CN116751278B - Preparation method of Glepaglutide - Google Patents
Preparation method of Glepaglutide Download PDFInfo
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- CN116751278B CN116751278B CN202310740246.1A CN202310740246A CN116751278B CN 116751278 B CN116751278 B CN 116751278B CN 202310740246 A CN202310740246 A CN 202310740246A CN 116751278 B CN116751278 B CN 116751278B
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- DOAUQKRTILFGHV-PDCMDPCFSA-N (4S)-5-[[2-[[(2S,3R)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S,3R)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S,3R)-1-[[(2S)-6-amino-1-[[(2S,3S)-1-[[(2S,3R)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-1,6-diamino-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-2-oxoethyl]amino]-4-[[2-[[(2S)-2-amino-3-(1H-imidazol-4-yl)propanoyl]amino]acetyl]amino]-5-oxopentanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)Cc1c[nH]cn1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O DOAUQKRTILFGHV-PDCMDPCFSA-N 0.000 title claims abstract description 84
- 229940057954 glepaglutide Drugs 0.000 title claims abstract description 82
- 238000002360 preparation method Methods 0.000 title abstract description 37
- 239000011347 resin Substances 0.000 claims abstract description 81
- 229920005989 resin Polymers 0.000 claims abstract description 81
- 238000010168 coupling process Methods 0.000 claims abstract description 40
- 238000005859 coupling reaction Methods 0.000 claims abstract description 37
- 150000001413 amino acids Chemical class 0.000 claims abstract description 34
- 230000008878 coupling Effects 0.000 claims abstract description 32
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 25
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims abstract description 21
- 239000012634 fragment Substances 0.000 claims abstract description 20
- 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 claims abstract description 16
- AYMLQYFMYHISQO-QMMMGPOBSA-N (2s)-3-(1h-imidazol-3-ium-5-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CN=CN1 AYMLQYFMYHISQO-QMMMGPOBSA-N 0.000 claims abstract description 9
- UPMGJEMWPQOACJ-UHFFFAOYSA-N 2-[4-[(2,4-dimethoxyphenyl)-(9h-fluoren-9-ylmethoxycarbonylamino)methyl]phenoxy]acetic acid Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(O)=O)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 UPMGJEMWPQOACJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 5
- 238000004108 freeze drying Methods 0.000 claims abstract description 5
- 125000006239 protecting group Chemical group 0.000 claims abstract description 4
- 239000007790 solid phase Substances 0.000 claims abstract description 4
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 13
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 13
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 230000002194 synthesizing effect Effects 0.000 claims description 9
- 238000005336 cracking Methods 0.000 claims description 8
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 claims description 6
- -1 fmoc-Ile-OH Chemical compound 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 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 4
- 241000024188 Andala Species 0.000 claims description 4
- 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
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 claims description 2
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 claims description 2
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 claims 1
- 230000001681 protective effect Effects 0.000 claims 1
- 239000012043 crude product Substances 0.000 abstract description 31
- 239000000047 product Substances 0.000 abstract description 22
- 238000000746 purification Methods 0.000 abstract description 14
- 229920001184 polypeptide Polymers 0.000 abstract description 6
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 6
- 238000001308 synthesis method Methods 0.000 abstract description 5
- 238000003776 cleavage reaction Methods 0.000 abstract description 4
- 230000007017 scission Effects 0.000 abstract description 4
- 238000006243 chemical reaction Methods 0.000 description 27
- 239000003795 chemical substances by application Substances 0.000 description 26
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 11
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 8
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 8
- 239000006166 lysate Substances 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 239000011521 glass Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 208000016261 weight loss Diseases 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 235000016236 parenteral nutrition Nutrition 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- LJOUDGUKJXHDQH-UHFFFAOYSA-N methyl 6-nitro-2,3-dihydro-1,4-benzodioxine-7-carboxylate Chemical compound O1CCOC2=C1C=C(C(=O)OC)C([N+]([O-])=O)=C2 LJOUDGUKJXHDQH-UHFFFAOYSA-N 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000004580 weight loss Effects 0.000 description 3
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 206010049416 Short-bowel syndrome Diseases 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 201000007651 Brooke-Spiegler syndrome Diseases 0.000 description 1
- HNDVDQJCIGZPNO-RXMQYKEDSA-N D-histidine Chemical compound OC(=O)[C@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-RXMQYKEDSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 101500028774 Homo sapiens Glucagon-like peptide 1 Proteins 0.000 description 1
- 206010025476 Malabsorption Diseases 0.000 description 1
- 208000004155 Malabsorption Syndromes Diseases 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012321 colectomy Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- JHWZADUIXGMAKM-UHFFFAOYSA-N methyl 4,5-difluoro-2-nitrobenzoate Chemical compound COC(=O)C1=CC(F)=C(F)C=C1[N+]([O-])=O JHWZADUIXGMAKM-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000006180 nutrition needs Nutrition 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 208000026775 severe diarrhea Diseases 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000003442 weekly effect Effects 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
- C07K14/605—Glucagons
-
- 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
- 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 invention discloses a preparation method of Glepaglutide, which relates to the technical field of preparation of pharmaceutical polypeptides and comprises the following steps: rink Amide Linker and solid phase carrier to obtain Rink Amide MBHA Resin; removing Fmoc protecting groups of each amino acid or fragment according to the sequence of Glepaglutide, and coupling the amino acids or fragments one by one, wherein His 1 ‑Gly 4 Obtaining Glepaglutide peptide resin by adopting Boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH fragments; after cleavage, purification and freeze-drying, the Glepaglutide fine product is obtained. The crude product purity, the crude product yield and the purification yield obtained by the synthesis method are all higher than those of the crude product obtained by coupling one by one, the quality and the purification yield of the product are improved, and the method has wide practical value and prospect.
Description
Technical Field
The invention belongs to the technical field of preparation of pharmaceutical polypeptides, and particularly relates to a preparation method of Glepaglutide.
Background
Short bowel syndrome (short bowel syndromle) refers to malabsorption syndrome in which residual functional intestinal tracts are unable to maintain the nutritional needs of a patient following extensive small bowel resection (including segmental colectomy). Clinically, the traditional Chinese medicine composition is characterized by severe diarrhea, weight loss, progressive malnutrition and water and electrolyte metabolic disturbance, influences the development of organisms and has higher mortality. At present, nutrition support and small intestine transplantation treatment methods are mainly adopted, but the curative effect cannot be confirmed, and the life quality of patients still depends on the length of the remained small intestine and the functional state thereof.
Glepaglutide is a long-acting human glucagon-like polypeptide-2 (GLP-2) analog that can be subcutaneously injected via an automatic injector to reduce the dependence of short bowel syndrome patients on parenteral nutrition support. The EASE 1 study recruited a total of 108 SBS patients and the Glepaglutide group received twice weekly treatment for a total of 24 weeks. The study results showed that the Glepaglutide group of patients reached the initial evaluation endpoint, with a significant decrease in parenteral nutrition support compared to baseline period, by 5.13 liters. In addition, 66% of patients in the Glepaglutide group show clinically significant improvement, and the parenteral nutrition support dosage is reduced by more than 20%. Of these 9 patients no longer use parenteral nutrition support. Glepaglutide has the Chinese name human glucagon-like peptide-1, CAS number 914009-86-2, molecular weight 4316.08, and has the following structure:
H-His 1 -Gly 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Ser 7 -Ser 8 -Glu 9 -Leu 10 -Ala 11 -Thr 12 -Ile 13 -Leu 14 -Asp 15 -Ala 16 -Leu 17 -Ala 18 -Ala 19 -Arg 20 -Asp 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Thr 29 -Lys 30 -Ile 31 -Thr 32 -Asp 33 -Lys 34 -Lys 35 -Lys 36 -Lys 37 -Lys 38 -Lys 39 -NH 2 。
because more hydrophobic amino acids are in the Glepaglutide sequence, the Glepaglutide is easy to fold when the peptide sequence is prolonged, so that the resin is seriously contracted, the problem of difficult coupling can occur, the cost is increased, the operation steps are complicated, the waste liquid is excessive, and the problem of being unfavorable for industrial production is solved. The method can effectively reduce D-His racemization impurity and +Gly impurity, improve the purity and yield of Glepaglutide, improve the purity of crude peptide, greatly reduce the material cost and the purification cost, and is beneficial to industrialized amplified production.
Disclosure of Invention
The invention aims to provide a preparation method of Glepaglutide, the resin yield, crude product purity, crude product yield and purification yield obtained by the synthesis method are all higher than those of crude products obtained by coupling one by one, the quality and purification yield of Glepaglutide products are greatly improved, and the preparation method has wide practical value and application prospect.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a method for preparing gleagalutide, comprising the following steps:
step 1: rink Amide Linker and solid phase carrier to obtain Rink Amide MBHA Resin;
step 2: removing Fmoc protecting groups of each amino acid or fragment according to the sequence of Glepaglutide, coupling amino acids one by one, detecting the end point of the reaction by using ninhydrin detection solution, wherein His 1 -Gly 4 Using Boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH fragment, thr 5 -Phe 6 Fmoc-Thr (tBu) -Phe-OH fragment, ala 11 -Thr 12 And Ala 28 -Thr 29 Glepaglutide peptide resins were obtained using Fmoc-Ala-Thr (tBu) -OH fragments. The invention adopts special protection amino acid and fragments of Boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH, fmoc-Thr (tBu) -Phe-OH and Fmoc-Ala-Thr (tBu) -OH polypeptide fragments to solve the problems of lower purity and higher coupling difficulty, and Arg20 adopts excessive feeding to solve the problem of high coupling difficulty. Glepaglutide is obtained through coupling preparation, and the coupling synthesis method, the crude product yield and the purification yield are obviously improved; the resin yield, the crude product purity, the crude product yield and the purification yield obtained by the synthetic method are all higher than those of the crude product obtained by coupling one by one, so that the quality and the purification yield of the product are greatly improved, and the synthetic method has wide practical value and application prospect. The Glepaglutide synthesis method disclosed by the invention can shorten the production period, greatly improve the purity of crude products, improve the yield of products and has great application prospect.
Step 3: and (3) obtaining a crude Glepaglutide product by cracking the peptide resin, and obtaining a refined Glepaglutide product by purifying and freeze-drying.
In the preparation method of Glepaglutide, the solid carrier comprises MBHA Resin.
In the preparation method of Glepaglutide, the substitution degree of MBHA Resin is 0.40-0.62 mmol/g, and MBHA Resin with the substitution degree of 0.58-0.62 mmol/g is preferentially selected.
The condensing reagents used in the coupling in the step 1 are DIC, HOBT and DIEA, and the solvent is DMF.
The molar ratio of the feed in step 1 was MBHA Resin: rink Amide Linker: DIC: HOBT: DIEA=1:1.5-2.5:1.5-2.5:0.8-1.2, preferably in a molar ratio of 1:2:2:2:1.
It should be noted that, in addition to other conventional protected amino acids, the following specific protected amino acids and fragments are used in the synthesis process of the gleagalutide peptide resin: boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH, fmoc-Thr (tBu) -Phe-OH, fmoc-Ala-Thr (tBu) -OH.
The Glepaglutide peptide resin has the following structure:
Boc-His(Trt)-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ala-Thr(tBu)-Ile-Leu-Asp(OtBu)-Ala-Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Ile-Ala-Thr(tBu)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-MBHA Resin。
the solvent used in the step 2 was DMF, and the coupling reagents were DIC and HOBT.
In the coupling of Fmoc amino acid in the step 2, the molar ratio of Rink Amide MBHA Resin, fmoc protected amino acid, DIC and HOBT is 1:2.5-3.5:2.5-3.5, preferably 1:3:3:3.
In step 2, arg was coupled 20 In the case of Rink Amide MBHA Resin, fmoc-Arg (Pbf) -OH, DIC and HOBT, the molar ratio is 1:5.5-6.5:5.5-6.5:5-6.5, and the preferred molar ratio is 1:6:6:6.
In step 2, ala was coupled 11 -Thr 12 And Ala 28 -Thr 29 In the case of Rink Amide MBHA Resin, fmoc-Ala-Thr (tBu) -OH, DIC and HOBT, the molar ratio is 1:3.5-4.5:3.5-4.5, preferably 1:4:4:4.
More preferably, as condensing agent, a compound represented by formula I or formula II:
i or->II；
At this time, the coupling reagent in the step 2 is a combination of a compound shown in the formula I or the formula II and EDC, and the molar ratio of the two is 1:1-1.5. The condensing agent is prepared by adopting 7-nitro-1, 4-benzodioxane-6-methyl formate or 3, 4-difluoro-6-methyl nitrobenzoate to react with hydrazine hydrate, has higher catalytic activity, can be applied to polypeptide synthesis, and can effectively promote the coupling reaction of amino acid or fragments, so that the purity and yield of the prepared product are obviously improved. The condensing agent with the novel structure can reduce the use amount of the condensing agent in the application process, or shorten the coupling reaction time and improve the reaction effect; the preparation method is simple, and the condensing agent is safe and easy to store, and has better atomic economy compared with other types of condensing agents.
A process for the preparation of a compound of formula I or formula II comprising: the preparation method adopts 7-nitro-1, 4-benzodioxane-6-methyl formate or 3, 4-difluoro-6-methyl nitrobenzoate and hydrazine hydrate to prepare the compound.
Specifically, the preparation method of the compound shown in the formula I or the formula II comprises the following steps:
dissolving 7-nitro-1, 4-benzodioxane-6-methyl formate or 3, 4-difluoro-6-methyl nitrobenzoate in toluene, adding hydrazine hydrate, refluxing under stirring for reaction, adding sodium hydroxide when the raw material point disappears, and performing TLC to monitor the reaction process, and separating by column chromatography to obtain a condensing agent.
The solid-to-liquid ratio of 7-nitro-1, 4-benzodioxane-6-methyl formate or 3, 4-difluoro-6-methyl nitrobenzoate to toluene is 0.3-0.4 g:1mL; the molar ratio of hydrazine hydrate to 7-nitro-1, 4-benzodioxane-6-methyl formate or 3, 4-difluoro-6-methyl nitrobenzoate is 1-1.2:1; the molar ratio of the sodium hydroxide to the 7-nitro-1, 4-benzodioxane-6-methyl formate or the 3, 4-difluoro-6-methyl nitrobenzoate is 0.2-0.4:1.
The reaction end point of the coupled amino acid in step 2 was detected by ninhydrin detection solution.
Further, the specific method for preparing Glepaglutide comprises the following steps: rink Amide Linker and MBHA Resin to obtain RinkAmide MBHA Resin, then according to the peptide sequence of Glepaglutide, sequentially coupling Fmoc-Lys (Boc) -OH, fmoc-Asp (OtBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Ile-OH, fmoc-Lys (Boc) -OH, fmoc-Ala-Thr (tBu) -OH, fmoc-Ile-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH Fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Ala-OH, fmoc-Leu-OH, fmoc-Ala-OH, fmoc-Asp (OtBu) -OH, fmoc-Leu-OH, fmoc-Ile-OH, fmoc-Ala-Thr (tBu) -OH, fmoc-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -Phe-OH, boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH to give Glepaglutide peptide resins, cleavage of the Glepaglutide peptide resin gives crude Glepaglutide:
H-His 1 -Gly 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Ser 7 -Ser 8 -Glu 9 -Leu 10 -Ala 11 -Thr 12 -Ile 13 -Leu 14 -Asp 15 -Ala 16 -Leu 17 -Ala 18 -Ala 19 -Arg 20 -Asp 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Thr 29 -Lys 30 -Ile 31 -Thr 32 -Asp 33 -Lys 34 -Lys 35 -Lys 36 -Lys 37 -Lys 38 -Lys 39 -NH 2 。
in the preparation method of Glepaglutide, the feeding amount of Fmoc-protected amino acid or fragment is 3-6 times of the total mole number of the resin; further preferably Arg 20 The amount of the added substances is 6 times of the total mole number of the added resin, ala 11 -Thr 12 And Ala 28 -Thr 29 The amount of the resin is 4 times of the total mole number of the resin, and the other is 3 times.
In the preparation method of Glepaglutide, the Fmoc-protecting group-removed solution is 20% pip/DMF, and the volume is 2-3 times of the volume of the resin; and after the removal is finished, washing the resin for 5 times by using a DMF solution, wherein the volume of each DMF washing is 2-3 times of that of the resin.
In the preparation method of Glepaglutide, after each amino acid or fragment is connected, ninhydrin detection solution is used for detection, resin is transparent, solution is yellowish to indicate that coupling is completed, then DMF is used for washing the resin for 3-5 times, and the volume of DMF washed each time is 2-3 times of that of the resin.
As a preferable scheme of the invention, the Glepaglutide peptide resin is subjected to acidolysis to simultaneously remove the resin and the side chain protecting group, so as to obtain a Glepaglutide crude product.
In the preparation method of Glepaglutide, the lysate adopted in the cleavage of Glepaglutide peptide resin comprises TFA, EDT, tis and H 2 O, the volume ratio is TFA, EDT, tis and H 2 O=88-92:4-5:2-3:2-3; preferably TFA: EDT: tis: H 2 O=90:5:2.5:2.5。
The amount of the lysate is 6-10 mL of the lysate per gram of the Glepaglutide peptide resin, preferably 8mL of the lysate per gram of the Glepaglutide resin.
The pyrolysis time of the pyrolysis liquid is 2-3.5 h, preferably 3h; the cracking temperature is 20-30 ℃, preferably 25 ℃.
The crude Glepaglutide obtained by cleavage was purified by a C18 column to obtain a refined Glepaglutide.
Further, the solvent for dissolving the crude Glepaglutide product in the purification method is acetonitrile and water.
Still another object of the present invention is to disclose the use of the compound of formula I or formula II as described above as condensing agent for the preparation of polypeptides.
The invention also discloses application of the compound shown in the formula I or the formula II as a condensing agent in preparation of Glepaglutide.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method adopts the special protected amino acid and the fragment to prepare the Glepaglutide through coupling, and the obtained resin yield, crude product purity, crude product yield and purification yield are all higher than those of the crude product obtained through coupling one by one, so that the quality and purification yield of the product are greatly improved, and the preparation method has wide practical value and application prospect. Meanwhile, the condensing agent is prepared by adopting the reaction of the 7-nitro-1, 4-benzodioxane-6-methyl formate or the 3, 4-difluoro-6-methyl nitrobenzoate and hydrazine hydrate, and can be applied to polypeptide synthesis, so that the coupling reaction of amino acid or fragments can be effectively promoted, and the purity and the yield of the prepared product are obviously improved.
Therefore, the invention provides a preparation method of Glepaglutide, the resin yield, crude product purity, crude product yield and purification yield obtained by the synthesis method are all higher than those of crude products obtained by coupling one by one, the quality and purification yield of Glepaglutide products are greatly improved, and the preparation method has wide practical value and application prospect.
Drawings
FIG. 1 is a high performance liquid chromatogram of crude Glepaglutide in example 1 of the present invention;
FIG. 2 is a high performance liquid chromatogram of crude Glepaglutide in example 2 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the following describes in detail various embodiments of the present invention with reference to the embodiments. However, those of ordinary skill in the art will understand that in various embodiments of the present invention, numerous technical details have been set forth in order to provide a better understanding of the present application. However, the technical solutions claimed in the present application can be implemented without these technical details and with various changes and modifications based on the following embodiments.
The chinese meaning corresponding to the abbreviations used in the present invention is shown in table 1 below:
table 1 abbreviations correspond to Chinese meanings
Example 1:
preparation of Glepaglutide:
step 1: preparation Rink Amide MBHA Resin
20g of MBHA resin with the substitution degree of 0.6mml/g is added into a jacketed glass reactor, 150mL of DMF is added, nitrogen is blown for swelling, the swelling time is 50min, DMF is drawn off, resin is washed 2 times by DMF, 12.94g Rink Amide Linker g and 3.24g of HOBT are weighed and added into the reactor, rink Amide Linker g and HOBT are dissolved after 20mL of LDMF is blown into nitrogen, 3.72mL of DIC and 2.1mL of DIEA are added into the reactor, the coupling temperature is controlled to be about 40 ℃, the reaction is carried out for about 2h, sampling is carried out by using ninhydrin solution, the detection is negative (transparent and bright yellow of the resin) and indicates that the connection is finished, the reaction solution is drawn off, resin is washed 3 times by DMF, 200mL of MeOH is used for washing the resin 2 times, DCM is used for washing the resin 2 times, 200mL of resin is used for washing, and then the resin is transferred into a constant-temperature vacuum drying box until the constant weight (continuous weight is reduced by less than 1% in one hour), and the weight is made into Rink Amide MBHA Resin, and the weight is 27.32g.
Step 2: ligating the 1 st to 39 th Fmoc-protected amino acids or amino acid fragments
A. Weighing 6mmol of Rink Amide MBHA Resin, adding the mixture into a jacketed glass reactor, then adding 200mL of 20% pip/DMF solution into the reactor, and blowing nitrogen to react for 30min, wherein the reaction temperature is set to be 30 ℃; after the completion, the solution is pumped out, the resin is washed by 200mL DMF for 5 times, a small amount of resin is taken and detected by ninhydrin solution (comprising 80wt% of phenol/ethanol solution of A, 2v% of 0.001M KCN/pyridine solution of B and 5wt% of ninhydrin/ethanol solution of C), and the positive (resin, solution deep blue) is detected;
B. weighing 8.42g of Fmoc-Lys (Boc) -OH and 2.43g of HOBT in a beaker, adding 25mL of DMF, shaking and dissolving, placing the mixture in a reaction bath with stirring at a low temperature and constant temperature, setting the temperature at 7 ℃, pre-cooling for 10min, adding 2.78mL of DIC for activation for 30min, adding the activated liquid into a jacketed glass reactor, blowing nitrogen for reaction, controlling the reaction temperature at 30 ℃ and the reaction time at 1.5h, taking a small amount of Resin, detecting with ninhydrin detection liquid, detecting negative (transparent Resin and bright yellow solution), indicating that the coupling is completed, pumping out the reaction liquid, and washing the Resin with 150mLDMF for 3 times to obtain Fmoc-Lys (Boc) -MBHA Resin.
According to the deprotection method of the step A and the coupling method of the step B, the rest amino acids or fragments are coupled in sequence according to the sequence of main chain amino acids, and different coupling time and coupling temperature are set, as shown in the table 2 in sequence:
TABLE 2 Experimental conditions for remaining amino acids or fragments
Obtaining Glepaglutide peptide resin:
Boc-His(Trt)-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ala-Thr(tBu)-Ile-Leu-Asp(OtBu)-Ala-Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Ile-Ala-Thr(tBu)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-MBHA Resin；
finally the resin was washed 2 times with MeOH, 2 times with DCM and 2 times with MTBE, then the resin was transferred to a thermostatted vacuum oven for 3 hours at-0.08 MPa and 30 ℃ until the weight was constant (two consecutive weight reductions of less than 1% in one hour) yielding 50.35g of peptide resin.
Step 3: preparation of crude Glepaglutide:
a. a250 mL Erlenmeyer flask was taken, 72mL TFA was added, followed by 4mL EDT, 2mL Tis, 2mL H 2 O, shaking up and putting into a refrigerator for precooling;
b. weighing 10g of Glepaglutide peptide resin prepared in the step 2, respectively adding the Glepaglutide peptide resin into the prepared lysate, and placing the lysate on a constant-temperature oscillator for shaking and cracking for 3 hours, wherein the cracking temperature is controlled to be about 25 ℃; after 3h the reaction mixture was filtered separately with a sand core funnel, the resin was washed with 5mL TFA 2 times respectively and filtered continuously with suction, the filtrates were collected and slowly added to 800mL MTBE (temperature-10 ℃) with stirring, the white solids were separated out and centrifuged, the supernatant was discarded, the solid precipitate was washed 3 times with MTBE, 800mL each time, and the solid precipitate was dried to constant weight (twice weight loss less than 1% in one hour) in a vacuum thermostated oven, wherein 4.98g of gleagalutide crude product was obtained, the crude product yield was 96.14%; 4.52g of Glepaglutide crude product is obtained by a coupling method one by one, and the crude product yield is 87.26%;
the crude product is taken to measure the molecular weight of 4316.08 +/-1, wherein the purity of the crude product of Glepaglutide obtained by the method is 72.51 percent, and the maximum single impurity is 4.8 percent, which is shown in figure 1.
Purifying the crude product: the crude product is dissolved by acetonitrile and water, purified under a C18 preparation column, and then is put into a freeze dryer for freeze drying, wherein the yield of the Glepaglutide refined product prepared by the method is 48.59%.
Example 2:
preparation of Glepaglutide:
step 1: rink Amide MBHA Resin was prepared in the same manner as in example 1.
Step 2: the 1 st to 39 th protected amino acid is accessed by adopting a coupling-by-coupling method, and the method specifically comprises the following steps:
C. 6mmol of Rink Amide MBHA Resin are weighed into a jacketed glass reactor, 200mL of 20% pip/DMF solution is added into the reactor, nitrogen is blown for reaction for 30min, and the reaction temperature is set at 30 ℃. After the completion, the solution is pumped out, the resin is washed by 200mL DMF for 5 times, a small amount of resin is taken and detected by ninhydrin solution, and the detection is positive (resin, solution is dark blue);
D. weighing 8.42g of Fmoc-Lys (Boc) -OH and 2.43g of HOBT in a beaker, adding 25mL of DMF, shaking and dissolving, placing the mixture in a reaction bath with stirring at a low temperature and constant temperature, setting the temperature at 7 ℃, pre-cooling for 10min, adding 2.78mL of DIC for activation for 30min, adding the activated liquid into a jacketed glass reactor, blowing nitrogen for reaction, controlling the reaction temperature at 30 ℃ and the reaction time at 1.5h, taking a small amount of Resin, detecting with ninhydrin detection liquid, detecting negative (transparent Resin and bright yellow solution), indicating that the coupling is completed, pumping out the reaction liquid, and washing the Resin with 150mLDMF for 3 times to obtain Fmoc-Lys (Boc) -MBHA Resin.
According to the deprotection method of the step C and the coupling method of the step D, the rest amino acids are coupled in sequence according to the sequence of the main chain amino acids, and different coupling time and coupling temperature are set, as shown in the following table 3 in sequence:
TABLE 3 Experimental conditions for remaining amino acids
Obtaining Glepaglutide peptide resin:
Boc-His(Trt)-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ala-Thr(tBu)-Ile-Leu-Asp(OtBu)-Ala-Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Ile-Ala-Thr(tBu)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-MBHA Resin；
finally the resin was washed 2 times with MeOH, 2 times with DCM and 2 times with MTBE, then the resin was transferred to a thermostatted vacuum oven for 3 hours at-0.08 MPa and 30 ℃ until constant weight (two consecutive weight reductions of less than 1% in one hour) gave 49.87g of peptide resin.
Step 3: preparation of crude Glepaglutide:
a. a250 mL Erlenmeyer flask was taken, 72mL TFA was added, followed by 4mL EDT, 2mL Tis, 2mL H 2 O, shaking up and putting into a refrigerator for precooling;
b. weighing 10g of Glepaglutide peptide resin prepared in the step 2, respectively adding the Glepaglutide peptide resin into the prepared lysate, and placing the lysate on a constant-temperature oscillator for shaking and cracking for 3 hours, wherein the cracking temperature is controlled to be about 25 ℃; after 3h the reaction mixture was filtered separately with a sand core funnel, the resin was washed 2 times with 5mL TFA, the filtrates were collected by combining, slowly added to 800mL MTBE (temperature-10 ℃) and stirred, the white solid precipitated and centrifuged, the supernatant was discarded, the solid precipitate was washed 3 times with MTBE, 800mL each time, and the solid precipitate was dried to constant weight (twice weight loss less than 1% in one hour) in a vacuum thermostatted oven; the crude Glepaglutide product was obtained in 87.26% yield by the coupling-by-coupling method.
Taking a crude product, and detecting to obtain a molecular weight of 4316.08 +/-1; the crude Glepaglutide product obtained by the coupling method has the purity of 55.63% and the maximum single impurity of 16.42%, and is shown in figure 2.
Purifying the crude product: the crude product is dissolved by acetonitrile and water, purified under a C18 preparation column, and then is put into a freeze dryer for freeze drying, and the yield of Glepaglutide refined products prepared by coupling one by one is 21.23 percent.
Example 3:
the preparation of Glepaglutide differs from example 1 in that:
in step 2, the condensing agent used in the amino acid coupling process is the condensing agent prepared in this example, specifically:
B. the condensing agent prepared in this example was weighed out as Fmoc-Lys (Boc) -OH 8.42g and equimolar equivalent, 25mL of DMF was added for shaking dissolution, the mixture was placed in a reaction bath stirred at a low temperature and constant temperature, the temperature was set at 7 ℃, pre-chilled for 10min, EDC with 1.2 times molar equivalent was added, the mixture was stirred for 30min, then the mixture was added into a jacketed glass reactor, nitrogen was blown in to react, the reaction temperature was controlled at 30 ℃, the reaction time was controlled at 1.5h, a small amount of Resin was detected with ninhydrin detection solution, the detection was negative (transparent Resin and bright yellow solution) and the coupling was completed, the reaction solution was withdrawn, and the Resin was washed 3 times with 150mL of DMF to obtain Fmoc-Lys (Boc) -MBHA Resin. Similarly, the condensing agent prepared in this example was used to couple the remaining amino acids or fragments in sequence according to the backbone amino acid sequence.
Preparation of condensing agent:
taking 7-nitro-1, 4-benzodioxane-6-methyl formate, dissolving in toluene, adding hydrazine hydrate, carrying out reflux reaction under stirring, monitoring the reaction process by TLC, adding sodium hydroxide when the raw material point disappears, monitoring the reaction process by TLC, and separating by column chromatography to obtain a condensing agent (the structure is shown as below), wherein the yield is 89.4% and the purity is 97.5%.
The solid-to-liquid ratio of methyl 7-nitro-1, 4-benzodioxane-6-carboxylate to toluene was 0.35g:1mL; the molar ratio of hydrazine hydrate to 7-nitro-1, 4-benzodioxane-6-methyl formate is 1.1:1; the molar ratio of sodium hydroxide to methyl 7-nitro-1, 4-benzodioxane-6-carboxylate was 0.3:1.
1 H NMR（400 MHz，CDCl 3 ）：δ：7.19、6.65（2H，Ar-H），4.31、4.23（4H，-CH 2 ）。
Example 4:
the preparation of Glepaglutide differs from that of example 3 in that:
condensing agent was prepared in this example.
The condensing agent was prepared differently from example 3:
3, 4-difluoro-6-nitrobenzoic acid methyl ester is adopted to replace 7-nitro-1, 4-benzodioxane-6-formic acid methyl ester. The chemical structure is as follows:
1 H NMR（400 MHz，CDCl 3 ）：δ：7.36、7.10（2H，Ar-H）。
example 5:
the preparation of Glepaglutide differs from example 2 in that:
in step 2, the condensing agent used in the amino acid coupling process is the condensing agent prepared in this example, specifically:
B. the condensing agent prepared in this example was weighed out as Fmoc-Lys (Boc) -OH 8.42g and equimolar equivalent, 25mL of DMF was added for shaking dissolution, the mixture was placed in a reaction bath stirred at a low temperature and constant temperature, the temperature was set at 7℃for 10min, EDC of 1.2 times molar equivalent was added, the mixture was stirred for 30min and then added into a jacketed glass reactor, nitrogen was blown to react, the reaction temperature was controlled at 30℃and the reaction time was controlled at 1.5h, a small amount of resin was detected with ninhydrin detection solution, the detection was negative (transparent resin and bright yellow solution) indicating that the coupling was completed, the reaction solution was withdrawn, and the resin was washed 3 times with 150mL of DMF to obtain Fmoc-Lys (Boc) -MBHAresin. Similarly, the condensing agent prepared in this example was used to couple the remaining amino acids in sequence with the backbone amino acids.
The condensing agent was prepared in the same manner as in example 3.
Example 6:
the preparation of Glepaglutide differs from example 5 in that:
condensing agent was prepared in this example.
The condensing agent was prepared in the same manner as in example 4.
Test example 1:
test results of crude product purity and Fine product yield
The crude Glepaglutide product prepared in examples 1-6 has the purity and yield of the refined Glepaglutide product shown in Table 4:
TABLE 4 purity and yield test results
As can be seen from the data analysis in Table 4, the crude product purity and the fine product yield of Glepaglutide prepared in example 1 are obviously higher than those of example 2, which shows that the method for synthesizing Glepaglutide by adopting special protected amino acid and fragments disclosed by the invention shows better reaction activity, and the purity and the yield of the prepared product are obviously improved. The effect of example 3 and example 4 is obviously better than that of example 1, the effect of example 5 and example 6 is obviously better than that of example 2, and the effect of using 7-nitro-1, 4-benzodioxane-6-methyl formate or 3, 4-difluoro-6-methyl nitrobenzoate to react with hydrazine hydrate to prepare the obtained compound, wherein the compound is used as a condensing agent for coupling reaction of amino acid or fragment, the occurrence of the coupling reaction can be obviously promoted, and the purity and yield of the prepared Glepaglutide product are obviously increased.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. A method for synthesizing Glepaglutide, comprising the following steps:
step 1: rink Amide Linker and solid phase carrier to obtain Rink Amide MBHA Resin;
step 2: removing Fmoc protecting groups of each amino acid or fragment according to the sequence of Glepaglutide, sequentially coupling Fmoc-Lys (Boc) -OH, fmoc-Asp (OtBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Ile-OH, fmoc-Lys (Boc) -OH, fmoc-Ala-Thr (tBu) -OH, fmoc-Ile-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH Fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Ala-OH, fmoc-Leu-OH, fmoc-Ala-OH, fmoc-Asp (OtBu) -OH, fmoc-Leu-OH, fmoc-Ile-OH, fmoc-Ala-Thr (tBu) -OH, fmoc-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -Phe-OH, boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH to obtain Glepaglutide peptide resin;
step 3: and (3) obtaining a crude Glepaglutide product by cracking the peptide resin, and obtaining a refined Glepaglutide product by purifying and freeze-drying.
2. The method for synthesizing Glepaglutide according to claim 1, wherein: the solid phase carrier in the step 1 is MBHA Resin.
3. The method for synthesizing Glepaglutide according to claim 2, wherein: the substitution degree of the MBHA Resin is 0.40 mmol/g-0.62 mmol/g.
4. The method for synthesizing Glepaglutide according to claim 1, wherein: the Fmoc-protecting group removal solution in the step 2 is 20% pip/DMF solution.
5. The method for synthesizing Glepaglutide according to claim 1, wherein: when Fmoc amino acid is coupled in the step 2, the molar ratio of Rink Amide MBHA Resin to Fmoc protective amino acid to DIC to HOBT is 1:2.5-3.5:2.5-3.5.
6. The method for synthesizing Glepaglutide according to claim 1, wherein: coupling Arg in said step 2 20 In the process, the molar ratio of Rink Amide MBHA Resin to Fmoc-Arg (Pbf) -OH to DIC to HOBT is 1:5.5-6.5:5.5-6.5.
7. The method for synthesizing Glepaglutide according to claim 1, wherein: coupling Ala in step 2 11 -Thr 12 And Ala 28 -Thr 29 In the process, the molar ratio of Rink Amide MBHA Resin to Fmoc-Ala-Thr (tBu) -OH to DIC to HOBT is 1:3.5-4.5:3.5-4.5.
8. The method for synthesizing Glepaglutide according to claim 1, wherein: the cracking liquid used in the step 3 is TFA, EDT, tis and H 2 O, TFA: EDT: tis: h 2 The O volume ratio is 72:4:2:2.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101171262A (en) * | 2005-05-04 | 2008-04-30 | 西兰制药公司 | Glucagon-like-peptide-2 (GLP-2) analogues |
| CN111560061A (en) * | 2020-05-26 | 2020-08-21 | 成都圣诺生物制药有限公司 | Preparation method of Gelpaglutide |
| CN112125970A (en) * | 2020-09-23 | 2020-12-25 | 深圳深创生物药业有限公司 | Synthesis method of semaglutide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101171262A (en) * | 2005-05-04 | 2008-04-30 | 西兰制药公司 | Glucagon-like-peptide-2 (GLP-2) analogues |
| CN111560061A (en) * | 2020-05-26 | 2020-08-21 | 成都圣诺生物制药有限公司 | Preparation method of Gelpaglutide |
| CN112125970A (en) * | 2020-09-23 | 2020-12-25 | 深圳深创生物药业有限公司 | Synthesis method of semaglutide |
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