CN112062811A - Synthetic method of vilacatide - Google Patents
Synthetic method of vilacatide Download PDFInfo
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- CN112062811A CN112062811A CN201910496387.7A CN201910496387A CN112062811A CN 112062811 A CN112062811 A CN 112062811A CN 201910496387 A CN201910496387 A CN 201910496387A CN 112062811 A CN112062811 A CN 112062811A
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- 238000010189 synthetic method Methods 0.000 title description 3
- 229920005989 resin Polymers 0.000 claims abstract description 104
- 239000011347 resin Substances 0.000 claims abstract description 104
- 238000006243 chemical reaction Methods 0.000 claims abstract description 82
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 53
- 238000000034 method Methods 0.000 claims abstract description 31
- 239000007790 solid phase Substances 0.000 claims abstract description 30
- 125000006239 protecting group Chemical group 0.000 claims abstract description 15
- 229920003002 synthetic resin Polymers 0.000 claims abstract description 14
- 239000000057 synthetic resin Substances 0.000 claims abstract description 14
- 238000010168 coupling process Methods 0.000 claims abstract description 13
- 238000005859 coupling reaction Methods 0.000 claims abstract description 13
- 230000008878 coupling Effects 0.000 claims abstract description 12
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000018417 cysteine Nutrition 0.000 claims abstract description 10
- 238000005336 cracking Methods 0.000 claims abstract description 9
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 9
- 239000003792 electrolyte Substances 0.000 claims abstract description 7
- 238000006056 electrooxidation reaction Methods 0.000 claims abstract description 6
- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 238000011097 chromatography purification Methods 0.000 claims abstract description 3
- 238000005406 washing Methods 0.000 claims description 33
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 27
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 20
- HNICLNKVURBTKV-MUUNZHRXSA-N (2r)-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-MUUNZHRXSA-N 0.000 claims description 12
- 235000001014 amino acid Nutrition 0.000 claims description 12
- 150000001413 amino acids Chemical class 0.000 claims description 12
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 12
- -1 tetrabutylammonium tetrafluoroborate Chemical compound 0.000 claims description 10
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- QWXZOFZKSQXPDC-LLVKDONJSA-N (2r)-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-LLVKDONJSA-N 0.000 claims description 8
- 239000007822 coupling agent Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 238000003746 solid phase reaction Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 238000003776 cleavage reaction Methods 0.000 claims description 6
- 230000003647 oxidation Effects 0.000 claims description 6
- 238000007254 oxidation reaction Methods 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- 229910052697 platinum Inorganic materials 0.000 claims description 6
- 230000007017 scission Effects 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- VXGGBPQPMISJCA-STQMWFEESA-N (2s)-2-[[(2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoyl]amino]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 VXGGBPQPMISJCA-STQMWFEESA-N 0.000 claims description 4
- 230000005526 G1 to G0 transition Effects 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 230000002441 reversible effect Effects 0.000 claims description 3
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 claims description 2
- 238000003487 electrochemical reaction Methods 0.000 claims description 2
- 238000004007 reversed phase HPLC Methods 0.000 claims description 2
- KBLZDCFTQSIIOH-UHFFFAOYSA-M tetrabutylazanium;perchlorate Chemical compound [O-]Cl(=O)(=O)=O.CCCC[N+](CCCC)(CCCC)CCCC KBLZDCFTQSIIOH-UHFFFAOYSA-M 0.000 claims description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 abstract description 16
- 239000012634 fragment Substances 0.000 abstract description 7
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical group N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 36
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 33
- 239000000243 solution Substances 0.000 description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 238000010511 deprotection reaction Methods 0.000 description 18
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical group CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 17
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 16
- 239000000706 filtrate Substances 0.000 description 15
- 229910052757 nitrogen Inorganic materials 0.000 description 15
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 14
- 239000005457 ice water Substances 0.000 description 14
- 229960002433 cysteine Drugs 0.000 description 9
- AHDSRXYHVZECER-UHFFFAOYSA-N 2,4,6-tris[(dimethylamino)methyl]phenol Chemical compound CN(C)CC1=CC(CN(C)C)=C(O)C(CN(C)C)=C1 AHDSRXYHVZECER-UHFFFAOYSA-N 0.000 description 8
- 229940024606 amino acid Drugs 0.000 description 8
- 238000001556 precipitation Methods 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 230000005587 bubbling Effects 0.000 description 6
- 229910001873 dinitrogen Inorganic materials 0.000 description 6
- 230000004584 weight gain Effects 0.000 description 6
- 235000019786 weight gain Nutrition 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 238000000861 blow drying Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 229960001305 cysteine hydrochloride Drugs 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 238000005691 oxidative coupling reaction Methods 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 description 3
- 229940126062 Compound A Drugs 0.000 description 3
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 description 2
- 150000008574 D-amino acids Chemical class 0.000 description 2
- 239000007821 HATU Substances 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 208000005770 Secondary Hyperparathyroidism Diseases 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
- 150000001412 amines Chemical class 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- VDHAWDNDOKGFTD-MRXNPFEDSA-N cinacalcet Chemical compound N([C@H](C)C=1C2=CC=CC=C2C=CC=1)CCCC1=CC=CC(C(F)(F)F)=C1 VDHAWDNDOKGFTD-MRXNPFEDSA-N 0.000 description 2
- 229960003315 cinacalcet Drugs 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002848 electrochemical method Methods 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000001631 haemodialysis Methods 0.000 description 2
- 230000000322 hemodialysis Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- WHMDPDGBKYUEMW-UHFFFAOYSA-N pyridine-2-thiol Chemical class SC1=CC=CC=N1 WHMDPDGBKYUEMW-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000002092 calcimimetic effect Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 208000012839 conversion disease Diseases 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- ANIAZGVDEUQPRI-ZJQCGQFWSA-N etelcalcetide Chemical compound NC(N)=NCCC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCCN=C(N)N)NC(=O)[C@@H](CCCN=C(N)N)NC(=O)[C@@H](CCCN=C(N)N)NC(=O)[C@@H](C)NC(=O)[C@@H](CSSC[C@H](N)C(O)=O)NC(C)=O ANIAZGVDEUQPRI-ZJQCGQFWSA-N 0.000 description 1
- 229950006502 etelcalcetide Drugs 0.000 description 1
- 108091022127 etelcalcetide hydrochloride Proteins 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical group CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a preparation method of vilacatide, which comprises the following steps: 1) preparing fully-protected vilacatide main chain-solid phase synthetic resin by a solid phase synthesis method; 2) cracking to remove side chain protecting group P of Cys; 3) solid-phase electrochemical oxidation, coupling cysteine through disulfide bond; 4) cracking all protecting groups and the solid-phase synthetic resin to obtain crude vilacatide; optionally, 5) carrying out chromatographic purification to obtain the vilacatide refined peptide; wherein, the solid-phase electrochemical oxidation method is that the polypeptide-solid-phase synthetic resin which is obtained in the step 2) and is removed with the side chain protecting group of Cys is electrolyzed in electrolyte and solution until the disulfide bond reaction is completed; p is a side chain protecting group of Cys. The method can improve the utilization rate of the fragment peptide resin, and improve the utilization rate of the main chain fragment and the atom utilization rate, thereby reducing the production cost, avoiding the use of the 2, 2-dipyridyl disulfide compound in the original patent, being more environment-friendly and advocating the concept of green chemistry.
Description
Technical Field
The invention relates to the field of pharmaceutical chemistry, and particularly relates to a synthetic method of vilacatide.
Background
Vilacatide (etelcalcide) is a calcium mimetic agent developed by AMGEN INC, and is mainly used as a polypeptide drug for secondary hyperparathyroidism in hemodialysis treatment of adult patients with chronic kidney diseases. Marketed in 2017 in the united states on day 07 of month 2 under the trade name Parsabiv. It is as good as cinacalcet in reducing parathyroid hormone levels when used in secondary hyperparathyroidism, and is superior to the existing standard calcimimetic therapeutic agent cinacalcet because it can be administered intravenously after hemodialysis.
The main chain of the vilacatide consists of seven D-type amino acids, and the side chain is connected with the L-cysteine through a disulfide bond. The peptide sequence is as follows:
peptide sequence of vilaca peptide
The key point of the conventional method for coupling and synthesizing the vilacatide is the construction of a disulfide bond, and the methods for constructing the disulfide bond used in the conventional peptide synthesis comprise an air oxidation method, an iodine/acetic acid system oxidation method and a hydrogen peroxide oxidation method, which cannot ensure the specific selectivity of the reaction, inevitably generate mismatched impurities, have low product purity and increase the purification difficulty. Patent CN105504012 introduces a pseudo-dilution reaction to avoid the problem of polypeptide inter-chain disulfide bond mismatching in liquid phase reaction, but the solid phase disulfide bond forming reaction is heterogeneous reaction, the reaction conversion rate is low, and the yield of the product vilacatide is lower than 30%. Patent CN106928320 and patent CN201580029560.2 report methods for synthesizing vilacatide by using 2, 2-dipyridyl disulfide activation method, but the generation of mercaptopyridine compounds with equivalent weight not only increases the purification difficulty, but also causes environmental pollution, and is not beneficial to environmental protection. Patent CN106928321 introduces a method for synthesizing vilacatide in a full liquid phase, but the treatment operation after reaction is complicated, and the total yield is low.
The development of atom economy reaction and environmental protection become the core content of green chemical research, and from the atom economy point of view, the effective utilization rate of seven D-amino acid main chain fragments is low, namely the atom utilization rate is low when the last L-type cysteine disulfide bond is constructed due to the fact that the conventional sequential coupling method cannot avoid the problem of specific selectivity of the construction of the disulfide bond of the linear peptide. In a general solid-phase synthesis operation process, the using amount of amino acid is generally 3-5 eqv, when the effective utilization rate of a main chain fragment is low, a large amount of corresponding amino acid is wasted, the price of D-amino acid is high, and the atom utilization rate is necessary to be improved, so that the production cost is reduced, and the energy waste is reduced. The adoption of the 2, 2-dipyridyl disulfide activation method for synthesizing the vilacatide can cause the generation of a large amount of mercaptopyridine byproducts, cause environmental pollution and be not beneficial to environmental protection.
Disclosure of Invention
In view of the above situation, an environment-friendly and efficient preparation method of vilaca peptide is needed, the invention uses Fmoc/tBu strategy to connect main chain amino acid residues in a solid phase manner, then uses an electrochemical method to couple side chain cysteine in a solid phase manner, and finally obtains the vilaca peptide by cracking and precipitation. Therefore, the problem of specific selectivity of the reaction can be avoided, the utilization rate of the main chain fragments is improved, the utilization rate of atoms is improved, the production cost is reduced, the use of a 2, 2-dithiodipyridine compound is avoided, the environment is protected, and the green chemical concept is met. The reaction process is as follows:
one aspect of the present invention provides a preparation method of vilacatide, which comprises the following steps:
1) preparing a fully-protected Vila peptide backbone-solid phase synthetic resin by a solid phase synthesis method, namely Ac-D-Cys (P) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -solid phase synthetic resin;
2) cracking to remove side chain protecting group P of Cys;
3) solid-phase electrochemical oxidation, coupling cysteine through disulfide bond;
4) cracking all protecting groups and the solid-phase synthetic resin to obtain crude vilacatide;
optionally, 5) carrying out chromatographic purification to obtain the vilacatide refined peptide;
wherein, the solid-phase electrochemical oxidation method is that the polypeptide-solid-phase synthetic resin which is obtained in the step 2) and is removed with the side chain protecting group of Cys is electrolyzed in electrolyte and solution until the disulfide bond reaction is completed;
p is a side chain protecting group of Cys.
In the technical scheme of the invention, the electrochemical reaction temperature in the step 3) is 25-65 ℃, and preferably 40-60 ℃.
In the technical scheme of the invention, the electrolyte in the step 3) is selected from tetrabutylammonium perchlorate, tetrabutylammonium tetrafluoroborate and tetrabutylammonium hexafluorophosphate, and tetrabutylammonium tetrafluoroborate is preferred.
In the technical scheme of the invention, a platinum electrode is adopted for electrifying and oxidizing in the step 3), and the current intensity is 5-20 mA; preferably 10-15 mA.
In the technical scheme of the invention, the reaction time of the step 3) is 2-100 hours.
In the technical scheme of the invention, the solid phase synthetic Resin in the step 1) is Rink Amide AM Resin, Rink Amide MBHA Resin or Rink Amide Resin, and the substitution degree of the Resin is 0.1-1.0mmol/g, preferably 0.2-0.8mmol/g, and more preferably 0.3-0.5 mmol/g.
In the technical scheme of the invention, the preparation of the solid phase synthesis method in the step 1) refers to the coupling of Fmoc-AA-OH from the C end to the N end, the coupling sequence of the Fmoc-AA-OH is Fmoc-D-Arg (Pbf) -OH, Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-D-Ala-OH, Fmoc-Cys (P) -OH and then acetic acid.
In the technical scheme of the invention, P in Fmoc-Cys (P) -OH is Trt, Mmt or Dpm.
In the technical scheme of the invention, the solid-phase synthesis method comprises the following steps: a) removing Fmoc, and then washing the resin by using a solvent until the Fmoc is completely removed by using a detection method; b) dissolving amino acid (or acetic acid) to be coupled and a coupling agent in a solvent, activating, and adding the amino acid (or acetic acid) to be coupled and the coupling agent into a solid phase reaction column together until the reaction termination is detected by a detection method; c) repeating a) and b).
In the technical scheme of the invention, the Fmoc removing reagent is 20% piperidine/DMF solution (DBLK), namely piperidine: DMF (volume ratio) is 1: 4.
In the technical scheme of the invention, the coupling agent in the step a) is a composition of DIPCDI and a compound A, wherein the compound A is HOBt or HOAt, the compound B is PyBOP, PyAOP, HATU, HBTU or TBTU, and the composition of DIPCDI and the compound A is preferred.
In the technical scheme of the invention, the reaction in the step 1) is carried out in a solid-phase reaction column. The solid-phase reaction column is not particularly limited, and may be any solid-phase reaction column capable of achieving the object. Further, the time for the coupling reaction of each amino acid is usually 1.5 to 4 hours, preferably 2 to 3 hours; the pressure is preferably normal pressure, and may be suitably increased or decreased; the temperature is preferably room temperature (i.e., 20. + -. 5 ℃ C.), and may be suitably elevated or reduced.
The reaction of step 1) is preferably carried out by swelling the solid phase synthetic resin prior to coupling, said steps of washing and swelling being carried out in the art using any reagent which accomplishes this purpose, including DMF, NMP, dichloromethane and the like, preferably DMF. The detection method employed in the reaction is any method known in the art for this purpose, such as chromatography or chemical calibration, preferably using a reagent that can determine the end of the reaction, preferably ninhydrin, which when used indicates a free amine in the polypeptide if the resin develops color, i.e., no protecting group on the amine. The coupling agent is DIPCDI + A or DIPEA + A + B, wherein A is HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU and TBTU.
The removing agent used in the step 2) is a mixed solution of TFA/DCM, the volume ratio of the solution is 1% -10%, preferably 1% -5%, and the reaction end point is that the solution is changed from red to colorless.
The cracking liquid in the step 4) is TFA, TIS and H2A mixture of O; preferably TFA, TIS, H2O＝95:2.5:2.5。
And 5) adopting reverse-phase high-pressure liquid chromatography for the purification step. Further, the reversed-phase high-pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting target peak fraction, concentrating, and lyophilizing.
Ac-D-Cys (Mmt) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -solid phase synthetic resin, D represents D configuration amino acid, Mmt and Pbf are side chain protecting groups, and the main chain is coupled through alpha amino group and carboxyl group of the amino acid.
Abbreviations and English meanings
Advantageous effects
The invention adopts Fmoc/tBu strategy to connect main chain amino acid residues in a solid phase manner, then adopts an electrochemical method to carry out solid phase oxidation coupling on cysteine of a side chain, and then obtains the vilacatide through cracking, precipitation and purification. The method can improve the utilization rate of the fragment peptide resin, the utilization rate of the main chain fragment and the utilization rate of atoms, thereby reducing the production cost, avoiding the use of the 2, 2-dipyridyl disulfide compound in the original patent, being more environment-friendly and advocating the concept of green chemistry.
Detailed Description
Example 1: backbone peptide resin Synthesis
Rink Amide resin (52g, substitution 0.48mmol/g) was weighed into a solid phase reaction column, washed with DMF 2 times, the resin was then swelled with DMF for 30 min, DBLK was added for deprotection (5min +7min), and the resin was washed with DMF 6 times. Weighing Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g,90mmol), adding the mixture into DMF (250mL) for dissolving, cooling to 0-5 ℃ in an ice-water bath, adding DIPCDI (12.75mL, 75mmol) for activation for 5min, adding the activated solution into a reaction column, bubbling nitrogen gas for 2 h at room temperature, and detecting the reaction end point by ninhydrin (the reaction is stopped if the resin is colorless and transparent; and the reaction is prolonged for 1 h if the resin is colored). After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin. Fmoc-D-Ala-OH (23.372g, 75mmol) and HOBT (12.159g,90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice water bath, DIPCDI (12.75mL, 75mmol) was added to activate for 5min, the activated solution was added to the reaction column, and the reaction end point was detected by ninhydrin after bubbling nitrogen gas at room temperature for 2 h. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g,90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 hours, and the end point of the reaction was detected with ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin. Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g,90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 hours, and the end point of the reaction was detected with ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin. Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g,90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 hours, and the end point of the reaction was detected with ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin. Fmoc-D-Ala-OH (23.372g, 75mmol) and HOBT (12.159g,90mmol) were dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice water bath, DIPCDI (12.75mL, 75mmol) was added to activate for 5min, the activated solution was added to the reaction column, and the reaction end point was detected by ninhydrin after bubbling nitrogen gas at room temperature for 2 h. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. Fmoc-D-Cys (Mmt) -OH (46.180g, 75mmol) and HOBT (12.159g,90mmol) are added into DMF (250mL) to be dissolved, cooled to 0-5 ℃ in ice water bath, DIPCDI (12.75mL, 75mmol) is added to activate for 5min, the activated solution is added into a reaction column, nitrogen is bubbled for 2 h at room temperature, and the reaction end point is detected by ninhydrin. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. 70mL of acetic anhydride and 60mL of pyridine were added and nitrogen bubbled at room temperature for 2 hours, and the end of the reaction was detected with ninhydrin. After the reaction was complete, the resin was washed 6 times with DMF. After coupling, the resin was shrunk with methanol and vacuum-dried to obtain 80.0g of peptide resin, the resin weight gain was 80.5%.
Example 2: solid phase removal of cysteine protecting groups
To the peptide resin obtained in example 1 was added 2% TFA/CH2Cl2Solution 250mL removed Mmt protecting group. The reaction appeared red, the reaction was carried out for 2 minutes, then the solution was pumped out and repeated 10 times until the red color disappeared, and CH was used2Cl2After 3 washes, the column was washed 3 times with DMF.
Example 3: solid phase oxidative coupling of cysteine
The peptide resin obtained in example 2 was charged into a 1000ml three-necked flask, and DMF500ml, 8.2g (25mmol) of tetrabutylammonium tetrafluoroborate and 11.7g (75mmol) of cysteine hydrochloride monohydrate were sequentially added, followed by insertion of a platinum electrode (anode: 15 mm. times.15 mm. times.0.3 mm, cathode: 15 mm. times.15 mm. times.0.3 mm)), and reaction was stirred at 40 ℃ for 48 hours with a controlled current of 12 mA. After the reaction, the energization was stopped, the filtration was carried out, DMF washing was carried out 6 times, DCM washing was carried out 3 times, methanol shrinkage was carried out, and vacuum drying was carried out to obtain 94.0g of peptide resin, the resin weight gain was 79.5%.
Example 4: cleavage peptide resin
94.0g of the peptide resin obtained in example 3 was put into a 2L single-necked flask, and a lysate (TFA: TIS: H) prefreezed to-15 ℃ was added2O95: 2.5:2.5(V: V), 940ml), stirred at room temperature for 4 hours, filtered the resinAnd collecting the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 9.4L of glacial ethyl ether for precipitation. Centrifugation, washing with glacial ethyl ether 5 times, and nitrogen blow-drying to obtain 26.20 g of crude peptide with purity of 89.7% and yield of 99.92%.
Example 5: preparation of refined peptide by reversed phase chromatography
The crude peptide obtained in example 4 was purified by high performance liquid chromatography using reverse-phase octadecylsilane as a stationary phase and 0.1% trifluoroacetic acid aqueous solution/acetonitrile as a mobile phase, and the desired peak fraction was collected, concentrated and lyophilized. 16.8g of refined peptide is obtained, the purity is 99.98 percent, and the yield is 64.5 percent.
Example 6: primary method comparative example backbone peptide resin Synthesis
Rink Amide resin (52g, substitution 0.48mmol/g) was weighed into a solid phase reaction column, washed with DMF 2 times, the resin was then swelled with DMF for 30 min, DBLK was added for deprotection (5min +7min), and the resin was washed with DMF 6 times. Weighing Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g,90mmol), adding the mixture into DMF (250mL) for dissolving, cooling to 0-5 ℃ in an ice-water bath, adding DIPCDI (12.75mL, 75mmol) for activation for 5min, adding the activated solution into a reaction column, bubbling nitrogen gas for 2 h at room temperature, and detecting the reaction end point by ninhydrin (the reaction is stopped if the resin is colorless and transparent; and the reaction is prolonged for 1 h if the resin is colored). After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin. Fmoc-D-Ala-OH (23.372g, 75mmol) and HOBT (12.159g,90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice water bath, DIPCDI (12.75mL, 75mmol) was added to activate for 5min, the activated solution was added to the reaction column, and the reaction end point was detected by ninhydrin after bubbling nitrogen gas at room temperature for 2 h. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g,90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 hours, and the end point of the reaction was detected with ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin. Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g,90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 hours, and the end point of the reaction was detected with ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin. Fmoc-D-Arg (Pbf) -OH (48.660g, 75mmol) and HOBT (12.159g,90mmol) were weighed and dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice-water bath, and then DIPCDI (12.75mL, 75mmol) was added to activate for 5min, and the activated solution was put into a reaction column and bubbled with nitrogen at room temperature for 2 hours, and the end point of the reaction was detected with ninhydrin. After the reaction is finished, washing the resin with DMF 3 times, adding DBLK to perform deprotection for 5min +7min, washing the resin with DMF 6 times, and detecting the color of the resin by ninhydrin. Fmoc-D-Ala-OH (23.372g, 75mmol) and HOBT (12.159g,90mmol) were dissolved in DMF (250mL), cooled to 0-5 ℃ in an ice water bath, DIPCDI (12.75mL, 75mmol) was added to activate for 5min, the activated solution was added to the reaction column, and the reaction end point was detected by ninhydrin after bubbling nitrogen gas at room temperature for 2 h. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. Fmoc-D-Cys (Mmt) -OH (46.180g, 75mmol) and HOBT (12.159g,90mmol) are added into DMF (250mL) to be dissolved, cooled to 0-5 ℃ in ice water bath, DIPCDI (12.75mL, 75mmol) is added to activate for 5min, the activated solution is added into a reaction column, nitrogen is bubbled for 2 h at room temperature, and the reaction end point is detected by ninhydrin. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. After the reaction is finished, the resin is washed by DMF for 3 times, DBLK is added for deprotection for 5min +7min, the resin is washed by DMF for 6 times, and ninhydrin detects that the resin has color. 70mL of acetic anhydride and 60mL of pyridine were added and nitrogen bubbled at room temperature for 2 hours, and the end of the reaction was detected with ninhydrin. After the reaction was complete, the resin was washed 6 times with DMF. After coupling, the resin was shrunk with methanol and vacuum-dried to obtain 80.0g of peptide resin, the resin weight gain was 80.5%.
Example 7: comparative example of the original Process, cleavage and 2, 2-Dithiodipyridine activation
51.3g of the peptide resin obtained in example 6 was put into a 2L single-necked flask, and a lysate (TFA: TIS: H) prefreezed to-15 ℃ was added2O95: 2.5:2.5(V: V), 900ml) and 2, 2-dithiodipyridine (35.2g, 6.4eqv) were stirred at room temperature for 4 hours, the resin was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 9L of glacial ethyl ether for precipitation. Centrifuging, washing with glacial ethyl ether for 5 times, and drying by nitrogen to obtain 10.7g of crude peptide as an activated intermediate, wherein the purity of the crude peptide is 84.3 percent, and the yield is 91.06 percent.
Example 8: comparative example of the original Process, Oxidation to give crude peptide
The activated crude peptide from example 7 (10.7g) was added to 1.5% aqueous TFA (3.5L), L-Cys-OH (1.55g,1.1eqv) was added and stirred for 2 hours. The crude peptide was 40.31% pure by HPLC.
Example 9: comparative example of original research method, reverse phase chromatography for preparing refined peptide
The crude peptide obtained in example 7 was purified by HPLC to obtain 4.3g of refined peptide with a purity of 98.7% and a yield of 32.4%.
EXAMPLE 10 solid-phase oxidative coupling of cysteine (condition screening-temperature)
The peptide resin obtained in example 2 was charged into a 1000ml three-necked flask, and DMF500ml, 8.2g (25mmol) of tetrabutylammonium tetrafluoroborate and 11.7g (75mmol) of cysteine hydrochloride monohydrate were sequentially added, followed by insertion of a platinum electrode (anode: 15 mm. times.15 mm. times.0.3 mm, cathode: 15 mm. times.15 mm. times.0.3 mm)), and reaction was stirred at 60 ℃ for 48 hours with a controlled current of 12 mA. After the reaction, the energization was stopped, the filtration was carried out, DMF washing was carried out 6 times, DCM washing was carried out 3 times, methanol shrinkage was carried out, and vacuum drying was carried out to obtain 93.0g of peptide resin, the resin weight gain rate was 78.6%.
Example 11: cleavage peptide resin (Condition screening-temperature)
93.0g of the peptide resin obtained in example 10 was put into a 2L single-necked flask, and a lysate (TFA: TIS: H) prefreezed to-15 ℃ was added2O95: 2.5:2.5(V: V), 940ml), stirred at room temperature for 4 hours, the resin was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates combined.The filtrate was slowly added to 9.3L of glacial ethyl ether for precipitation. Centrifugation, washing with glacial ethyl ether 5 times, and nitrogen blow-drying gave 25.20 g of crude peptide, 90.1% pure, 96.18% yield.
EXAMPLE 12 solid-phase oxidative coupling of cysteine (Condition Screen-electrolyte)
The peptide resin obtained in example 2 was charged into a 1000ml three-necked flask, and DMF500ml, 9.3g (25mmol) of tetrabutylammonium hexafluorophosphate and 11.7g (75mmol) of cysteine hydrochloride monohydrate were sequentially added, followed by insertion of a platinum electrode (anode: 15 mm. times.15 mm. times.0.3 mm, cathode: 15 mm. times.15 mm. times.0.3 mm)), and the reaction was stirred at 40 ℃ for 48 hours with a current of 12 mA. After the reaction, the energization was stopped, the filtration was carried out, DMF washing was carried out 6 times, DCM washing was carried out 3 times, methanol shrinkage was carried out, and vacuum drying was carried out to obtain 94.1g of peptide resin with a resin weight gain of 79.5%.
Example 13: cleavage peptide resin (Condition screening-electrolyte)
94.1g of the peptide resin obtained in example 12 was put into a 2L single-neck bottle, a lysate (TFA: TIS: H2O ═ 95:2.5:2.5(V: V), 940ml) which was prefrozen to-15 ℃ was added, and the mixture was stirred at room temperature for 4 hours, followed by filtration of the resin and collection of the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 9.4L of glacial ethyl ether for precipitation. Centrifugation, washing with glacial ethyl ether 5 times, and nitrogen blow-drying gave 25.50 g of crude peptide, 89.80% pure, 97.30% yield.
EXAMPLE 14 solid-phase oxidative coupling of cysteine (conditional screen-Current)
The peptide resin obtained in example 2 was charged into a 1000ml three-necked flask, and DMF500ml, 8.2g (25mmol) of tetrabutylammonium tetrafluoroborate and 11.7g (75mmol) of cysteine hydrochloride monohydrate were sequentially added, followed by insertion of a platinum electrode (anode: 15 mm. times.15 mm. times.0.3 mm, cathode: 15 mm. times.15 mm. times.0.3 mm)), and reaction was stirred at 40 ℃ for 48 hours with a controlled current of 15 mA. After the reaction, the energization was stopped, the filtration was carried out, DMF washing was carried out 6 times, DCM washing was carried out 3 times, methanol shrinkage was carried out, and vacuum drying was carried out to obtain 93.9g of peptide resin with a resin weight gain of 79.5%.
Example 15: cleavage peptide resin (Condition screening-Current)
93.9g of the peptide resin obtained in example 14 was charged into a 2L single-necked flask, and a prefreezing to-15 ℃ split was addedHydrolyzed solution (TFA: TIS: H)2O95: 2.5:2.5(V: V), 940ml), stirred at room temperature for 4 hours, the resin was filtered, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 9.4L of glacial ethyl ether for precipitation. Centrifugation, washing with glacial ethyl ether 5 times, and nitrogen blow-drying gave 25.70 g of crude peptide, 89.7% pure, 98.12% yield.
Claims (10)
1. A preparation method of vilacatide comprises the following steps:
1) preparing a fully-protected Vila peptide backbone-solid phase synthetic resin by a solid phase synthesis method, namely Ac-D-Cys (P) -D-Arg (Pbf) -D-Ala-D-Arg (Pbf) -solid phase synthetic resin;
2) cracking to remove side chain protecting group P of Cys;
3) solid-phase electrochemical oxidation, coupling cysteine through disulfide bond;
4) cracking all protecting groups and the solid-phase synthetic resin to obtain crude vilacatide;
optionally, 5) carrying out chromatographic purification to obtain the vilacatide refined peptide;
wherein, the solid-phase electrochemical oxidation method is that the polypeptide-solid-phase synthetic resin which is obtained in the step 2) and is removed with the side chain protecting group of Cys is electrolyzed in electrolyte and solution until the disulfide bond reaction is completed;
p is a side chain protecting group of Cys, preferably Trt, Mmt or Dpm.
2. The method according to claim 1, wherein the electrochemical reaction temperature in step 3) is 25-65 ℃, preferably 40-60 ℃.
3. The production method according to any one of claims 1 to 2, wherein the electrolyte in step 3) is selected from the group consisting of tetrabutylammonium perchlorate, tetrabutylammonium tetrafluoroborate, tetrabutylammonium hexafluorophosphate, preferably tetrabutylammonium tetrafluoroborate.
4. The production method according to any one of claims 1 to 3, wherein the platinum electrode is used for the electrification and oxidation in the step 3), and the current intensity is 5 to 20 mA; preferably 10-15 mA.
5. The method of any one of claims 1 to 4, wherein the solid phase synthesis in step 1) is performed by coupling Fmoc-AA-OH in the order of C-terminus to N-terminus, Fmoc-AA-OH in the order of Fmoc-D-Arg (Pbf) -OH, Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-D-Ala-OH, Fmoc-Cys (P) -OH, and then coupling acetic acid.
6. The method of any one of claims 1 to 5, wherein the solid phase synthesis comprises: a) removing Fmoc, and then washing the resin by using a solvent until the Fmoc is completely removed by using a detection method; b) dissolving amino acid (or acetic acid) to be coupled and a coupling agent in a solvent, activating, and adding the amino acid (or acetic acid) to be coupled and the coupling agent into a solid phase reaction column together until the reaction termination is detected by a detection method; c) repeating a) and b).
7. The method of any one of claims 1 to 6, wherein the solid phase synthesis comprises: a) removing Fmoc, and then washing the resin by using a solvent until the Fmoc is completely removed by using a detection method; b) dissolving amino acid (or acetic acid) to be coupled and a coupling agent in a solvent, activating, and adding the amino acid (or acetic acid) to be coupled and the coupling agent into a solid phase reaction column together until the reaction termination is detected by a detection method; c) repeating a) and b).
8. The process according to any one of claims 1 to 7, wherein the stripping agent used in step 2) is a mixture of TFA/DCM in a volume ratio of 1% to 10%, preferably 1% to 5%, and the reaction is terminated by a change of the solution from red to colorless.
9. The method according to any one of claims 1 to 8, wherein the cleavage solution in step 4) is TFA, TIS or H2A mixture of O; preferably TFA, TIS, H2O＝95:2.5:2.5。
10. The method according to any one of claims 1 to 9, wherein the purification step of step 5) is performed by reverse phase high pressure liquid chromatography. Further, the reversed-phase high-pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting target peak fraction, concentrating, and lyophilizing.
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| CN112552376A (en) * | 2020-12-31 | 2021-03-26 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying vilacatide |
| CN114524860A (en) * | 2021-12-29 | 2022-05-24 | 深圳翰宇药业股份有限公司 | Synthesis method and application of Etelcalcetide |
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| DK3126373T3 (en) * | 2014-04-03 | 2020-05-04 | Amgen Inc | PROCEDURE FOR MANUFACTURING AMG 416 |
| CN105504012A (en) * | 2014-09-30 | 2016-04-20 | 深圳翰宇药业股份有限公司 | Preparation method of polypeptide |
| TW201915009A (en) * | 2017-10-03 | 2019-04-16 | 中化合成生技股份有限公司 | Method for synthesizing etelcalcetide or salts thereof |
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- 2019-08-20 WO PCT/CN2019/101609 patent/WO2020248360A1/en active Application Filing
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| CN112552376A (en) * | 2020-12-31 | 2021-03-26 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying vilacatide |
| CN114524860A (en) * | 2021-12-29 | 2022-05-24 | 深圳翰宇药业股份有限公司 | Synthesis method and application of Etelcalcetide |
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| CN112062811B (en) | 2022-03-25 |
| WO2020248360A1 (en) | 2020-12-17 |
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