CN107056927B - Preparation method of liraglutide - Google Patents
Preparation method of liraglutide Download PDFInfo
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- CN107056927B CN107056927B CN201710030103.6A CN201710030103A CN107056927B CN 107056927 B CN107056927 B CN 107056927B CN 201710030103 A CN201710030103 A CN 201710030103A CN 107056927 B CN107056927 B CN 107056927B
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- liraglutide
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- 108010019598 Liraglutide Proteins 0.000 title claims abstract description 52
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 title claims abstract description 46
- 229960002701 liraglutide Drugs 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 150000001413 amino acids Chemical group 0.000 claims abstract description 66
- 239000011347 resin Substances 0.000 claims abstract description 41
- 229920005989 resin Polymers 0.000 claims abstract description 41
- 238000005859 coupling reaction Methods 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 22
- 230000008878 coupling Effects 0.000 claims abstract description 20
- 238000010168 coupling process Methods 0.000 claims abstract description 20
- DBTMQODRSDEGRZ-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-(2-oxoethyl)carbamate Chemical compound C1=CC=C2C(COC(=O)NCC=O)C3=CC=CC=C3C2=C1 DBTMQODRSDEGRZ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 17
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 16
- 102000007079 Peptide Fragments Human genes 0.000 claims abstract description 14
- 108010033276 Peptide Fragments Proteins 0.000 claims abstract description 14
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 12
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 8
- 238000005336 cracking Methods 0.000 claims abstract description 8
- OYXZPXVCRAAKCM-SANMLTNESA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C1=NC(C[C@H](NC(=O)OC(C)(C)C)C(O)=O)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 OYXZPXVCRAAKCM-SANMLTNESA-N 0.000 claims abstract description 6
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 23
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 14
- -1 benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Chemical compound 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 9
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 claims description 8
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- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 6
- FJVAQLJNTSUQPY-CIUDSAMLSA-N Ala-Ala-Lys Chemical group C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN FJVAQLJNTSUQPY-CIUDSAMLSA-N 0.000 claims description 5
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical group CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 4
- 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 description 3
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 claims description 3
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 3
- 239000003875 Wang resin Substances 0.000 claims description 3
- 230000009089 cytolysis Effects 0.000 claims description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 3
- 125000006239 protecting group Chemical group 0.000 claims description 3
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical group C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 2
- 229910052763 palladium Inorganic materials 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 claims description 2
- 239000012634 fragment Substances 0.000 abstract description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 22
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
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- 238000005406 washing Methods 0.000 description 6
- 238000001914 filtration Methods 0.000 description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 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 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
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- 239000012071 phase Substances 0.000 description 2
- 125000003386 piperidinyl group Chemical group 0.000 description 2
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- 229910002027 silica gel Inorganic materials 0.000 description 2
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 description 1
- 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
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 1
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-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
- 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 1
- 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 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
- WDGICUODAOGOMO-DHUJRADRSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino)pentanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)CC(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 WDGICUODAOGOMO-DHUJRADRSA-N 0.000 description 1
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- 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
- 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 1
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 101500028774 Homo sapiens Glucagon-like peptide 1 Proteins 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
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- 238000010353 genetic engineering Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
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- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 1
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- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical class C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
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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
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- 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 liraglutide, which comprises the following steps: deprotecting by using a deprotection reagent Fmoc-Gly-Wang resin, and removing an Fmoc protection group; the three protected amino acids at positions 12 to 14 are coupled with a protected peptide fragment X; the three protected amino acids at positions 20 to 22 are coupled with a protected peptide fragment Y; coupling at the 31 th position in a Boc-His (trt) -OH protected form; removing alloc protection of lysine side chains from the liraglutide main chain peptide resin, and coupling the side chains one by one to obtain the liraglutide peptide resin; cracking the liraglutide peptide resin in a cracking solution to obtain a crude liraglutide product; the crude liraglutide is purified to obtain a fine liraglutide product, and the amino site which is most easily folded in the synthetic process of the main chain of the liraglutide is subjected to fragment coupling, so that the problems that amino acid needs to be coupled for many times and the access rate is low in the synthetic process due to folding are solved, the production period of the liraglutide is shortened, and the synthetic yield of the liraglutide is improved.
Description
Technical Field
The invention relates to synthesis of polypeptide, in particular to a preparation method of liraglutide.
Background
At present, with the improvement of living standard, the number of people suffering from diabetes is gradually increased, and liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue and is used for treating diabetes. The structure of liraglutide is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (Na-PAL-gamma-Glu) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. The demand for liraglutide is large in the market. The existing synthesis methods are more. The method of the genetic engineering has great difficulty and the technical difficulty is great; during the synthesis process, amino folding phenomena are easily caused by amino sites (20 th-22 th sites and 12 th-14 th sites) which are most easily folded during the main chain synthesis process, so that byproducts and other impurities are generated, and the yield and the purity of the product are influenced; many synthetic methods involve the main chain being synthesized one by one, which results in easy folding of the most easily folded amino site or in the amino acid requiring multiple couplings, further resulting in low product productivity or increased synthetic steps.
Disclosure of Invention
The invention aims to: aiming at the problems that the amino acid needs to be coupled for many times due to amino folding in the synthesis process, the access rate is low, the yield is further reduced, and the purity of the product is further reduced, the invention provides the preparation method of the liraglutide, which has the advantages of high yield, low cost, mild reaction conditions and contribution to realizing industrialization.
The technical scheme adopted by the invention is as follows:
(1) deprotecting Fmoc-Gly-Wang resin by using a deprotection reagent, and removing an Fmoc protection group; the deprotection reagent is piperidine/DMF solution, and the volume percentage of piperidine in the deprotection reagent is 30-50%; the deprotection reaction temperature is 10-25 ℃;
(2) coupling the deprotected Fmoc-Gly-Wang resin with protected amino acid one by one to obtain liraglutide main chain resin; wherein
The three protected amino acids at positions 12 to 14 are coupled with a protected peptide fragment X;
the three protected amino acids at positions 20 to 22 are coupled with a protected peptide fragment Y;
coupling at the 31 th position in a Boc-His (trt) -OH protected form;
the coupling amino acid fragment is as follows:
Boc-His (trt) -Ala-Glu (otbu) -Gly-Thr (tbu) -Phe-Thr (tbu) -Ser (tbu) -Asp (otbu) -Y (tbu) -Tyr (tbu) -Leu-Glu (otbu) -Gly-Gln (trt) -X (alloc) -Glu (otbu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Wang resin,
wherein X is Ala-Ala-Lys; y is Val-Ser-Ser;
(3) removing alloc protection of lysine side chains from the liraglutide main chain peptide resin, and coupling the side chains one by one to obtain the liraglutide peptide resin;
(4) cracking the liraglutide peptide resin in a cracking solution to obtain a crude liraglutide product;
(5) and purifying the crude liraglutide to obtain a refined liraglutide.
Preferably, the substitution value of Fmoc-Gly-Wang resin is less than or equal to 0.35 mmol/g.
Most preferably, the Fmoc-Gly-Wang resin has a substitution value of 0.25 mmol/g.
In the invention, Gly amino acid in Fmoc-Gly-Wang resin is used as the 1 st amino acid, the number of digits is calculated by reverse deduction in sequence, and His amino acid is used as the 31 st amino acid.
Preferably, an activating agent is added in the coupling reaction in the step (2), and the activating agent is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole.
Preferably, the condensing agent selected in the step (2) is one of N, N-diisopropylcarbodiimide, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 2- (7-aza-1H-benzotriazole-1-yl) -1,1,3, 3-tetramethylurea hexafluorophosphate, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate or O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate; the molar consumption of the condensing agent is 1.2 to 6 times of the total molar number of the Fmoc-Gly-Wang resin, and preferably 2.5 to 4.5 times.
Preferably, the Fmoc-removing protective reagent selected in the step (2) is a piperidine/N, N-dimethylformamide mixed solution, and the piperidine content in the mixed solution is 10% -50% (v/v). The amount of the activating reagent is 1.2 to 6 times of the total mole number of the Fmoc-Gly-Wang resin, preferably 2.5 to 4.5 times.
Preferably, in step (2), the protected peptide fragment corresponding to access X is Fmoc-Ala-Ala-Lys (alloc) -OH; the protective peptide fragment corresponding to the accessed Y is Fmoc-Val-Ser (tbu) -OH; the corresponding protection form of the accessed histidine is Boc-His (trt) -OH; preferably, the amount of the protected amino acid or the protected peptide fragment is 2 to 6 times the total molar amount of the resin; more preferably 2 to 4 times. Preferably, the coupling reaction time is 2 to 6 hours.
Preferably, in the step (2), when the Fmoc-Gly-Wang resin is coupled with the protected amino acid and the protected peptide fragment, the Fmoc protecting group of the protected amino acid resin obtained by reaction is removed, and then the protected amino acid resin is coupled with the next protected amino acid; after the coupling reaction is finished and the Fmoc protection is removed, the coupling reaction is detected by Kaiser Test.
Preferably, in the step (3), the alloc protective reagent for removing the lysine side chain from the liraglutide backbone peptide resin is a mixed solution of tetrakiss-phosphine palladium and morpholine/THF; the dosage of the tetrakistriphenylphosphorated palladium is 0.3-0.5 times of the mole number of the peptide resin; the dosage of morpholine is 5-10 times of the mole number of the peptide resin; deprotection is carried out under the condition of nitrogen; the deprotection time is 5-10 hours.
Preferably, in the step (3), the alloc-protected backbone of the liraglutide is removed, side chains are coupled one by one to obtain liraglutide resin, and the side chains are applied in the forms of Fmoc-Glu (OH) -otbu and palmitic acid respectively; preferably, side chain coupling reaction, liraglutide backbone resin: side chain: the molar ratio of the coupling reagents is 1:4: 6; the coupling condition is the same as that in the step (2); the coupling reaction time is 6-8 hours. After the coupling reaction is finished one by one and the Fmoc protection is removed, Kaiser Test is carried out.
Preferably, in the step (4), the lysis solution contains 80-95% (v/v) of trifluoroacetic acid, 1-10% (v/v) of 1, 2-ethanedithiol, 1-5% (v/v) of triisopropylsilane, and the solvent is water; the volume ratio of the more preferable mixed solvent is as follows: TFA 90%, EDT 4%, TIS 2%, and balance water.
Preferably, in step (5), the purified chromatographic column is an octadecyl bonded silica gel with a diameter of 10cm and a packing material with a particle size of 10um and a pore diameter of
The mobile phase of the C18 column is respectively diluted ammonia water solution and acetonitrile, the flow rate is 120ml/min, the loading amount is 5-10 g, and the detection wavelength of a chromatograph is 230 nm. After purification, the purity of the obtained product is more than 99.0 percent.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. according to the method, the most easily folded amino sites (12 th to 14 th sites and 20 th to 22 th sites) are adopted in the main chain synthesis process of the liraglutide for segment coupling, the folding sites are easily coupled in the main chain synthesis process, and therefore the problems that the amino acid caused by folding in the synthesis process needs to be coupled for multiple times and the access rate is low are solved. The two short and small protective amino acid fragments are easy to prepare and synthesize, and have high purity and low preparation cost, so that the production cost of the liraglutide is reduced;
2. proper deprotection conditions are adopted, so that the deprotection reaction is more thorough;
3, the substitution degree of the Fmoc-Gly-Wang resin is less than or equal to 0.35mmol/g, and the spatial structure of a later-stage product is uncontrollable and the product is easy to be impure due to the overhigh substitution degree of the Fmoc-Gly-Wang resin.
4. An activating agent is added in the coupling reaction, so that the coupling reaction speed is increased, and the yield of the protected amino acid is higher;
5. the dosage of reactants is reasonably controlled, and excessive impurities are avoided;
6. the reasonable reaction sequence avoids the occurrence of unnecessary by-products;
7. the cracking liquid consisting of trifluoroacetic acid, 1, 2-ethanedithiol and water is adopted, so that the cracking is more thorough.
The meanings of the abbreviations used in the present invention are listed in the following table:
the corresponding protected amino acid of the amino acid used in the invention is shown as the following:
| abbreviations | Protected amino acid forms |
| Gly | Fmoc-Gly-OH |
| Arg | Fmoc-Arg(pbf)-OH |
| Val | Fmoc-Val-OH |
| Leu | Fmoc-Leu-OH |
| Trp | Fmoc-Trp(boc)-OH |
| Ala | Fmoc-Ala-OH |
| Ile | Fmoc-Ile-OH |
| Phe | Fmoc-Phe-OH |
| Glu | Fmoc-Glu(OtBu)-OH |
| Ala-Ala-Lys | Fmoc-Ala-Ala-Lys(alloc)-OH |
| Gln | Fmoc-Gln(Trt)-OH |
| Tyr | Fmoc-Tyr(tBu)-OH |
| Val-Ser-Ser | Fmoc-Val-Ser(tbu)-Ser(tbu)-OH |
| Asp | Fmoc-Asp(otBu)-OH |
| Ser | Fmoc-Ser(tBu)-OH |
| Thr | Fmoc-Thr(tBu)-OH |
| His | Boc-His(Trt)-OH |
| Side chain Glu | Fmoc-Glu(OH)-OtBu |
All features disclosed in this specification may be combined in any combination, except features and/or steps that are mutually exclusive.
Detailed Description
Example 1
Preparation of liraglutide peptide backbone resin:
Fmoc-Gly-Wang resin is used as a starting carrier, protected amino acids are sequentially coupled through Fmoc protection removal and coupling reaction to prepare the liraglutide peptide main chain resin, Gly is used as the 1 st coupled amino acid, His is used as the 31 st coupled amino acid for sequencing, and the coupled amino acid fragment is as follows:
Boc-His (trt) -Ala-Glu (otbu) -Gly-Thr (tbu) -Phe-Thr (tbu) -Ser (tbu) -Asp (otbu) -Y (tbu) -Tyr (tbu) -Leu-Glu (otbu) -Gly-Gln (trt) -X (alloc) -Glu (otbu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Wang resin,
wherein X is Ala-Ala-Lys and Y is Val-Ser-Ser;
when X is connected, the corresponding protective amino acid is Fmoc-Ala-Ala-Lys (alloc) -OH;
when Y is accessed, the corresponding protected amino acid is Fmoc-Val-Ser (tbu) -OH;
when His is inoculated, the corresponding protected amino acid is Boc-His (trt) -OH.
The method comprises the following specific steps:
(1) coupling Arg on Fmoc-Gly-Wang resin
Dissolving 0.15mol of 2 nd protected amino acid Fmoc-Arg (pbf) -OH and 0.15mol of HOBt by using a proper amount of DMF; and adding 0.15mol DIC slowly into DMF solution containing protected amino acid under stirring, and reacting under stirring at room temperature for 30 min to obtain activated protected amino acid solution.
And taking 200g of Fmoc-Gly-Wang resin with the substitution value of 0.25mmol/g, deprotecting by using 2000mL of 30% PIP/DMF solution for 30 minutes, washing and filtering to obtain the Fmoc-removed resin.
And adding the activated 2 nd protected amino acid solution into the Fmoc-removed resin, performing coupling reaction for 4 hours, and performing suction filtration and washing to obtain the resin containing 2 protected amino acids.
(2) And inoculating 3-11 protected amino acids
Coupling the 3 rd to 11 th amino acids on the resin by using the method in the step (1).
(3) And inoculating 12 th to 14 th protected amino acid fragments
Dissolving 0.2mol of protected amino acid fragment, namely Fmoc-Ala-Ala-Lys (alloc) -OH and 0.2mol of HOBt in proper amount of DMF; and adding 0.2mol DIC slowly into the protected amino acid DMF solution under stirring, and stirring for reaction for 30 minutes at room temperature to obtain an activated Fmoc-Ala-Ala-Lys (alloc) -OH fragment solution.
And adding the activated Fmoc-Ala-Ala-Lys (alloc) -OH fragment solution into the Fmoc-removed 11 peptide resin, performing coupling reaction for 3-5 hours, filtering and washing to obtain the resin containing the 14 th protected amino acid.
(4) And 15 th to 19 th protected amino acid is inoculated
And sequentially accessing the corresponding 15 th to 19 th protected amino acids by adopting the same method for accessing the 2 nd protected amino acid.
(5) And inoculating the 20 th to 22 th protected amino acid fragments
Dissolving 0.2mol of protected amino acid fragment, namely Fmoc-Val-Ser (tbu) -OH and 0.2mol of HOAt in proper amount of DMF; and adding 0.2mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated Fmoc-Val-Ser (tbu) -OH fragment solution.
Adding the activated Fmoc-Val-Ser (tbu) -OH fragment solution into the Fmoc-removed 19-peptide resin, performing coupling reaction for 3-5 hours, filtering and washing to obtain the resin containing the 22 nd protected amino acid.
In the present invention, HOAT/DIC is used as an activator to activate a protected amino acid to allow coupling to a resin
The method is easier to carry out, the reaction rate is high, and the yield is high. As can be seen from comparative experiments, the activity at the addition step (5)
After the reagent, the yield of the protected amino acid is higher.
(6) And the 23 th to 29 th protected amino acid is inoculated
And sequentially accessing the corresponding 23 rd to 29 th protected amino acids by adopting the same method for accessing the 2 nd protected amino acid.
(7) And inoculating 30-31 th protected amino acid
And sequentially inoculating the corresponding 30 th to 31 th protected amino acids by adopting the same method for inoculating the protected amino acid fragments. Obtaining the liraglutide main chain peptide resin.
(8) Dealloc protection of liraglutide backbone peptide resins
0.02mol of tetrakistriphenylphosphine palladium and 0.3mol of morpholine were dissolved in 3000ml of THF solution to prepare a deprotection reagent for use. The liraglutide backbone peptide resin is respectively washed 3 times by proper amount of 15 percent triethylamine/DCM, washed 3 times by THF/DCM and pumped for standby; adding a deprotection reagent into the liraglutide main chain peptide resin, and introducing nitrogen to carry out alloc protection removal reaction. The deprotection time is 5-10 hours, and Kaiser Test is carried out to detect whether alloc protection is removed.
(9) Grafting of side chains Glu and Pal
The corresponding side chains Glu and Pal are sequentially grafted by the same method of grafting protected amino acid fragments. And obtaining the liraglutide peptide resin.
Example 2
On the basis of example 1, crude liraglutide was prepared:
taking liraglutide peptide resin, adding the mixture into the mixture at a volume ratio of TFA to EDT: TIS: water: 90: 4: 2: 4, the using amount of the lysis solution is 5mL/g resin, and the solution is uniformly stirred. Stirring and reacting for 3 hours at room temperature, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, merging the filtrate, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, and pumping to dry to obtain white-like powder, namely the crude liraglutide.
Example 3
On the basis of example 2, the purification process of the crude liraglutide comprises the following steps:
dissolving the crude liraglutide with 20% acetonitrile water solution of which the pH value is adjusted to 10 by ammonia water, filtering by a 0.45 mu m mixed microporous filter membrane, and purifying for later use;
purifying by high performance liquid chromatography with octadecyl bonded silica gel having diameter of 10cm and filler diameter of 10um
The mobile phase of the C18 column is respectively diluted ammonia water solution and acetonitrile solution, the flow rate is 120ml/min, the loading amount is 5-10 g, and the detection wavelength of a chromatograph is 230 nm.
And performing multiple purification, collecting qualified main peaks, analyzing a liquid phase to detect the purity of the main peaks, performing reduced pressure concentration to obtain a dilute ammonia solution of the liraglutide, and performing freeze drying to obtain 18.8g of the liraglutide with the total yield of 10.1%.
Single isobaric molecular weight: 3750.98 (100% M + H); purity: 99.01 percent.
In the above examples, the condensing agent is one of N, N-diisopropylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate, benzotriazol-N, N '-tetramethyluronium hexafluorophosphate, or O-benzotriazol-N, N' -tetramethyluronium tetrafluoroborate; the selected Fmoc-removing protective reagent is piperidine/N, N-dimethylformamide mixed solution, and the piperidine contained in the mixed solution is 10-50% (v/v). Has wide practical value and application prospect.
The present application can be preferably implemented by using the above-described embodiments, but the present application is not limited to the above-described embodiments.
Claims (6)
1. A preparation method of liraglutide is characterized by comprising the following steps:
(1) deprotecting Fmoc-Gly-Wang resin by using a deprotection reagent, and removing an Fmoc protection group;
(2) coupling the deprotected Fmoc-Gly-Wang resin with protected amino acid one by one to obtain liraglutide main chain resin; wherein
The three protected amino acids at positions 12 to 14 are coupled with a protected peptide fragment X;
the three protected amino acids at positions 20 to 22 are coupled with a protected peptide fragment Y;
coupling at the 31 th position in a Boc-His (trt) -OH protected form;
x is Ala-Ala-Lys; the Y is Val-Ser-Ser;
(3) removing alloc protection of lysine side chains from the liraglutide main chain peptide resin, and coupling the side chains one by one to obtain the liraglutide peptide resin;
(4) cracking the liraglutide peptide resin in a cracking solution to obtain a crude liraglutide product;
(5) purifying the crude liraglutide to obtain a refined liraglutide;
adding an activating agent in the coupling reaction in the step (2), wherein the activating agent is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole;
the protective peptide fragment corresponding to the access X is Fmoc-Ala-Ala-Lys (alloc) -OH; the protective peptide fragment corresponding to the accessed Y is Fmoc-Val-Ser (tbu) -OH; when Fmoc-Gly-Wang resin is coupled with protected amino acid and protected peptide fragments, the Fmoc protecting group of the protected amino acid resin obtained by reaction is removed, and then the protected amino acid resin is coupled with the next protected amino acid;
the substitution degree of the Fmoc-Gly-Wang resin is less than or equal to 0.35 mmol/g;
the coupling amino acid fragment is as follows:
Boc-His (trt) -Ala-Glu (otbu) -Gly-Thr (tbu) -Phe-Thr (tbu) -Ser (tbu) -Asp (otbu) -Y (tbu) -Tyr (tbu) -Leu-Glu (otbu) -Gly-Gln (trt) -X (alloc) -Glu (otbu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Wang resin,
wherein X is Ala-Ala-Lys; y is Val-Ser-Ser.
2. The method for preparing liraglutide according to claim 1, wherein the deprotection reagent in the step (1) is piperidine/DMF solution, and the volume percentage of piperidine in the deprotection reagent is 30-50%; the deprotection reaction temperature is 10-25 ℃.
3. The method for preparing liraglutide according to claim 1, wherein the coupling reagent used in the step (2) is one of N, N-diisopropylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate, benzotriazol-N, N '-tetramethyluronium hexafluorophosphate, O-benzotriazol-N, N' -tetramethyluronium tetrafluoroborate.
4. The method of claim 1, wherein the amount of protected amino acid or protected peptide fragment used during the coupling is 2-6 times the total molar amount of resin.
5. The method of claim 1, wherein the liraglutide is prepared by the following steps: in the step (3), the reagent for removing the alloc protecting group of the lysine side chain is a mixed solution of tetrakiss-cresyl palladium and morpholine/THF.
6. The method for preparing liraglutide according to claim 1, wherein in the step (4), the lysis solution comprises trifluoroacetic acid in an amount of 80-95% by volume, 1-10% by volume of 1, 2-ethanedithiol, 1-5% by volume of triisopropylsilane, and water as a solvent.
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