent; behind six peptide resins that must protect; take off the side chain protected group synchronously and cut peptide; obtain sea sand Rayleigh crude product; and carry out separation and purification through the C18 chromatographic column; obtain said sea sand Rayleigh; lyophilize be can pass through again then, sea sand nafarelin acetate salt or trifluoroacetic acid product salt obtained.
According to the present invention, connect amino acid successively with blocking group, obtain protection six peptide resins, the method for sloughing the Fmoc blocking group therebetween successively comprises the steps:
(1) preparation Fmoc-Lys (Boc)-Linker AM Resin
Select for use Fmoc-Linker-AM-Resin or Fmoc-Linker-MBHA-Resin to pour into and connect the peptide bottle, the DCM that adds 2.5 times of volumes of big reducible resin, soaking 10 minutes (normal temperature) back drains with vacuum pump, add the reagent of raising one's hat and put into constant temperature oscillator reaction 5 minutes, drain with vacuum pump, again add 15 minutes (30 ℃～40 ℃) of agent reaction of raising one's hat, drain with vacuum pump, DMF with technical grade washs 3 times, anhydrous methanol washing 3 times, the DCM that heavily steamed washing 3 times adds Fmoc-Lys (BOC)-OH, HOBt, the DMF of AG, DIC, Collidine reacts 40 minutes (30 ℃～50 ℃), drain with vacuum pump, DMF with technical grade washs 3 times, anhydrous methanol washing 3 times, the DCM that heavily steamed washing 3 times, drain, obtain Fmoc-Lys (Boc)-Linker AM Resin;
The component of the said here reagent of raising one's hat and volume ratio are: PIP: DMF (technical grade)=1: 4; Down together;
The weight of Fmoc-Linker-AM-Resin or Fmoc-Linker-MBHA-Resin is 8-12ml/g with the ratio of the add-on of the reagent of raising one's hat; Down together;
The mole number of Fmoc-Lys (Boc)-OH is 2-3 a times of resin; Down together;
The mole number of HOBt is 2-3 a times of resin; Down together;
The mole number of DIC is 2-3 a times of resin; Down together;
The ratio of the add-on of the weight of Fmoc-Linker-AM Resin or Fmoc-Linker-MBHA Resin and the DMF of AG is 8-10mi/g; Down together;
The weight of Fmoc-Linker-AM Resin or Fmoc-Linker-MBHA Resin is 8-10ml/g with the ratio of the add-on of the DCM that washs, heavily steamed with the DMF of technical grade, anhydrous methanol; Down together;
(2) preparation Fmoc-D-Phe-Lys (Boc)-Linker AM Resin
In the Fmoc-Lys of step (1) (Boc)-Linker AM Resin resin, add the reagent of raising one's hat, 30～60 ℃ were reacted 5 minutes, and drained, again add 30～60 ℃ of reactions of agent 15 minutes of raising one's hat, drain, washing, adding Fmoc-D-Phe-OH, HOBt, DMF, the DIC of AG, Collidine react 40 minutes (30 ℃～50 ℃), drain, washing is drained, and obtains Fmoc-D-Phe-Lys (Boc)-Linker AM Resin;
The mole number of Fmoc-D-Phe-OH is 2-3 a times of resin; Down together
All the other manipulations and processing condition are the same;
(3) preparation Fmoc-Trp (Boc)-D-Phe-Lys (Boc)-Linker AM Resin
At the Fmoc-D-Phe-Lys of step (2) (Boc)-Linker AM Resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain washing, adding Fmoc-Trp (BOC)-OH, HOBt, DMF, the DIC of AG, Collidine react 40 minutes (30 ℃～50 ℃), drain washing, drain, obtain Fmoc-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin;
The mole number of Fmoc-Trp (BOC)-OH is 2-3 a times of resin; Down together
All the other manipulations and processing condition are the same;
(4) preparation Fmoc-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin
In the Fmoc-Trp of step (3) (BOC)-D-Phe-Lys (Boc)-Linker AM Resin, add the reagent react 5 minutes of raising one's hat, drain, add the agent reaction 15 minutes of raising one's hat again, drain, washing, adding Fmoc-Ala-OH, HOBt, DMF, the DIC of AG, Collidine react 40 minutes (30 ℃～50 ℃), drain washing, drain, obtain Fmoc-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin;
The mole number of Fmoc-Ala-OH is 2-3 a times of resin; Down together
All the other manipulations and processing condition are the same
(5) preparation Fmoc-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin
In the Fmoc-Ala-Trp of step (4) (BOC)-D-Phe-Lys (Boc)-Linker AM Resin, add the reagent react 5 minutes of raising one's hat, drain, add the agent reaction 15 minutes of raising one's hat again, drain, washing, adding Fmoc-D-2-Me-Trp-OH, HOBt, DMF, the DIC of AG, Collidine react 40 minutes (30 ℃～50 ℃), drain washing, drain, obtain Fmoc-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AMResin;
The mole number of Fmoc-D-2-Me-Trp-OH is 2-3 a times of resin; Down together
All the other manipulations and processing condition are the same
(6) preparation Fmoc-His (Trt)-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AMResin is in the Fmoc-D-2-Me-Trp-Ala-Trp of step (5) (BOC)-D-Phe-Lys (Boc)-Linker AM Resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, washing, adding Fmoc-His (Trt)-OH, HOBt, DMF, the DIC of AG, Collidine react 40 minutes (30 ℃～50 ℃), drain, washing, drain, obtain
The mole number of Fmoc-His (Trt)-OH is 2-3 a times of resin; Down together
All the other manipulations and processing condition are the same
(7) in the Fmoc-His of step (6) (Trt)-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-LinkerAM Resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, use the DMF of technical grade to wash 3 times, anhydrous methanol washing 3 times, the DCM washing of heavily steaming 3 times is drained (taking out half an hour approximately with vacuum pump) with anhydrous diethyl ether always, and resin becomes very dried particle;
(8) Fmoc-His that drains (Trt)-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AMResin is poured into the eggplant type bottle of glass, (the TFA/EDT/p-Cresol/DCM=80/2.5/2.5/15 in the peptide reagent that cuts that adds precooling-5 ℃～-10 ℃, volume ratio), cut reactive polypeptide 2～3 hours for 20 ℃～40 ℃, remove by filter resin, precipitation adds diethyl ether, centrifugal, with ether repetitive scrubbing 6 times, in room temperature, put decompressing and extracting in the moisture eliminator (being generally 5～8 hours), obtain sea sand Rayleigh crude product.
Cut in the peptide reagent, the concentration of resin is: 8～12ml/g;
According to the present invention, crude product comprises the steps: through the method for C18 column separating purification
Sea sand Rayleigh crude product is dissolved in the 25%ACN aqueous solution, filters, filtrate is through C18 chromatographic column purifying, and moving phase has two kinds: A:0.1%TFA+99.9%ACN; B:0.1%TFA+99.9%H2O; Gradient: 22%～30%; Flow velocity is 110ml/min; The detection wavelength is 220nm; Follow the tracks of needed effluent liquid with liquid chromatograph, the sample peak merges the concentrated ACN that goes in back, changes acetate, concentrates the small volume freeze-drying, obtains finished product (MW:887.0), and total recovery is 46.73% (in the mmol of resin).
The invention has the beneficial effects as follows: sea sand of the present invention Rayleigh synthesis route has following characteristics: possess the large-scale production ability; process stabilizing; the raw and auxiliary material convenient sources, with short production cycle, production cost is low; the yield height; steady quality, production cost is low, has the market competitiveness; per step connects the peptide yield and can reach more than 98%, has reduced cost.
The Fmoc route of this process using gentleness, per step takes off Fmoc and only uses 20% PIP, and per step condensation reaction needed only 40 minutes, had shortened the production cycle greatly, and TFA is adopted in cutting, avoids using hydrogen fluoride, has significantly reduced the three wastes, helps suitability for industrialized production.
The raw material Chinese and English that is adopted in the aforementioned process is listed as follows:
Embodiment:
Embodiment 1
(1) preparation Fmoc-Lys (Boc)-Linker AM Resin
Taking by weighing Fmoc-Linker-AM Resin 1g (Loadding:0.6mmol/g) pours 30ml into and connects the peptide bottle, add big reducible resin 15mlDCM, soaking 10 minutes (normal temperature) back drains with vacuum pump, add the reagent 10ml that raises one's hat and put into constant temperature oscillator reaction 5 minutes, drain with vacuum pump, again add 15 minutes (30 ℃～40 ℃) of agent 10ml reaction of raising one's hat, drain with vacuum pump, DMF with technical grade washs 3 times, anhydrous methanol washing 3 times, the DCM that heavily steamed washing 3 times adds Fmoc-Lys (BOC)-OH0.702g, HOBt0.337g, the DMF8ml of AG, DIC0.2ml, Collidine0.3ml reacts 40 minutes (30 ℃～50 ℃), drain with vacuum pump, DMF with technical grade washs 3 times, anhydrous methanol washing 3 times, the DCM that heavily steamed washing 3 times, drain, obtain Fmoc-Lys (Boc)-LinkerAM Resin;
(2) preparation Fmoc-D-Phe-Lys (Boc)-Linker AM Resin
In the Fmoc-Lys of step (1) (Boc)-LinkerAM Resin, add the reagent of raising one's hat, 30～60 ℃ were reacted 5 minutes, and drained, again add 30～60 ℃ of reactions of agent 15 minutes of raising one's hat, drain, washing, adding Fmoc-D-Phe-OH0.581g, HOBt0.337g, DMF8ml, the DIC0.2ml of AG, Collidine0.3ml react 40 minutes (30 ℃～50 ℃), drain, washing is drained, and obtains Fmoc-D-Phe-Lys (Boc)-Linker AM Resin;
All the other manipulations and processing condition are the same;
(3) preparation Fmoc-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin
In the Fmoc-D-Phe-Lys of step (2) (Boc)-Linker AM Resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain washing, adding Fmoc-Trp (BOC)-OH0.790g, HOBt0.337g, DMF8ml, the DIC0.2ml of AG, Collidine0.3ml react 40 minutes (30 ℃～50 ℃), drain washing, drain, obtain Fmoc-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin;
All the other manipulations and processing condition are the same;
(4) preparation Fmoc-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin
In the Fmoc-Trp of step (3) (BOC)-D-Phe-Lys (Boc)-Linker AM Resin, add the reagent react 5 minutes of raising one's hat, drain, add the agent reaction 15 minutes of raising one's hat again, drain, washing, adding Fmoc-Ala-OH0.467g, HOBt0.337g, DMF8ml, the DIC0.2ml of AG, Collidine0.3ml react 40 minutes (30 ℃～50 ℃), drain washing, drain, obtain Fmoc-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin;
All the other manipulations and processing condition are the same
(5) preparation Fmoc-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin
In the Fmoc-Ala-Trp of step (4) (BOC)-D-Phe-Lys (Boc)-Linker AM Resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, washing, adding Fmoc-D-2-Me-Trp-OH0.635g, HOBt0.337g, DMF8ml, the DIC0.2ml of AG, Collidine0.3ml react 40 minutes (30 ℃～50 ℃), drain, washing, drain, obtain Fmoc-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin;
All the other manipulations and processing condition are the same.
(6) preparation Fmoc-His (Trt)-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AMResin
In the Fmoc-D-2-Me-Trp-Ala-Trp of step (5) (BOC)-D-Phe-Lys (Boc)-Linker AM Resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, washing, add Fmoc-His (Trt)-OH0.929g, HOBt0.337g, the DMF8ml of AG, DIC0.2ml, Collidine0.3ml reacts 40 minutes (30 ℃～50 ℃), drain, washing, drain, it is the same to obtain Fmoc-His (Trt)-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-all the other manipulations of Linker AM Resin and processing condition
(7) in the Fmoc-His of step (6) (Trt)-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-LinkerAM Resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, DMF with technical grade washs 3 times, anhydrous methanol washing 3 times, the DCM washing of heavily steaming 3 times, drain (taking out half an hour approximately) with anhydrous diethyl ether with vacuum pump always, resin becomes very dried particle, and described resin is Fmoc-His (Trt)-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AM Resin
(8) Fmoc-His that drains (Trt)-D-2-Me-Trp-Ala-Trp (BOC)-D-Phe-Lys (Boc)-Linker AMResin is poured into the eggplant type bottle of 25ml glass, what add 8ml precooling-5 ℃～-10 ℃ cuts peptide reagent (TFA/EDT/p-Cresol/DCM=80/2.5/2.5/15, volume ratio), cut reactive polypeptide 2～3 hours for 20 ℃～40 ℃, remove by filter resin, precipitation adds diethyl ether, centrifugal, with ether repetitive scrubbing 6 times, in room temperature, put decompressing and extracting in the moisture eliminator (being generally 5～8 hours), obtain sea sand Rayleigh crude product 0.520g.
According to the present invention, crude product comprises the steps: sea sand Rayleigh crude product is dissolved in the 25%ACN aqueous solution through the method for C18 column separating purification, filters, and filtrate is through C18 chromatographic column purifying, and moving phase has two kinds: A:0.1%TFA+99.9%ACN; B:0.1%TFA+99.9%H2O; Gradient: 22%～30%; Flow velocity is 110ml/min; The detection wavelength is 220nm; Follow the tracks of needed effluent liquid with liquid chromatograph, the sample peak merges the concentrated ACN that goes in back, changes acetate, concentrates the small volume freeze-drying, obtains finished product 0.243g (MW:887.0), and total recovery is 46.73% (in the mmol of resin).
Embodiment 2
Adopt method and the processing condition identical with embodiment 1, wherein:
Make starting raw material with Fmoc-Linker-MBHA Resin, add 30～50 ℃ of reactions of reagent 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, washing, DMF, Collidine, DIC/HOBt or DIC/HOAt or BOP/HOBt or BOP/HOAt or HBTU/HOBt or HBTU/HOAt or HATU/HOBt or HATU/HOAt or HCTU/HOBt or the HUTC/HOAt or the TBTU/HOBt of adding Fmoc-amino acid, AG reacted 40 minutes, with embodiment 1 operation, directly end six peptides and connect.Through reactions such as cuttings, obtain 0.540g white powder product again.
Embodiment 3
Taking by weighing Fmoc-Linker-AM Resin or Fmoc-Linker-MBHA Resin50g (Loadding:0.7mmol/g) pours 600ml into and connects the peptide bottle, add 30～50 ℃ of reactions of reagent 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, washing, DMF, Collidine, DIC/HOBt or the DIC/HOAt or BOP/HOBt or BOP/HOAt or HBTU/HOBt or HBTU/HOAt or HATU/HOBt or HATU/HOAt or HCTU/HOBt or the TBTU/HOBt that add Fmoc-amino acid, AG, reacted 40 minutes, with embodiment 1 operation, directly end six peptides and connect.Through reactions such as cuttings, obtain 33g white powder product again.Can be better with HBTU/HOBt crude reaction purity.
Claims (6)
1. the method for a solid phase polypeptide synthesis of hexarelin, it is characterized in that comprising the steps: that with fluorenes methoxy carbonyl acyl group-Linker-aminomethyl resin or fluorenes methoxy carbonyl acyl group-Linker-4-toluene hydrogen polyimide resin be starting raw material, method according to solid phase synthesis connects the amino acid with the protection of fluorenes methoxy carbonyl acyl group successively, obtain six peptide resins of protection, slough fluorenes methoxy carbonyl acyl group blocking group therebetween successively, connect reactive polypeptide with condensing agent, behind six peptide resins that must protect, take off the side chain protected group synchronously and cut peptide, obtain sea sand Rayleigh crude product, and carry out separation and purification through the C18 chromatographic column, obtain said sea sand Rayleigh;
When described six peptide resins are fluorenylmethyloxycarbonyl-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, comprise the steps: to select for use fluorenes methoxy carbonyl acyl group-Linker-aminomethyl resin or fluorenes methoxy carbonyl acyl group-Linker-4-toluene hydrogen polyimide resin to pour into and connect the peptide bottle, the methylene dichloride that adds 2.5 times of volumes of resin, soak at room temperature was drained with vacuum pump after 10 minutes, add the reagent of raising one's hat and put into constant temperature oscillator reaction 5 minutes, drain with vacuum pump, again add 30 ℃～40 ℃ reactions of agent 15 minutes of raising one's hat, drain with vacuum pump, dimethyl formamide with technical grade washs 3 times, anhydrous methanol washing 3 times, the washed with dichloromethane of heavily steaming 3 times, add fluorenylmethyloxycarbonyl-N-epsilon-tertbutyloxycarbonyl-L-Methionin, 1-hydroxyl-benzo-triazole, the dimethyl formamide of AG, N, N-di-isopropyl carbodiimide, 2,3,30 ℃～50 ℃ reactions of 5-trimethylpyridine 40 minutes, drain with vacuum pump, DMF with technical grade washs 3 times, anhydrous methanol washing 3 times, the dimethyl formamide washing of heavily steaming 3 times, drain, obtain fluorenylmethyloxycarbonyl-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin;
When described six peptide resins are fluorenylmethyloxycarbonyl-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, in fluorenylmethyloxycarbonyl-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, add the reagent of raising one's hat, 30～60 ℃ were reacted 5 minutes, drain, again add 30～60 ℃ of reactions of agent 15 minutes of raising one's hat, drain, washing, add fluorenylmethyloxycarbonyl-D-phenylamino acid, 1-hydroxyl-benzo-triazole, the dimethyl formamide of AG, N, N-di-isopropyl carbodiimide, 2,3,30 ℃～50 ℃ reactions of 5-trimethylpyridine 40 minutes, drain, washing is drained, obtain fluorenylmethyloxycarbonyl-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, wherein the mole number of fluorenylmethyloxycarbonyl-D-phenylamino acid be resin 2-3 doubly;
When described six peptide resins are fluorenylmethyloxycarbonyl-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, comprise the steps: in fluorenylmethyloxycarbonyl-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, washing, add fluorenylmethyloxycarbonyl-N-in-tert-butoxycarbonyl-l-l-tryptophan, 1-hydroxyl-benzo-triazole, the dimethyl formamide of AG, N, N-di-isopropyl carbodiimide, 2,3,30 ℃～50 ℃ reactions of 5-trimethylpyridine 40 minutes, drain, washing, drain, obtain fluorenylmethyloxycarbonyl-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, wherein the mole number of fluorenylmethyloxycarbonyl-N-in-tert-butoxycarbonyl-l-l-tryptophan be resin 2-3 doubly;
When described six peptide resins are fluorenylmethyloxycarbonyl-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl tree, comprise the steps: in fluorenylmethyloxycarbonyl-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, washing, add fluorenylmethyloxycarbonyl-L-L-Ala, 1-hydroxyl-benzo-triazole, the dimethyl formamide of AG, N, N-di-isopropyl carbodiimide, 2,3,30 ℃～50 ℃ reactions of 5-trimethylpyridine 40 minutes, drain, washing, drain, obtain fluorenylmethyloxycarbonyl-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, wherein the mole number of fluorenylmethyloxycarbonyl-L-L-Ala be resin 2-3 doubly;
When described six peptide resins are fluorenylmethyloxycarbonyl-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, comprise the steps: in fluorenylmethyloxycarbonyl-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, washing, add fluorenylmethyloxycarbonyl-D-2-methyl-tryptophane, 1-hydroxyl-benzo-triazole, the dimethyl formamide of AG, N, N-di-isopropyl carbodiimide, 2,3,30 ℃～50 ℃ reactions of 5-trimethylpyridine 40 minutes, drain, washing, drain, obtain fluorenylmethyloxycarbonyl-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, the mole number of fluorenylmethyloxycarbonyl-D-2-methyl-tryptophane is 2-3 a times of resin;
When described six peptide resins are fluorenylmethyloxycarbonyl-L-Histidine-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, comprise the steps: in obtaining fluorenylmethyloxycarbonyl-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, washing, add fluorenylmethyloxycarbonyl-L-Histidine, 1-hydroxyl-benzo-triazole, the dimethyl formamide of AG, N, N-di-isopropyl carbodiimide, 2,3,30 ℃～50 ℃ reactions of 5-trimethylpyridine 40 minutes, drain, washing, drain, obtain fluorenylmethyloxycarbonyl-L-Histidine-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, the mole number of fluorenylmethyloxycarbonyl-L-Histidine is 2-3 a times of resin;
When described six peptide resins are fluorenylmethyloxycarbonyl-L-Histidine-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, comprise the steps: in fluorenylmethyloxycarbonyl-L-Histidine-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, add the reagent react 5 minutes of raising one's hat, drain, again add the agent reaction 15 minutes of raising one's hat, drain, DMF with technical grade washs 3 times, anhydrous methanol washing 3 times, the DCM washing of heavily steaming 3 times, become very dried particle with the anhydrous diethyl ether dry adsorbent, resulting resin is: L-Histidine-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin;
Described condensing agent is N, N-di-isopropyl carbodiimide/1-hydroxyl-benzo-triazole, N, N-di-isopropyl carbodiimide/N-hydroxyl-7-azo benzotriazole, 7-azepine benzothiazole-1-base-oxygen-(three-(dimethylin) phosphines) hexafluorophosphate/1-hydroxyl-benzo-triazole, 7-azepine benzothiazole-1-base-oxygen-(three-(dimethylin) phosphines) hexafluorophosphate/1-hydroxyl-benzo-triazole/N-hydroxyl-7-azo benzotriazole, benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester/1-hydroxyl-benzo-triazole, benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester/N-hydroxyl-7-azo benzotriazole, 2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester/1-hydroxyl-benzo-triazole, 2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester/N-hydroxyl-7-azo benzotriazole or 6-chlorobenzene and triazole-1,1,3, wherein a kind of of 3-tetramethyl-urea phosphofluoric acid ester/1-hydroxyl-benzo-triazole.
2. the method for a kind of solid phase polypeptide synthesis of hexarelin according to claim 1 is characterized in that the sea sand Rayleigh that obtains passes through lyophilize again, obtains sea sand nafarelin acetate salt or trifluoroacetic acid product salt.
3. the method for a kind of solid phase polypeptide synthesis of hexarelin according to claim 1; the ratio that it is characterized in that the add-on of the weight of fluorenes methoxy carbonyl acyl group-Linker-aminomethyl resin or fluorenes methoxy carbonyl acyl group-Linker-4-toluene hydrogen polyimide resin and the reagent of raising one's hat is 8-12ml/g; the mole number of fluorenylmethyloxycarbonyl-N-epsilon-tertbutyloxycarbonyl-L-Methionin is 2-3 a times of resin; the mole number of 1-hydroxyl-benzo-triazole is 2-3 a times of resin; N; the mole number of N-di-isopropyl carbodiimide is 2-3 a times of resin; the ratio of the add-on of fluorenes methoxy carbonyl acyl group-Linker-aminomethyl resin or the fluorenes methoxy carbonyl acyl group-weight of Linker-4-toluene hydrogen polyimide resin and the dimethyl formamide of AG is 8-10ml/g, the weight of fluorenes methoxy carbonyl acyl group-Linker-aminomethyl resin or fluorenes methoxy carbonyl acyl group-Linker-4-toluene hydrogen polyimide resin and the dimethyl formamide of using technical grade; the anhydrous methanol washing; the ratio of the add-on of the methylene dichloride that heavily steamed is 8-10ml/g.
4. the method for a kind of solid phase polypeptide synthesis of hexarelin according to claim 1 is characterized in that the component of the said reagent of raising one's hat and volume ratio are: hexahydropyridine: dimethyl formamide=1: 4.
5. the method for a kind of solid phase polypeptide synthesis of hexarelin according to claim 1, it is characterized in that: when described six peptide resins are fluorenylmethyloxycarbonyl-L-Histidine-D-2-methyl-tryptophane-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin, L-Histidine-D-2-methyl-tryptophane of draining-L-L-Ala-N-in-tert-butoxycarbonyl-l-l-tryptophan-D-phenylamino acid-N-epsilon-tertbutyloxycarbonyl-L-Methionin-Linker aminomethyl resin is poured into the eggplant type bottle of glass, add the cutting in the peptide reagent of precooling-5 ℃～-10 ℃, cut in the peptide reagent, the concentration of resin is: 8～12ml/g, cut the volume ratio trifluoroacetic acid/dithioglycol/p-cresol/methylene dichloride=80/2.5/2.5/15 of its component of peptide reagent, cut reactive polypeptide 2～3 hours for 20 ℃～40 ℃, remove by filter resin, precipitation adds diethyl ether, centrifugal, with ether repetitive scrubbing 6 times, in room temperature, put decompressing and extracting in the moisture eliminator, obtain sea sand Rayleigh crude product.
6. the method for a kind of solid phase polypeptide synthesis of hexarelin according to claim 1, it is characterized in that: it is in 25% the acetonitrile solution that crude product comprises the steps: sea sand Rayleigh crude product is dissolved in concentration through the method for C18 column separating purification, filter, filtrate is through C18 chromatographic column purifying, and moving phase has two kinds: the A:0.1% trifluoroacetic acid adds 99.9% acetonitrile; The B:0.1% trifluoroacetic acid adds 99.9% water; Gradient: 22% to 30%; Flow velocity is per minute 110ml, and the detection wavelength is 220nm, follows the tracks of needed effluent liquid with liquid chromatograph, and the sample peak merges the concentrated acetonitrile that goes in back, changes acetate, concentrates the small volume freeze-drying, obtains finished product.
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| CN1858060A (en) * | 2005-05-08 | 2006-11-08 | 周达明 | Process for preparing solid phase polypeptide synthetic eptifibatide |
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| CN1858060A (en) * | 2005-05-08 | 2006-11-08 | 周达明 | Process for preparing solid phase polypeptide synthetic eptifibatide |
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| US11795646B2 (en) | 2019-11-27 | 2023-10-24 | Ironclad Siege, LLC | System for reducing contaminants in a body of water |
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