CN106519008B - The method of one species specificity synthesis group amine Rayleigh - Google Patents
The method of one species specificity synthesis group amine Rayleigh Download PDFInfo
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- CN106519008B CN106519008B CN201610927306.0A CN201610927306A CN106519008B CN 106519008 B CN106519008 B CN 106519008B CN 201610927306 A CN201610927306 A CN 201610927306A CN 106519008 B CN106519008 B CN 106519008B
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- fmoc
- chlorine resin
- supprelin
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 15
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 15
- 150000001412 amines Chemical class 0.000 title claims abstract description 8
- 229920005989 resin Polymers 0.000 claims abstract description 108
- 239000011347 resin Substances 0.000 claims abstract description 108
- 238000006243 chemical reaction Methods 0.000 claims abstract description 95
- 239000000460 chlorine Substances 0.000 claims abstract description 49
- 229910052801 chlorine Inorganic materials 0.000 claims abstract description 49
- 108700020746 histrelin Proteins 0.000 claims abstract description 40
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 39
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000005406 washing Methods 0.000 claims abstract description 28
- HHXHVIJIIXKSOE-QILQGKCVSA-N histrelin Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC(N=C1)=CN1CC1=CC=CC=C1 HHXHVIJIIXKSOE-QILQGKCVSA-N 0.000 claims abstract description 27
- 229940036197 supprelin Drugs 0.000 claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 24
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000012043 crude product Substances 0.000 claims abstract description 21
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000005520 cutting process Methods 0.000 claims abstract description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 15
- 150000001413 amino acids Chemical class 0.000 claims abstract description 13
- 238000006482 condensation reaction Methods 0.000 claims abstract description 13
- KAIVPTGNIAHCTM-UHFFFAOYSA-N C(C)N.[Cl] Chemical compound C(C)N.[Cl] KAIVPTGNIAHCTM-UHFFFAOYSA-N 0.000 claims abstract description 11
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 10
- 239000002904 solvent Substances 0.000 claims abstract description 8
- 238000005516 engineering process Methods 0.000 claims abstract description 7
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 4
- 238000009833 condensation Methods 0.000 claims abstract description 4
- 230000005494 condensation Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 45
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- 238000004062 sedimentation Methods 0.000 claims description 14
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 11
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 11
- 229920001184 polypeptide Polymers 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 150000003053 piperidines Chemical class 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- 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 claims description 2
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 claims 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 claims description 2
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 claims description 2
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 claims description 2
- AZBCBZPTQNYCPF-UHFFFAOYSA-N 3-(1-benzylimidazol-4-yl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)NC(C(=O)O)CC(N=C1)=CN1CC1=CC=CC=C1 AZBCBZPTQNYCPF-UHFFFAOYSA-N 0.000 claims description 2
- -1 Fmoc-Leu- OH Chemical compound 0.000 claims description 2
- 239000012190 activator Substances 0.000 claims description 2
- 229920003180 amino resin Polymers 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 238000010532 solid phase synthesis reaction Methods 0.000 abstract description 4
- 239000011435 rock Substances 0.000 description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 19
- 239000007788 liquid Substances 0.000 description 13
- 239000002994 raw material Substances 0.000 description 12
- 230000003252 repetitive effect Effects 0.000 description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 7
- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 description 6
- 230000008961 swelling Effects 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical class C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 4
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 4
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 238000009777 vacuum freeze-drying Methods 0.000 description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940046085 endocrine therapy drug gonadotropin releasing hormone analogues Drugs 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 229930182833 estradiol Natural products 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 125000005519 fluorenylmethyloxycarbonyl group Chemical group 0.000 description 1
- 230000001456 gonadotroph Effects 0.000 description 1
- 229960002193 histrelin Drugs 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- HHXHVIJIIXKSOE-UHFFFAOYSA-N n-[1-[[1-[[1-[[1-[[3-(1-benzylimidazol-4-yl)-1-[[1-[[5-(diaminomethylideneamino)-1-[2-(ethylcarbamoyl)pyrrolidin-1-yl]-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1 Chemical compound CCNC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CC=1C=CC(O)=CC=1)NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC(N=C1)=CN1CC1=CC=CC=C1 HHXHVIJIIXKSOE-UHFFFAOYSA-N 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 201000010065 polycystic ovary syndrome Diseases 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 208000010485 smooth muscle tumor Diseases 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The method that the present invention discloses a species specificity synthesis group amine Rayleigh, includes the following steps: using chlorine resin as initial resin carrier, dissolves mercaptoethylmaine with basic solvent, mixes under constant temperature conditions with chlorine resin, reaction overnight, obtains ethylamine chlorine resin；The methanol and DIEA of isometric ratio are added in ethylamine chlorine resin, carries out end socket reaction；Ethylamine chlorine resin after washing end socket with DMF, the Fmoc protected amino acid after then successively investment activates into reaction vessel, carries out continuous peptide chain condensation reaction；The chlorine resin of reaction is handled, with microwave technology with the peptide chain after DMF solution washing reaction-chlorine resin；Full guard Supprelin-chlorine resin is subjected to cleavage reaction with cutting reagent, obtains Supprelin crude product peptide.The present invention uses Fmoc solid-phase synthesis, enormously simplifies process flow, shortens the production time, reduce production cost, improves reaction condensation efficiency, and crude yield has reached 90%, has the considerable market competitiveness and application prospect.
Description
Technical field
The invention belongs to the synthesis preparation method technical fields of polypeptide drugs, are related to a kind of synthetic method of Supprelin,
In particular to the method for a species specificity synthesis group amine Rayleigh.
Background technique
Chinese: Supprelin
English name: Histrelin
Structural formula: Pyr-His-Trp-Ser-Tyr-D-His (Bzl)-Leu-Arg-Pro-NHEt
Molecular formula: C66H86N18O12
Molecular weight: 1323.52
Supprelin is a kind of nonapeptide of artificial chemistry synthesis, is a kind of non-peptide gonadotropin releasing hormone analogues,
For treating central puberty precocity, it can also be used to treat mullerianosis, polycystic ovarian disease, uterine smooth muscle
Tumor can treat serious premenopausal syndrome with estrogen or progesterone use in conjunction.
Supprelin is more more effective than endogenous gonadotropin-releasing hormone (GRH), and gonadotropin-releasing hormone (GRH) is used for a long time
Agonist can reduce the number of pituitary gonadotropic hormone receptor, inhibit reaction of the promoting sexual gland hormone to receptor for stimulating.Clinical research
Show that the Supprelin for giving daily dosage appropriate can make promoting sexual gland hormone water in the boy with central puberty precocity
Flat decline and the decline of testicular nucleic acid amount, and estradiol concentration then drops to prepuberal concentration in girl's serum, ischomenia,
Linear growth, skeletal maturation is slack-off and adult height increases.
The method of synthesis Supprelin currently on the market, needs by cleavage reaction twice or more, technique is numerous more
It is trivial, yield is low, and high production cost, the wilderness demand for being unfavorable for Supprelin and quickly production.
Summary of the invention
The purpose of the present invention is to provide the method for a species specificity synthesis group amine Rayleigh, which is condensed yield
Height, simple process period are short, at low cost, have extensive market application prospect.
The purpose of the present invention can be achieved through the following technical solutions:
The method of one species specificity synthesis group amine Rayleigh, this method comprise the following specific steps that:
(1) using chlorine resin as initial resin carrier, with basic solvent dissolve mercaptoethylmaine, under constant temperature conditions with chlorine resin
Mixing, reaction overnight, obtain ethylamine chlorine resin；
(2) methanol and DIEA of isometric ratio are added in ethylamine chlorine resin, carries out end socket reaction, reaction time 20-
30min；
(3) the ethylamine chlorine resin after washing end socket with DMF, the Fmoc after then successively investment activates into reaction vessel
Protected amino acid carries out continuous peptide chain condensation reaction using 20% piperidines/DMF solution as deprotecting regent；
(4) chlorine of reaction is handled with microwave technology after the Fmoc protected amino acid reaction 20-30min of investment condensation every time
Resin, with the peptide chain after DMF solution washing reaction-chlorine resin；
(5) full guard Supprelin-chlorine resin is subjected to cleavage reaction with cutting reagent, the second being pre-chilled with -20 DEG C of low temperature
Ether is settled, and Supprelin crude product peptide is obtained.
In the technical solution of above-mentioned specificity synthesis Supprelin, step (1) the chlorine resin is preferably 2-Cl
(Trt)-Cl Resin；Using DCM/DIEA as solvent in the ethylamine reaction of chlorine resin, reacted under 30 DEG C of constant temperatures
Night.
Step (2) the end socket reaction, the end socket reagent of reaction is methanol: the mixing of DIEA=1:1 (volume ratio) configuration
Solution, reaction vessel, which is placed on shaking table, rocks reaction, shaking speed 20-30r/min.
Fmoc- protected amino acid described in step (3) is followed successively by Fmoc-Pro-OH, Fmoc-Arg (pbf)-OH, Fmoc-
Leu-OH、Fmoc-D-His(Bzl)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Trp(Boc)-OH、
Fmoc-His (Trt)-OH, Fmoc-Pyr-OH, reaction temperature is 28 DEG C in the condensation reaction, reaction time 20-60min.
The condensation reaction of step (3) the Supprelin peptide chain, using DIC/HOBT as condensing agent and activator in reaction, with
The chlorine resin of DMF solution washing reaction, reaction time 20-60min, reaction temperature are 28 DEG C of -30 DEG C of conditions.
Step (4) microwave technology reaction, reaction condition refer to after the normal condensation reaction of Fmoc- amino acid, to anti-
It answers and suitable DIEA is added in container, then by amino resins microwave 15s, microwave power 25%.
Cutting, the sedimentation reaction of step (5) full guard Supprelin-chlorine resin, cutting reagent TFA:TIS:H2O
=95:2.5:2.5 (volume ratio), every gram of resin use 8-10ml cutting reagent, and low temperature ether is added to the polypeptide after cutting
In, centrifugal sedimentation is carried out, Supprelin crude product peptide is obtained.
Beneficial effects of the present invention: the present invention uses Fmoc solid-phase synthesis, using 2-Cl resin as solid phase carrier, in alkalinity
Under the conditions of, the ethylamine reaction of chlorine resin is carried out, then using the HOBT/DIC of low cost as condensing agent, under specific microwave condition
It is successively condensed Fmoc- protected amino acid according to Supprelin peptide chain sequence, obtains full guard Supprelin peptide chain-chlorine resin；With
Cutting reagent and ether are cut, are settled, and Supprelin crude product peptide is obtained.The present invention enormously simplifies process flow, shortens
Production time, production cost is reduced, reaction condensation efficiency is improved, crude yield has reached 90%, has considerable
The market competitiveness and application prospect.
Specific embodiment
It will be helpful to understand the present invention by following embodiments, but the contents of the present invention cannot be limited.For institute of the present invention
Belong to those skilled in the art for, without departing from the inventive concept of the premise, can also make several simple replacements or
It deduces, is all considered as belonging to protection scope of the present invention.
The method of one species specificity synthesis group amine Rayleigh, using Fmoc solid-phase synthesis, selecting 2-Cl resin is that starting carries
Body carries out the ethylamine reaction of chlorine resin under alkaline condition, then using the DIC/HOBT of low cost as condensing agent, in specificity
It is successively condensed Fmoc- protected amino acid according to Supprelin peptide chain sequence under microwave condition, obtains full guard Supprelin peptide chain-
Chlorine resin；Full guard Supprelin peptide chain-chlorine resin is cut, is settled, Supprelin crude product peptide is obtained.
Embodiment 1
The swelling of 1.2-Cl resin
Weighing degree of substitution is the 2-Cl resin 0.1g of 0.9-1.1mmol/g in Peptide systhesis reactor, and DCM solvent is added
So that it is totally submerged chlorine resin, is swollen 30min.
2. chlorine resin is ethylamine
10mg mercaptoethylmaine is weighed, is dissolved with 1ml DCM, 0.2ml DIEA is added and is completely dissolved, is then added to
In chlorine resin after swelling, reacted overnight at 30 DEG C.
3. the end socket of ethylamine chlorine resin reacts
0.5ml methanol and 0.5ml DIEA solution are added into reaction vessel, making the resin in container in the solution is in stream
Sand-like state, reaction vessel, which is placed on shaking table, rocks reaction 20min.
4. the synthesis of full guard Supprelin peptide chain-chlorine resin
3ml DMF solution is added into reaction vessel, rocks washing 1min, then drains the liquid in reaction vessel,
3ml DMF solution washing resin is added, rocks and washs 1min, repetitive operation 4 times.
The Fmoc-Pro-OH raw material of 41mg and the HOBT raw material of 20mg are weighed, is dissolved with 2ml DMF, 0.5ml is added
DIC solution is completely dissolved, and is then added in reaction vessel, is reacted 35min under the conditions of 28 DEG C, is drained, obtains Fmoc-
Pro-NHEt-2-Cl resin.
3ml DMF solution is added into reaction vessel, rocks washing 1min, then drains the liquid in reaction vessel,
3ml DMF solution washing resin is added, washing 1min is rocked, repetitive operation 3 times, drains.
20% piperidines of 4ml/DMF solution is added into reaction vessel, rocks reaction 20min, drains；Then 3ml is added
DMF solution rocks washing 1min, then drains the liquid in reaction vessel, add 3ml DMF solution into reaction vessel
Resin is washed, washing 1min is rocked, repetitive operation 4 times, drains.
Fmoc-Arg (the pbf)-OH raw material of 78mg and the HOBT raw material of 20mg are weighed, is dissolved with 2ml DMF, is added
0.5ml DIC solution is completely dissolved, and is then added in reaction vessel, is reacted 35min under the conditions of 28 DEG C, is drained, obtains
Fmoc-Arg (pbf)-Pro-NHEt-2-Cl resin.
According to aforesaid operations method, the 3-9, end of 2-Cl resin Fmoc- amino acid is successively put into, condensation reaction obtains
Fmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tbu)-Tyr(tbu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-
NHEt-2-Cl resin, is drained.
Protected amino acid used in the present invention starts the corresponding protected amino acid of the 1-9 amino acid from 2-Cl resin
And molecular weight is as shown in the table:
Some common abbreviations have following meanings in the present invention
2-Cl resin: 2- chlorine trityl chloride resin
DIC: diisopropylcarbodiimide
HOBT:1- hydroxybenzotriazole
Fmoc: fluorenylmethyloxycarbonyl
DIEA:N, N- diisopropylethylamine
DMF:N, dinethylformamide
DCM: methylene chloride
TFA: trifluoroacetic acid
TIS: tri isopropyl silane
5. the cutting of full guard Supprelin peptide chain-chlorine resin settles
Configuration cuts reagent TFA:TIS:H2The mixed solution of O=4.75ml:0.125ml:0.125ml, is added to and drains
In reaction vessel afterwards, reaction 40-50min is rocked.Two 10ml centrifuge tubes are taken, 6ml is separately added into and is pre-chilled under the conditions of -20 DEG C
Ether reagent, then cutting liquid is averagely added in centrifuge tube, is mixed well, then carry out centrifugal sedimentation reaction；Sedimentation
Centrifuge tube is taken out after primary, then adds 8ml low temperature ether, is mixed, centrifugal sedimentation, repetitive operation 2 times；Sediment is collected,
Vacuum freeze drying obtains Supprelin crude product peptide.
6. the yield of Supprelin crude product peptide calculates
The Supprelin total 62.5mg of crude product peptide after weighing freeze-drying is computed crude yield and (surveys resin to take for 82.2%
Dai Du is 0.4mmol/g), reach 70% with high-efficient liquid phase chromatogram technique analysis crude product purity, the receipts with the prior art only 50-70%
Rate and the crude product purity of 50-65% compare, and product yield is significantly increased, and product purity also greatly improves.
Embodiment 2
The swelling of 1.2-Cl resin
Weighing degree of substitution is the 2-Cl resin 0.1g of 0.9-1.1mmol/g in Peptide systhesis reactor, and DCM solvent is added
So that it is totally submerged chlorine resin, is swollen 30min.
2. chlorine resin is ethylamine
10mg mercaptoethylmaine is weighed, is dissolved with 1ml DCM, 0.2ml DIEA is added and is completely dissolved, is then added to
In chlorine resin after swelling, reacted overnight at 30 DEG C.
3. the end socket of ethylamine chlorine resin reacts
0.5ml methanol and 0.5ml DIEA solution are added into reaction vessel, making the resin in container in the solution is in stream
Sand-like state, reaction vessel, which is placed on shaking table, rocks reaction 20min.
4. the synthesis of full guard Supprelin peptide chain-chlorine resin
3ml DMF solution is added into reaction vessel, rocks washing 1min, then drains the liquid in reaction vessel,
3ml DMF solution washing resin is added, rocks and washs 1min, repetitive operation 4 times.
The Fmoc-Pro-OH raw material of 41mg and the HOBT raw material of 20mg are weighed, is dissolved with 2ml DMF, 0.5ml is added
DIC solution is completely dissolved, and is then added in reaction vessel, after reacting 25min under the conditions of 28 DEG C, is added 0.5ml's
DIEA, microwave peptide chain-chlorine resin 15s, the DMF solution that 3ml is added into reaction vessel wash resin, drain, obtain Fmoc-
Pro-NHEt-2-Cl resin.
3ml DMF solution is added into reaction vessel, rocks washing 1min, then drains the liquid in reaction vessel,
3ml DMF solution washing resin is added, washing 1min is rocked, repetitive operation 3 times, drains.
20% piperidines of 4ml/DMF solution is added into reaction vessel, rocks reaction 20min, drains；Then 3ml is added
DMF solution rocks washing 1min, then drains the liquid in reaction vessel, add 3ml DMF solution into reaction vessel
Resin is washed, washing 1min is rocked, repetitive operation 4 times, drains.
Fmoc-Arg (the pbf)-OH raw material of 78mg and the HOBT raw material of 20mg are weighed, is dissolved with 2ml DMF, is added
0.5ml DIC solution is completely dissolved, and is then added in reaction vessel, after reacting 25min under the conditions of 28 DEG C, is added
The DIEA of 0.5ml, microwave peptide chain-chlorine resin 15s, the DMF solution that 3ml is added into reaction vessel wash resin, drain, obtain
Fmoc-Arg (pbf)-Pro-NHEt-2-Cl resin.
According to aforesaid operations method, the 3-9, end of 2-Cl resin Fmoc- amino acid is successively put into, condensation reaction obtains
Fmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tbu)-Tyr(tbu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-
NHEt-2-Cl resin, is drained.
5. the cutting of full guard Supprelin peptide chain-chlorine resin settles
Configuration cuts reagent TFA:TIS:H2The mixed solution of O=4.75ml:0.125ml:0.125ml, is added to and drains
In reaction vessel afterwards, reaction 40-50min is rocked.Two 10ml centrifuge tubes are taken, 6ml is separately added into and is pre-chilled under the conditions of -20 DEG C
Ether reagent, then cutting liquid is averagely added in centrifuge tube, is mixed well, then carry out centrifugal sedimentation reaction；Sedimentation
Centrifuge tube is taken out after primary, then adds 8ml low temperature ether, is mixed, centrifugal sedimentation, repetitive operation 2 times；Sediment is collected,
Vacuum freeze drying obtains Supprelin crude product peptide.
6. the yield of Supprelin crude product peptide calculates
The Supprelin total 68.9mg of crude product peptide after weighing freeze-drying, is computed the polypeptide crude yield after microwave treatment
For 90.7% (actual measurement resin degree of substitution is 0.4mmol/g), 8.5 are higher by without the polypeptide yield of microwave treatment than embodiment 1
Percentage point；Reach 71% with high-efficient liquid phase chromatogram technique analysis crude product purity.
Embodiment 3
The swelling of 1.2-Cl resin
Weighing degree of substitution is the 2-Cl resin 1g of 0.9-1.1mmol/g in Peptide systhesis reactor, and DCM solvent, which is added, to be made
It is totally submerged chlorine resin, is swollen 40min.
2. chlorine resin is ethylamine
100mg mercaptoethylmaine is weighed, is dissolved with 12ml DCM, 4ml DIEA is added and is completely dissolved, is then added to
In chlorine resin after swelling, reacted overnight at 30 DEG C.
3. the end socket of ethylamine chlorine resin reacts
Resin in reaction vessel is drained, 10ml methanol and 10ml DIEA solution is then added, makes the resin in container
It is in the solution in drift sand state, reaction vessel, which is placed on shaking table, rocks reaction 20min.
4. the synthesis of full guard Supprelin peptide chain-chlorine resin
30ml DMF solution is added into reaction vessel, rocks washing 1min, then drains the liquid in reaction vessel,
30ml DMF solution washing resin is added, rocks and washs 1min, repetitive operation 4 times.
The Fmoc-Pro-OH raw material of 410mg and the HOBT raw material of 200mg are weighed, is dissolved with 20ml DMF, 3ml is added
DIC solution is completely dissolved, and is then added in reaction vessel, and after reacting 30min under the conditions of 28 DEG C, the DIEA of 3ml is added,
Microwave peptide chain-chlorine resin 15s, the DMF solution that 30ml is added into reaction vessel wash resin, drain, obtain Fmoc-Pro-
NHEt-2-Cl resin.
30ml DMF solution is added into reaction vessel, rocks washing 1min, then drains the liquid in reaction vessel,
30ml DMF solution washing resin is added, washing 1min is rocked, repetitive operation 3 times, drains.
20% piperidines of 40ml/DMF solution is added into reaction vessel, rocks reaction 20min, drains；Then 30ml is added
DMF solution rocks washing 1min, then drains the liquid in reaction vessel, it is molten to add 30ml DMF into reaction vessel
Liquid washs resin, rocks washing 1min, repetitive operation 4 times, drains.
Fmoc-Arg (the pbf)-OH raw material of 780mg and the HOBT raw material of 200mg are weighed, is dissolved with 20ml DMF, is added
3ml DIC solution is completely dissolved, and is then added in reaction vessel, after reacting 30min under the conditions of 28 DEG C, is added 3ml's
DIEA, microwave peptide chain-chlorine resin 15s, the DMF solution that 30ml is added into reaction vessel wash resin, drain, obtain Fmoc-
Arg (pbf)-Pro-NHEt-2-Cl resin.
According to aforesaid operations method, the 3-9, end of 2-Cl resin Fmoc- amino acid is successively put into, condensation reaction obtains
Fmoc-Pyr-His(Trt)-Trp(Boc)-Ser(tbu)-Tyr(tbu)-D-His(Bzl)-Leu-Arg(pbf)-Pro-
NHEt-2-Cl resin, is drained.
5. the cutting of full guard Supprelin peptide chain-chlorine resin settles
Configuration cuts reagent TFA:TIS:H2The mixed solution of O=38ml:1ml:1ml is added to the appearance of the reaction after draining
In device, reaction 50-60min is rocked.Five 50ml centrifuge tubes are taken, the ether examination that 35ml is pre-chilled under the conditions of -20 DEG C is separately added into
Then cutting liquid is averagely added in centrifuge tube, mixes well by agent, then carry out centrifugal sedimentation reaction；It is taken after sedimentation is primary
Then centrifuge tube out adds 35ml low temperature ether, mix, centrifugal sedimentation, repetitive operation 3 times；Collect sediment, vacuum refrigeration
It is dry, obtain Supprelin crude product peptide.
6. the yield of Supprelin crude product peptide calculates
The Supprelin total 681.7mg of crude product peptide after weighing freeze-drying, is computed, after output amplifies 10 times, by microwave
Treated, and polypeptide crude yield is 89.7% (actual measurement resin degree of substitution is 0.4mmol/g), more with 2 small-scale production of embodiment
The yield 90.7% of peptide is not much different；Reach 68% with high-efficient liquid phase chromatogram technique analysis crude product purity；Confirm present invention experiment number
According to stability and correctness.
It is tested through above-described embodiment 1 and example 2, obtains after normal condensation reaction, then carry out microwave technology reaction, synthesize
The yield of the Supprelin crude product peptide arrived is higher, better effect.
The present invention uses Fmoc solid-phase synthesis, introduces innovative technology, under alkaline condition, it is ethylamine to carry out chlorine resin
Reaction, and it is successively condensed Fmoc- protected amino acid using specific microwave reaction, it is anti-that product is carried out disposable cutting sedimentation
It answers, obtains the thick peptide of Supprelin；Process flow is enormously simplified, the yield of target product is improved, is conducive to quickly, at low cost
Large-scale production.
Above content is only citing made for the present invention and explanation, affiliated those skilled in the art are to being retouched
The specific embodiment stated does various modifications or additions or is substituted in a similar manner, and without departing from invention or surpasses
More range defined in the claims, is within the scope of protection of the invention.
Claims (1)
1. the method for a species specificity synthesis group amine Rayleigh, which is characterized in that method includes the following steps:
(1) using chlorine resin as initial resin carrier, mercaptoethylmaine is dissolved with basic solvent, it is mixed with chlorine resin under constant temperature conditions
It closes, reaction overnight, obtains ethylamine chlorine resin；
(2) methanol and DIEA of isometric ratio are added in ethylamine chlorine resin, carries out end socket reaction, reaction time 20-
30min；
(3) the ethylamine chlorine resin after washing end socket with DMF, the then successively Fmoc protection into reaction vessel after investment activation
Amino acid carries out continuous peptide chain condensation reaction using 20% piperidines/DMF solution as deprotecting regent；
(4) the chlorine resin of reaction is handled with microwave technology after the Fmoc protected amino acid reaction 20-30min of investment condensation every time,
With the peptide chain after DMF solution washing reaction-chlorine resin；
(5) full guard Supprelin-chlorine resin is subjected to cleavage reaction with cutting reagent, with -20 DEG C of low temperature be pre-chilled ether into
Row sedimentation, obtains Supprelin crude product peptide；
Step (1) the chlorine resin is 2-Cl (Trt)-Cl Resin；It is with DCM/DIEA in the ethylamine reaction of chlorine resin
Solvent reacts overnight under 30 DEG C of constant temperatures；
Step (2) the end socket reaction reagent volume ratio is methanol: the mixed solution of DIEA=1:1 configuration, reaction vessel are placed in
Reaction, shaking speed 20-30r/min are rocked on shaking table；
Fmoc- protected amino acid described in step (3) is followed successively by Fmoc-Pro-OH, Fmoc-Arg (pbf)-OH, Fmoc-Leu-
OH、Fmoc-D-His(Bzl)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Trp(Boc)-OH、
Fmoc-His (Trt)-OH, Fmoc-Pyr-OH, reaction temperature is 28 DEG C in the condensation reaction, reaction time 20-60min；
Using DIC/HOBT as condensing agent and activator in the condensation reaction of step (3) the Supprelin peptide chain, washed with DMF solution
The chlorine resin of reaction, reaction time 20-60min are washed, reaction temperature is 28 DEG C of -30 DEG C of conditions；
Step (4) the microwave technology reaction condition refers to after the normal condensation reaction of Fmoc- amino acid, adds into reaction vessel
Enter suitable DIEA, then by amino resins microwave 15s, microwave power 25%；
The cutting reagent volume ratio of step (5) full guard Supprelin-chlorine resin is TFA:TIS:H2O=95:2.5:
2.5, every gram of resin uses 8-10ml cutting reagent, and low temperature ether is added in the polypeptide after cutting, centrifugal sedimentation is carried out, obtains
To Supprelin crude product peptide.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699404A (en) * | 2005-05-17 | 2005-11-23 | 南京工业大学 | Process for solid-phase microwave synthesis of polypeptide |
| CN102850437A (en) * | 2012-09-12 | 2013-01-02 | 上海吉尔多肽有限公司 | Histrelin synthesizing method |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699404A (en) * | 2005-05-17 | 2005-11-23 | 南京工业大学 | Process for solid-phase microwave synthesis of polypeptide |
| CN102850437A (en) * | 2012-09-12 | 2013-01-02 | 上海吉尔多肽有限公司 | Histrelin synthesizing method |
Non-Patent Citations (2)
| Title |
|---|
| Enhanced Coupling Efficiency in Solid-Phase Peptide Synthesis by Microwave Irradiation;Hui Ming Yu et al.;《The Journal of Organic Chemistry》;19920828;4781-4784 |
| 微波作用下醋酸亮丙瑞林的固相合成;张俊等;《化学试剂》;20090630;401-404 |
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