CN118005766A - Solid phase synthesis method of Glepaglutide - Google Patents
Solid phase synthesis method of Glepaglutide Download PDFInfo
- Publication number
- CN118005766A CN118005766A CN202410220867.1A CN202410220867A CN118005766A CN 118005766 A CN118005766 A CN 118005766A CN 202410220867 A CN202410220867 A CN 202410220867A CN 118005766 A CN118005766 A CN 118005766A
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- glepaglutide
- fmoc
- thr
- resin
- ala
- Prior art date
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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 89
- 229940057954 glepaglutide Drugs 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000010532 solid phase synthesis reaction Methods 0.000 title claims description 10
- 239000011347 resin Substances 0.000 claims abstract description 80
- 229920005989 resin Polymers 0.000 claims abstract description 80
- 239000012043 crude product Substances 0.000 claims abstract description 39
- 150000001413 amino acids Chemical class 0.000 claims abstract description 35
- 238000010168 coupling process Methods 0.000 claims abstract description 35
- 238000005859 coupling reaction Methods 0.000 claims abstract description 33
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 29
- 230000008878 coupling Effects 0.000 claims abstract description 28
- 239000012634 fragment Substances 0.000 claims abstract description 23
- 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
- 239000000047 product Substances 0.000 claims abstract description 19
- 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 15
- 238000005336 cracking Methods 0.000 claims abstract description 10
- 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
- 239000007790 solid phase Substances 0.000 claims abstract description 5
- 125000006239 protecting group Chemical group 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
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 claims description 12
- 239000006166 lysate Substances 0.000 claims description 8
- 241000024188 Andala Species 0.000 claims description 6
- 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 3
- -1 Fmoc amino acid Chemical class 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 29
- 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
- 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
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 15
- 238000001514 detection method Methods 0.000 description 14
- 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
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 11
- 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
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 7
- 239000011521 glass Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 5
- 208000016261 weight loss Diseases 0.000 description 5
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 4
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 4
- 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 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 238000004090 dissolution Methods 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
- 230000008569 process Effects 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 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 description 3
- 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
- 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
- 238000000197 pyrolysis Methods 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
- 238000005406 washing Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 230000004580 weight loss Effects 0.000 description 3
- 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 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 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
- 238000004440 column chromatography Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000010511 deprotection reaction 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
- 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
- 239000006228 supernatant Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- ADOHASQZJSJZBT-SANMLTNESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[1-[(2-methylpropan-2-yl)oxycarbonyl]indol-3-yl]propanoic acid Chemical compound C12=CC=CC=C2N(C(=O)OC(C)(C)C)C=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ADOHASQZJSJZBT-SANMLTNESA-N 0.000 description 1
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 description 1
- 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 description 1
- 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 description 1
- 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
- 150000001408 amides Chemical class 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000005587 bubbling Effects 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
- 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
- 230000009257 reactivity 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
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 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 a solid phase carrier are coupled 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-Gly4 adopts Boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH fragments to obtain Glepaglutide peptide resin; the Glepaglutide fine product is obtained after cracking, purification and freeze-drying. 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
The application relates to a split application of a Chinese patent application with the application date of 2023, 06 and 21, the application number of 202310740246.1 and the name of 'a preparation method of Glepaglutide'.
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 bowel is 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 Glepaglutide groups received twice weekly treatment for a total of 24 weeks. The study results showed that Glepaglutide groups of patients reached the initial evaluation endpoint, and the parenteral nutrition support dose was significantly reduced by 5.13 liters compared to the baseline period. In addition, the Glepaglutide groups of patients have 66 percent of clinical improvement, and the parenteral nutrition support dosage is reduced by more than 20 percent. Of these 9 patients no longer use parenteral nutrition support. Glepaglutide Chinese name human glucagon-like peptide-1, CAS number 914009-86-2, molecular weight 4316.08, glepaglutide has the following structure:
H-His1-Gly2-Glu3-Gly4-Thr5-Phe6-Ser7-Ser8-Glu9-Leu10-Ala11-Thr12-Ile13-Leu14-Asp15-Ala16-Leu17-Ala18-Ala19-Arg20-Asp21-Phe22-Ile23-Ala24-Trp25-Leu26-Ile27-Ala28-Thr29-Lys30-Ile31-Thr32-Asp33-Lys34-Lys35-Lys36-Lys37-Lys38-Lys39-NH2.
Because of more hydrophobic amino acids in Glepaglutide sequence, the method is easy to form folding when peptide sequence is prolonged, so that resin shrinkage is serious, coupling difficulty can occur, cost is increased, operation steps are complicated, waste liquid is excessive, and industrial production is not facilitated. 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 of preparing Glepaglutide comprising the steps of:
Step 1: RINK AMIDE LINKER and a solid phase carrier are coupled to obtain RINK AMIDE MBHA Resin;
Step 2: according to Glepaglutide sequences, fmoc protecting groups of each amino acid or fragment are removed, amino acids are coupled one by one, the reaction end point is detected by ninhydrin detection solution, wherein His 1-Gly4 adopts Boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH fragments, thr 5-Phe6 adopts Fmoc-Thr (tBu) -Phe-OH fragments, ala 11-Thr12 and Ala 28-Thr29 adopt Fmoc-Ala-Thr (tBu) -OH fragments, and Glepaglutide peptide resin is obtained. 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 the crude product, improve the product yield and has a huge application prospect.
Step 3: and (3) cracking the peptide resin to obtain Glepaglutide crude products, and purifying and freeze-drying to obtain Glepaglutide refined products.
In the preparation method Glepaglutide, the solid phase carrier includes MBHA Resin.
In the Glepaglutide preparation method, the MBHA Resin with the substitution degree range of 0.40 mmol/g-0.62 mmol/g is preferably selected from MBHA Resin with the substitution degree of 0.58 mmol/g-0.62 mmol/g.
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 Glepaglutide peptide resin: boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH, fmoc-Thr (tBu) -Phe-OH, fmoc-Ala-Thr (tBu) -OH.
The Glepaglutide peptide resin had 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 step2 was DMF, and the coupling reagents were DIC and HOBT.
In the step 2, when Fmoc amino acid is coupled, 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 the step 2, when Arg 20 is coupled, the molar ratio of RINK AMIDE MBHA Resin, fmoc-Arg (Pbf) -OH, DIC and HOBT is 1:5.5-6.5:5.5-6.5, preferably 1:6:6:6.
In the step 2, when Ala 11-Thr12 and Ala 28-Thr29 are coupled, the molar ratio of RINK AMIDE MBHA Resin, fmoc-Ala-Thr (tBu) -OH, DIC and HOBT 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 are coupled to obtain RINK AMIDE MBHA Resin, then Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、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-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-Ser(tBu)-OH、Fmoc-Thr(tBu)-Phe-OH、Boc-His(Trt)-Gly-Glu(OtBu)-Gly-OH is coupled in sequence according to the peptide sequence of Glepaglutide to obtain Glepaglutide peptide Resin, and Glepaglutide peptide Resin is cracked to obtain Glepaglutide crude product:
H-His1-Gly2-Glu3-Gly4-Thr5-Phe6-Ser7-Ser8-Glu9-Leu10-Ala11-Thr12-Ile13-Leu14-Asp15-Ala16-Leu17-Ala18-Ala19-Arg20-Asp21-Phe22-Ile23-Ala24-Trp25-Leu26-Ile27-Ala28-Thr29-Lys30-Ile31-Thr32-Asp33-Lys34-Lys35-Lys36-Lys37-Lys38-Lys39-NH2.
In the preparation method of Glepaglutide, the dosage of Fmoc-protected amino acid or fragment is 3-6 times of the total mole number of the resin; further preferably, arg 20 is fed in an amount of 6 times the total moles of resin fed, ala 11-Thr12 and Ala 28-Thr29 are fed in an amount of 4 times the total moles of resin fed, and the others are all 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, the resin is transparent, the solution is slightly yellow to indicate that the coupling is finished, 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 cracking liquid adopted in the Glepaglutide peptide resin cracking process comprises TFA, EDT, tis and H 2 O, wherein the volume ratio of TFA to EDT to Tis to H 2 O=88-92 to 4-5 to 2-3; preferably TFA: EDT: tis: H 2 O=90:5:2.5:2.5.
It should be noted that the amount of the lysate is 6-10 mL of the lysate per gram Glepaglutide of the peptide resin, preferably 8mL of the lysate per gram Glepaglutide of the 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 the pyrolysis was purified by using a C18 column to obtain a refined Glepaglutide.
Further, the solvent for dissolving Glepaglutide crude 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 invention adopts special protected amino acid and fragment to prepare Glepaglutide through coupling, the yield, the crude product purity, the crude product yield and the purification yield of the obtained resin are all higher than those of the crude product obtained through coupling one by one, the quality and the purification yield of the product are greatly improved, and the invention 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 Glepaglutide crude product in example 1 of the present invention;
FIG. 2 is a high performance liquid chromatogram of Glepaglutide crude product 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 application more apparent, the following describes in detail various embodiments of the present application with reference to the embodiments. However, those of ordinary skill in the art will understand that in various embodiments of the present application, numerous technical details have been set forth in order to provide a better understanding of the present application. The claimed application may be practiced without these specific 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 and 3.24g of HOBT are weighed and added into the reactor, after 20mL of DMF is blown into nitrogen, RINK AMIDE LINKER and HOBT are dissolved, 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) to indicate that the connection is finished, the reaction solution is drawn off, the Resin is washed 3 times by DMF, 200mL of each time is washed 2 times by MeOH, resin is washed 2 times by DCM, 200mL of MTBE is washed 2 times, and then the Resin is transferred into a constant-temperature vacuum drying box until the constant weight (the constant weight is reduced by less than 1% in one hour), 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 RINK AMIDE mmol of RINK AMIDE MBHA Resin, adding the Resin 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. Fmoc-Lys (Boc) -OH 8.42g and HOBT 2.43g are weighed into a beaker, 25mL of DMF is added for shaking dissolution, the mixture is placed into a reaction bath with stirring at a low temperature and constant temperature, the temperature is set at 7 ℃, the pre-cooling is carried out for 10min, 2.78mL of DIC is added for activation for 30min, the activated solution is added into a jacketed glass reactor, nitrogen is blown for reaction, the reaction temperature is controlled at 30 ℃, the reaction time is controlled at 1.5h, a small amount of Resin is detected by ninhydrin detection solution, the detection is negative (transparent Resin and bright yellow solution), the coupling is completed, the reaction solution is pumped out, and the Resin is washed 3 times by 150mL of DMF 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
The Glepaglutide peptide resin was obtained:
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: preparing Glepaglutide crude products:
a. Taking a 250mL conical flask, adding 72mL of TFA respectively, adding 4mL of EDT, 2mL of Tis and 2mL of H 2 O, shaking uniformly, 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 vibrating 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 while white solids were separated out, 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, wherein the invention gives Glepaglutide crude 4.98g, crude yield 96.14%; the method of coupling one by one obtains Glepaglutide crude products of 4.52g, and the yield of the crude products is 87.26%;
the crude product is taken to have molecular weight of 4316.08 +/-1, wherein the purity of the Glepaglutide crude product 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 percent.
Example 2:
Preparation of Glepaglutide:
step 1: the procedure for preparation RINK AMIDE MBHA Resin was 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 RINK AMIDE MBHA Resin was weighed into a jacketed glass reactor, 200mL of 20% pip/DMF solution was then added to the reactor and reacted for 30min with nitrogen bubbling, the reaction temperature was 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. Fmoc-Lys (Boc) -OH 8.42g and HOBT 2.43g are weighed into a beaker, 25mL of DMF is added for shaking dissolution, the mixture is placed into a reaction bath with stirring at a low temperature and constant temperature, the temperature is set at7 ℃, the pre-cooling is carried out for 10min, 2.78mL of DIC is added for activation for 30min, the activated solution is added into a jacketed glass reactor, nitrogen is blown for reaction, the reaction temperature is controlled at 30 ℃, the reaction time is controlled at 1.5h, a small amount of Resin is detected by ninhydrin detection solution, the detection is negative (transparent Resin and bright yellow solution), the coupling is completed, the reaction solution is pumped out, and the Resin is washed 3 times by 150mL of DMF 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
The Glepaglutide peptide resin was obtained:
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: preparing Glepaglutide crude products:
a. Taking a 250mL conical flask, adding 72mL of TFA, adding 4mL of EDT, 2mL of Tis and 2mL of H 2 O, shaking uniformly, 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 vibrating 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 product yield of Glepaglutide was 87.26% 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 Glepaglutide refined products prepared by coupling one by one are obtained with the yield of 21.23 percent.
Example 3:
glepaglutide was prepared in a manner different from that of example 1:
In step2, 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.
1H NMR（400 MHz,CDCl3）：δ：7.19、6.65（2H,Ar-H）,4.31、4.23（4H,-CH2）。
Example 4:
Glepaglutide was prepared in a manner different from that of example 3:
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:
1H NMR（400 MHz,CDCl3）：δ：7.36、7.10（2H，Ar-H）。
example 5:
glepaglutide was prepared in a manner different from that of example 2:
In step2, 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 in sequence with the backbone amino acids.
The condensing agent was prepared in the same manner as in example 3.
Example 6:
glepaglutide was prepared in a manner different from that of example 5:
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 product purity and Glepaglutide refined product yield results of Glepaglutide prepared in examples 1-6 are 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 refined product yield of Glepaglutide prepared in example 1 are significantly higher than those of example 2, which indicates that the method disclosed by the invention for synthesizing Glepaglutide by using the special protected amino acid and fragments shows better reactivity, and the purity and the yield of the prepared product are significantly 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 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 products 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 of solid phase synthesis of Glepaglutide comprising the steps of:
Step 1: RINK AMIDE LINKER and a solid phase carrier are coupled to obtain RINK AMIDE MBHA Resin;
Step 2: 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-Gly4 adopts Boc-His (Trt) -Gly-Glu (OtBu) -Gly-OH fragments, thr 5-Phe6 adopts Fmoc-Thr (tBu) -Phe-OH fragments, and Ala 11-Thr12 and Ala 28-Thr29 adopt Fmoc-Ala-Thr (tBu) -OH fragments to obtain Glepaglutide peptide resin;
Step 3: and (3) cracking the peptide resin to obtain Glepaglutide crude products, and purifying and freeze-drying to obtain Glepaglutide refined products.
2. The solid phase synthesis method of Glepaglutide according to claim 1, wherein: the solid phase carrier in the step 1 comprises MBHA Resin.
3. The solid phase synthesis method of Glepaglutide according to claim 2, wherein: the substitution degree of the MBHA Resin is 0.40 mmol/g-0.62 mmol/g.
4. The solid phase synthesis method of Glepaglutide according to claim 1, wherein: the Fmoc-protecting group removal solution in step 2 comprises a 20% pip/DMF solution.
5. The solid phase synthesis method of Glepaglutide according to claim 1, wherein: when Fmoc amino acid is coupled 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.
6. The solid phase synthesis method of Glepaglutide according to claim 1, wherein: when Arg 20 is coupled in the step 2, the molar ratio of RINK AMIDE MBHA Resin, fmoc-Arg (Pbf) -OH, DIC and HOBT is 1:5.5-6.5:5.5-6.5.
7. The solid phase synthesis method of Glepaglutide according to claim 1, wherein: in the step 2, when Ala 11-Thr12 and Ala 28-Thr29 are coupled, the molar ratio of RINK AMIDE MBHA Resin, fmoc-Ala-Thr (tBu) -OH, DIC and HOBT is 1:3.5-4.5:3.5-4.5.
8. The solid phase synthesis method of Glepaglutide according to claim 1, wherein: the lysates used for cleavage in step 3 included TFA, EDT, tis and H 2 O, TFA: EDT: tis: the volume ratio of H 2 O is 88-92:4-5:2-3:2-3.
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|---|---|---|---|---|
| EP2314616A1 (en) * | 2009-10-23 | 2011-04-27 | Ferring B.V. | Peptidic GLP-2 agonists |
| CN111560061A (en) * | 2020-05-26 | 2020-08-21 | 成都圣诺生物制药有限公司 | Preparation method of Gelpaglutide |
| WO2021198195A1 (en) * | 2020-03-30 | 2021-10-07 | Zealand Pharma A/S | Agonist combination |
| CN115746102A (en) * | 2021-09-02 | 2023-03-07 | 深圳市健翔生物制药有限公司 | Method for preparing Bulevirtide by combining solid phase with liquid phase |
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| EP2314616A1 (en) * | 2009-10-23 | 2011-04-27 | Ferring B.V. | Peptidic GLP-2 agonists |
| WO2021198195A1 (en) * | 2020-03-30 | 2021-10-07 | Zealand Pharma A/S | Agonist combination |
| CN111560061A (en) * | 2020-05-26 | 2020-08-21 | 成都圣诺生物制药有限公司 | Preparation method of Gelpaglutide |
| CN115746102A (en) * | 2021-09-02 | 2023-03-07 | 深圳市健翔生物制药有限公司 | Method for preparing Bulevirtide by combining solid phase with liquid phase |
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