CN114057862A - 一种glp-1(1-37)多肽的合成方法 - Google Patents
一种glp-1(1-37)多肽的合成方法 Download PDFInfo
- Publication number
- CN114057862A CN114057862A CN202111531468.XA CN202111531468A CN114057862A CN 114057862 A CN114057862 A CN 114057862A CN 202111531468 A CN202111531468 A CN 202111531468A CN 114057862 A CN114057862 A CN 114057862A
- Authority
- CN
- China
- Prior art keywords
- fmoc
- glp
- tbu
- ser
- trt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound 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(N)=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 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 title claims abstract description 44
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 39
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 26
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 26
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 title claims abstract 18
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 title claims abstract 18
- 238000010189 synthetic method Methods 0.000 title abstract description 7
- 239000011347 resin Substances 0.000 claims abstract description 58
- 229920005989 resin Polymers 0.000 claims abstract description 58
- 238000000034 method Methods 0.000 claims abstract description 20
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 claims abstract description 18
- 238000005859 coupling reaction Methods 0.000 claims abstract description 14
- 230000008878 coupling Effects 0.000 claims abstract description 13
- 238000010168 coupling process Methods 0.000 claims abstract description 13
- 150000001413 amino acids Chemical class 0.000 claims abstract description 12
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 12
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 8
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 8
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 6
- 230000003213 activating effect Effects 0.000 claims abstract description 5
- 238000005336 cracking Methods 0.000 claims abstract description 3
- 125000006239 protecting group Chemical group 0.000 claims abstract description 3
- 150000003839 salts Chemical class 0.000 claims abstract description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 17
- KJYAFJQCGPUXJY-UMSFTDKQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-(tritylamino)butanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KJYAFJQCGPUXJY-UMSFTDKQSA-N 0.000 claims description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- 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 13
- 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 claims description 12
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 239000012071 phase Substances 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 238000003746 solid phase reaction Methods 0.000 claims description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 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 claims description 8
- 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 claims description 7
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 7
- 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 6
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 claims description 5
- 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 claims description 5
- 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 claims description 5
- -1 9-fluorenylmethyloxycarbonyl Chemical group 0.000 claims description 5
- 238000006482 condensation reaction Methods 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 5
- OXEDICXRSSILSN-GOTSBHOMSA-N (4s)-3-[(2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoyl]-2,2-dimethyl-1,3-oxazolidine-4-carboxylic acid Chemical compound O=C([C@@H](NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)COC(C)(C)C)N1[C@H](C(O)=O)COC1(C)C OXEDICXRSSILSN-GOTSBHOMSA-N 0.000 claims description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 4
- 230000008961 swelling Effects 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 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 claims description 3
- 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 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 claims description 3
- 230000005526 G1 to G0 transition Effects 0.000 claims description 3
- 238000007792 addition Methods 0.000 claims description 3
- 150000001408 amides Chemical class 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 108010016626 Dipeptides Proteins 0.000 claims description 2
- NGWKGSCSHDHHAJ-YPFQVHCOSA-N Liquoric acid Chemical compound C1C[C@H](O)C(C)(C)C2CC[C@@]3(C)[C@]4(C)C[C@H]5O[C@@H]([C@](C6)(C)C(O)=O)C[C@@]5(C)[C@@H]6C4=CC(=O)C3[C@]21C NGWKGSCSHDHHAJ-YPFQVHCOSA-N 0.000 claims description 2
- XZKQVQKUZMAADP-IMJSIDKUSA-N Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(O)=O XZKQVQKUZMAADP-IMJSIDKUSA-N 0.000 claims description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 2
- 239000004473 Threonine Substances 0.000 claims description 2
- 239000012190 activator Substances 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims 7
- XXMYDXUIZKNHDT-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(1-tritylimidazol-4-yl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(N=C1)=CN1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XXMYDXUIZKNHDT-QNGWXLTQSA-N 0.000 claims 1
- 210000004899 c-terminal region Anatomy 0.000 claims 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 1
- 239000000047 product Substances 0.000 abstract description 6
- 239000006227 byproduct Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 abstract 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 abstract 1
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 description 27
- 102100040918 Pro-glucagon Human genes 0.000 description 27
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- JZTKZVJMSCONAK-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-hydroxypropanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CO)C(O)=O)C3=CC=CC=C3C2=C1 JZTKZVJMSCONAK-INIZCTEOSA-N 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- UCARTONYOJORBQ-UMSFTDKQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-trityloxypropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)OC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 UCARTONYOJORBQ-UMSFTDKQSA-N 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 230000001276 controlling effect Effects 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 3
- 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 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102100037505 Secretin Human genes 0.000 description 2
- 108010086019 Secretin Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 239000000859 incretin Substances 0.000 description 2
- 230000003914 insulin secretion Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 229960002101 secretin Drugs 0.000 description 2
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 1
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical class C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000032928 Dyslipidaemia Diseases 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 208000002705 Glucose Intolerance Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000017170 Lipid metabolism disease Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)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(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- MGXWVYUBJRZYPE-YUGYIWNOSA-N incretin Chemical class C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O)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=1C=CC(O)=CC=1)[C@@H](C)O)[C@@H](C)CC)C1=CC=C(O)C=C1 MGXWVYUBJRZYPE-YUGYIWNOSA-N 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 210000002571 pancreatic alpha cell Anatomy 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 201000009104 prediabetes syndrome Diseases 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound 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
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Diabetes (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Endocrinology (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Zoology (AREA)
- Child & Adolescent Psychology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Emergency Medicine (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Nutrition Science (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种GLP‑1(1‑37)多肽的合成方法，属于多肽药物合成技术领域，包括以下步骤：以Rink‑Resin为载体树脂，在添加活化剂和缩合剂的条件下，使载体树脂和酪氨酸偶联得到Fmoc‑Tyr(tBu)‑Rink‑Resin；通过固相合成法，根据GLP‑1(1‑37)的氨基酸序列依次偶联其它氨基酸；脱除保护基和裂解载体树脂后，得到GLP‑1(1‑37)粗肽；纯化、转盐、冻干，得到GLP‑1(1‑37)多肽。该方法合成周期短，成本低，后处理容易，副产物少，产品收率高，利于GLP‑1(1‑37)的大规模生产，具有可观的经济适用价值和广泛的应用前景。
Description
技术领域
本发明属于多肽药物合成技术领域，具体地，涉及一种GLP-1(1-37)多肽的合成方法。
背景技术
肠促胰素是人体内一种肠源性激素，肠促胰素以葡萄糖浓度依赖性方式促进胰岛β细胞分泌胰岛素，并减少胰岛α细胞分泌胰高血糖素(glucagon)，从而降低血糖。正常人在进餐后，肠促胰素开始分泌，进而促进胰岛素分泌，以减少餐后血糖的波动。但对于2型糖尿病患者，其“肠促胰素效应”受损，主要表现为进餐后胰高血糖素样肽-1(GLP-1)浓度升高幅度较正常人有所减小，但其促进胰岛素分泌以及降血糖的作用并无明显受损，因此GLP-1及其类似物可以作为2型糖尿病治疗的一个重要靶点。
GLP-1(1-37)多肽为GLP-1多肽的一种，其氨基酸序列缩写为：
Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr；
单字母符号表示为：KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY；
现有技术中，GLP-1(1-37)的合成多采用常规依次偶联氨基酸的方式，但该合成方式会使GLP-1(1-37)肽链形成β折叠，导致合成结束时肽链纯度差，产品收率低。
发明内容
本发明的目的在于提供一种GLP-1(1-37)多肽的合成方法，该合成方法操作简单，合成周期短，成本低，减少了废液的产生，后处理容易，副产物少，产品收率高。
本发明的目的可以通过以下技术方案实现：
一种GLP-1(1-37)多肽的合成方法，具体包括以下步骤：
步骤S1、制备GLP-1(1-37)前体树脂：以载体树脂为起始，将载体树脂加入到固相反应柱中，用DMF洗涤、溶胀，然后将Fmoc-Tyr(tBu)-OH用DMF溶解并与活化剂和缩合剂混合后，加入到上述装有载体树脂的反应柱中，进行缩合反应，使固相的载体树脂和N端Fmoc保护的Fmoc-Tyr(tBu)-OH偶联得到Fmoc-Tyr(tBu)-载体树脂；
步骤S2、根据GLP-1(1-37)多肽的氨基酸序列，按照从C端到N端的偶联顺序，采用固相合成法依次将具有N端Fmoc保护且侧链保护的氨基酸与Fmoc-Tyr(tBu)-载体树脂进行偶联，得到Fmoc-GLP-1(1-37)-载体树脂；
步骤S3、将Fmoc-GLP-1(1-37)-载体树脂脱除Fmoc保护后，使用混合液和三氟乙酸切割裂解Fmoc-GLP-1(1-37)-载体树脂，得到GLP-1(1-37)粗品肽；
步骤S4、将GLP-1(1-37)粗品肽纯化、转盐、冻干后，得到GLP-1(1-37)多肽成品。
进一步地，载体树脂为酰胺树脂(Rink-Resin)，取代度为0.37mmol/g。
进一步地，Fmoc为9-芴甲氧羰基，Fmoc与氨基相连或与羧基所连的碳原子上的氮原子相连。
进一步地，步骤S2中的固相合成法包括以下步骤：
步骤A1：采用DBLK使所述Fmoc-Tyr(tBu)-载体树脂脱除Fmoc保护基，得到NH-Tyr(tBu)-载体树脂，所述DBLK为哌啶和DMF按照体积比为1:3混合组成；
步骤A2：在添加活化剂和缩合剂系统的条件下，NH-Tyr(tBu)-载体树脂和Fmoc保护且侧链保护的苏氨酸偶联，得到Fmoc-Thr(tBu)-Tyr(tBu)-载体树脂；
步骤A3：根据GLP-1(1-37)多肽的氨基酸序列，重复步骤A1和步骤A2。
进一步地，活化剂为DIC，缩合剂为HOBt，缩合反应时间为2h。
进一步地，GLP-1(1-37)多肽中的氨基酸Ser-Ser采用伪二肽Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH。
进一步地，步骤S2中C端到N端的偶联顺序为Fmoc-Asn(Trt)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Gly-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH、Fmoc-Leu-OH、Fmoc-Ile-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Thr(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Lys(Boc)-OH。
进一步地，步骤S4中纯化的条件为：采用反相高效液相色谱，以反相十八烷基硅烷为固定相，柱温为35℃，流动相A为体积分数为0.1％的醋酸/水溶液，流动相B为乙腈，流动相A和流动相B按照体积比70:30等梯度洗脱，流速为70-80mL/min，检测波长为214nm。
通过上述合成方法制得的GLP-1(1-37)多肽具有调节血糖，保护胰岛β细胞，降低体重，抗炎降脂降压，保护心血管等多重生理作用，应用于制备治疗或预防高血糖症、2型糖尿病、葡萄糖耐量降低、1型糖尿病、肥胖症、高血压、X综合征、血脂障碍、动脉粥样硬化、心肌梗塞、冠心病和其它心血管疾病、中风、炎性肠综合征、消化不良和胃溃疡的药物。
本发明的有益效果：
本发明提供了一种固相合成法进行GLP-1(1-37)的合成方法，操作简单，合成周期短，成本较低，减少了废液的产生，后处理容易，副产物少，产品收率高，有利于GLP-1(1-37)多肽的大规模生产，具有可观的经济适用价值和广泛的应用前景。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
对本发明中所用英文缩写及其含义说明如下表1：
表1
| 英文缩写 | 含义 |
| Rink-Resin | 酰胺树脂 |
| Fmoc | 9-芴甲氧羰基 |
| DMF | N,N-二甲基甲酰胺 |
| DIC | 1,3-二异丙基碳二亚胺 |
| HOBt | 1-羟基苯并三唑 |
| DCM | 二氯甲烷 |
| TFA | 三氟乙酸 |
| PhOMe | 苯甲醚 |
| PhSMe | 苯甲硫醚 |
本文中，“替代度”指的是单位量的树脂负载的物质的数量，单位为“mmol/g”。
实施例1
Fmoc-Tyr(tBu)-Rink-Resin的合成：
称取20g替代度为0.37mmol/g的Rink-Resin，加入到固相反应柱中，用DMF洗涤2次，用DMF溶胀Rink-Resin 25min后，称取4.45gFmoc-Tyr(tBu)-OH用DMF溶解，冰水浴下加入3.62gHOBt和4.75mL DIC充分混合后，加入上述装有Rink-Resin的反应柱中，缩合反应2h，然后用DMF洗涤，洗涤后再加入8mL醋酸酐封闭1h，然后用DMF洗涤3次，DCM洗3次，甲醇收缩抽干，得到Fmoc-Tyr(tBu)-Rink-Resin。
实施例2
Fmoc-Tyr(tBu)-Rink-Resin肽链的延长反应：
称取Fmoc-Tyr(tBu)-Rink-Resin10g，加入固相反应柱中，用DMF洗涤2次，用DMF溶胀Fmoc-Tyr(tBu)-Rink-Resin20min后，用20％DBLK脱除Fmoc保护，然后用DMF洗涤4次，DCM洗2次，用茚三酮法检测树脂颜色，树脂有颜色，表示Fmoc已脱除；
将3.89g Fmoc-Thr(tBu)-OH、0.97g HOBt和0.91g DIC溶于体积比为1:1的DCM和DMF混合溶液中，然后加入固相反应柱中，室温反应2h。以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需再偶联反应1h，此判断标准适用于后续内容中以茚三酮法检测判断反应终点；
重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，按照GLP-1(7-37)主链肽序，依次完成Fmoc-Asn(Trt)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Gly-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH、Fmoc-Leu-OH、Fmoc-Ile-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Thr(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH和Fmoc-Lys(Boc)-OH的偶联，得到Fmoc-GLP-1(1-37)-Rink-Resin。
实施例3
侧链全保护GLP-1(-37)-Rink-Resin的抽干：
取实施例2中得到的Fmoc-GLP-1(1-37)-Rink-Resin，用20％DBLK脱除Fmoc保护，然后用DMF洗涤4次，DCM洗2次，用茚三酮法检测树脂颜色，树脂有颜色，表示Fmoc已脱除。然后用甲醇洗涤2次，树脂真空干燥过夜。
称重得到GLP-1(1-37)-Rink-Resin16.5g(树脂增重率81.1％)。
实施例4
GLP-1(1-37)粗品肽的制备：
将实施例3得到的130g侧链全保护的GLP-1(1-37)-Rink-Resin加入到1L三口瓶中，加入900mL预先配置好的TFA:H2O:PhOMe:PhSMe＝90:5:4:1(V:V)，室温反应2h，减压过滤树脂，收集滤液，再用少量TFA洗涤滤渣，合并滤液，将合并的滤液缓慢加入10L冰乙醚中沉淀，离心，冰乙醚洗涤5次(5L/次)，减压干燥得到GLP-1(1-37)粗品肽42.7g，通过高效液相色谱检测分析，GLP-1(1-37)粗品肽的纯度为86.6％。
实施例5
GLP-1(1-37)精肽醋酸盐的制备：
称取42.7g GLP-1(1-37)粗品肽溶于500mL体积分数为50％的乙腈/水溶液中，将该混合物直接上样；
纯化条件：以反相十八烷基硅烷为固定相；流动A相为0.1％(v/v)醋酸/水溶液，流动B相为乙腈，A:B＝70:30(v:v)等梯度洗脱；流速：75mL/min；检测波长：214nm；
收集目标峰馏分，通过液氮浓缩冻干，得纯品14.5g，纯度96％，收率90％。
在说明书的描述中，参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上内容仅仅是对本发明所作的举例和说明，所属本技术领域的技术人员对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代，只要不偏离发明或者超越本权利要求书所定义的范围，均应属于本发明的保护范围。
Claims (8)
1.一种GLP-1(1-37)多肽的合成方法，其特征在于：包括以下步骤：
步骤S1、将载体树脂加入到固相反应柱中洗涤、溶胀，然后将Fmoc-Tyr(tBu)-OH用DMF溶解并与活化剂和缩合剂混合后，加入到固相反应柱中，进行缩合反应，使载体树脂和Fmoc-Tyr(tBu)-OH偶联得到Fmoc-Tyr(tBu)-载体树脂；
步骤S2、按照从C端到N端的偶联顺序，采用固相合成法将氨基酸与Fmoc-Tyr(tBu)-载体树脂进行偶联，得到Fmoc-GLP-1(1-37)-载体树脂；
步骤S3、将Fmoc-GLP-1(1-37)-载体树脂脱除Fmoc保护后，使用混合液和三氟乙酸切割裂解Fmoc-GLP-1(1-37)-载体树脂，得到GLP-1(1-37)粗品肽；
步骤S4、将GLP-1(1-37)粗品肽纯化、转盐、冻干后，得到GLP-1(1-37)多肽。
2.根据权利要求1所述的一种GLP-1(1-37)多肽的合成方法，其特征在于：所述载体树脂为酰胺树脂，取代度为0.37mmol/g。
3.根据权利要求1所述的一种GLP-1(1-37)多肽的合成方法，其特征在于：Fmoc为9-芴甲氧羰基，Fmoc与氨基相连或与羧基所连的碳原子上的氮原子相连。
4.根据权利要求1所述的一种GLP-1(1-37)多肽的合成方法，其特征在于，所述固相合成法包括以下步骤：
步骤A1：使用DBLK脱除Fmoc-Tyr(tBu)-载体树脂的Fmoc保护基，得到NH-Tyr(tBu)-载体树脂，所述DBLK为哌啶和DMF按照体积比为1:3混合组成；
步骤A2：在添加活化剂和缩合剂系统的条件下，NH-Tyr(tBu)-载体树脂和Fmoc保护且侧链保护的苏氨酸偶联，得到Fmoc-Thr(tBu)-Tyr(tBu)-载体树脂；
步骤A3：根据GLP-1(1-37)多肽的氨基酸序列，重复步骤A1和步骤A2。
5.根据权利要求4所述的一种GLP-1(1-37)多肽的合成方法，其特征在于：活化剂为DIC，缩合剂为HOBt，缩合反应时间为2h。
6.根据权利要求4所述的一种GLP-1(1-37)多肽的合成方法，其特征在于：氨基酸Ser-Ser采用伪二肽Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH。
7.根据权利要求6所述的一种GLP-1(1-37)多肽的合成方法，其特征在于：所述C端到N端的偶联顺序为Fmoc-Asn(Trt)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Gly-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH、Fmoc-Leu-OH、Fmoc-I le-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Thr(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Lys(Boc)-OH。
8.根据权利要求1所述的一种GLP-1(1-37)多肽的合成方法，其特征在于，纯化的条件为：以反相十八烷基硅烷为固定相，柱温为35℃，流动相A为体积分数为0.1％的醋酸/水溶液，流动相B为乙腈，流动相A和流动相B按照体积比70:30等梯度洗脱，流速为70-80mL/min，检测波长为214nm。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111531468.XA CN114057862A (zh) | 2021-12-14 | 2021-12-14 | 一种glp-1(1-37)多肽的合成方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111531468.XA CN114057862A (zh) | 2021-12-14 | 2021-12-14 | 一种glp-1(1-37)多肽的合成方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114057862A true CN114057862A (zh) | 2022-02-18 |
Family
ID=80229526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111531468.XA Pending CN114057862A (zh) | 2021-12-14 | 2021-12-14 | 一种glp-1(1-37)多肽的合成方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114057862A (zh) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108948179A (zh) * | 2018-07-23 | 2018-12-07 | 安徽省国平药业有限公司 | 一种glp-1（7-37）的合成方法 |
| CN112074289A (zh) * | 2018-04-25 | 2020-12-11 | 詹森药业有限公司 | 硫醚环状肽胰淀素受体调节剂 |
| CN113121673A (zh) * | 2021-04-08 | 2021-07-16 | 润辉生物技术（威海）有限公司 | 一种固液结合法制备依降钙素的方法 |
| CN113521255A (zh) * | 2021-07-13 | 2021-10-22 | 中国药科大学 | Iapp-ft在制备胰岛淀粉样多肽聚集和纤维化抑制剂中的应用 |
-
2021
- 2021-12-14 CN CN202111531468.XA patent/CN114057862A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112074289A (zh) * | 2018-04-25 | 2020-12-11 | 詹森药业有限公司 | 硫醚环状肽胰淀素受体调节剂 |
| CN108948179A (zh) * | 2018-07-23 | 2018-12-07 | 安徽省国平药业有限公司 | 一种glp-1（7-37）的合成方法 |
| CN113121673A (zh) * | 2021-04-08 | 2021-07-16 | 润辉生物技术（威海）有限公司 | 一种固液结合法制备依降钙素的方法 |
| CN113521255A (zh) * | 2021-07-13 | 2021-10-22 | 中国药科大学 | Iapp-ft在制备胰岛淀粉样多肽聚集和纤维化抑制剂中的应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110372785B (zh) | 一种索马鲁肽的合成方法 | |
| CN111253475B (zh) | Glp-1激动多肽化合物及其盐与合成方法及用途 | |
| CN103497245B (zh) | 一种合成胸腺法新的方法 | |
| CN109369798A (zh) | 一种合成索玛鲁肽的方法 | |
| CN114437181B (zh) | 一种glp-1r/gcgr/gipr三重受体激动剂及其应用 | |
| WO2024159569A1 (zh) | 一种司美格鲁肽的合成方法 | |
| CN103087181A (zh) | 一种利拉鲁肽的固相合成方法 | |
| CN113135991B (zh) | 一种制备索玛鲁肽的方法 | |
| CN110922470A (zh) | 一种索玛鲁肽的制备方法 | |
| CN102977204A (zh) | 一种固相合成glp-1类似物的方法 | |
| CN112679602A (zh) | 索马鲁肽的固相合成方法 | |
| CN114736271B (zh) | 一种Tirzepatide的合成方法 | |
| CN115536739A (zh) | 一种glp-1受体和gcg受体共激动多肽衍生物的制备方法 | |
| CN1552728B (zh) | 一种生长抑素的合成方法 | |
| US9637517B2 (en) | Method for preparing exenatide | |
| CN117106055A (zh) | 一种替尔泊肽的合成方法 | |
| CN111944038B (zh) | 一种索玛鲁肽的合成方法 | |
| CN114057862A (zh) | 一种glp-1(1-37)多肽的合成方法 | |
| WO2023279323A1 (zh) | 一种合成glp-1类似物的方法 | |
| CN112175067B (zh) | 一种替度鲁肽的制备方法 | |
| CN113637064B (zh) | 一种索玛鲁肽的合成方法 | |
| CN108948179A (zh) | 一种glp-1（7-37）的合成方法 | |
| CN111944037B (zh) | 一种索玛鲁肽的合成方法 | |
| CN110845600B (zh) | 一种制备利拉鲁肽的方法 | |
| CN113173987B (zh) | 一种合成利司那肽的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220218 |