CN116333091A - Method for preparing HGH (176-191) by continuous flow solid phase synthesis - Google Patents
Method for preparing HGH (176-191) by continuous flow solid phase synthesis Download PDFInfo
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- CN116333091A CN116333091A CN202310335967.4A CN202310335967A CN116333091A CN 116333091 A CN116333091 A CN 116333091A CN 202310335967 A CN202310335967 A CN 202310335967A CN 116333091 A CN116333091 A CN 116333091A
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- LSTCHEVTSMJOCE-YWWHUHEESA-N 66004-57-7 Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)OC(=O)CC[C@H](NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@@H](N)CCC(N)=O)C=S)C(C)C)C(=O)NCC(=O)OC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)CNC(=O)[C@H](CS)NC(=O)[C@@H](N)CO)C1=CC=CC=C1 LSTCHEVTSMJOCE-YWWHUHEESA-N 0.000 title claims abstract description 67
- 108010002097 somatotropin (176-191) Proteins 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 54
- 238000010532 solid phase synthesis reaction Methods 0.000 title claims abstract description 38
- 239000011347 resin Substances 0.000 claims abstract description 61
- 229920005989 resin Polymers 0.000 claims abstract description 61
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 57
- 150000001413 amino acids Chemical class 0.000 claims abstract description 50
- 239000012634 fragment Substances 0.000 claims abstract description 45
- 238000005859 coupling reaction Methods 0.000 claims abstract description 39
- 238000010168 coupling process Methods 0.000 claims abstract description 32
- 230000008878 coupling Effects 0.000 claims abstract description 30
- 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 29
- 239000002904 solvent Substances 0.000 claims abstract description 26
- 239000003875 Wang resin Substances 0.000 claims abstract description 21
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 19
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 19
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 claims abstract description 18
- 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 abstract description 16
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 16
- 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 abstract description 13
- 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 abstract description 10
- -1 fmoc-Gly-OH Chemical compound 0.000 claims abstract description 10
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 claims abstract description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 74
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 50
- 239000000243 solution Substances 0.000 claims description 49
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 43
- 238000006243 chemical reaction Methods 0.000 claims description 32
- 238000005406 washing Methods 0.000 claims description 32
- 125000006239 protecting group Chemical group 0.000 claims description 29
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 20
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical group CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 18
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 18
- 239000003795 chemical substances by application Substances 0.000 claims description 17
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 14
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 14
- 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 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 238000011068 loading method Methods 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- 229920001184 polypeptide Chemical group 0.000 claims description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 12
- 230000008961 swelling Effects 0.000 claims description 12
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 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 10
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 10
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 9
- 238000005520 cutting process Methods 0.000 claims description 9
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- 230000035484 reaction time Effects 0.000 claims description 8
- 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 6
- 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 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 230000010933 acylation Effects 0.000 claims description 6
- 238000005917 acylation reaction Methods 0.000 claims description 6
- 239000003054 catalyst Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
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- 238000011049 filling Methods 0.000 claims description 6
- 238000005086 pumping Methods 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 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
- QSECPQCFCWVBKM-UHFFFAOYSA-N 2-iodoethanol Chemical compound OCCI QSECPQCFCWVBKM-UHFFFAOYSA-N 0.000 claims description 4
- TZCYLJGNWDVJRA-UHFFFAOYSA-N 6-chloro-1-hydroxybenzotriazole Chemical compound C1=C(Cl)C=C2N(O)N=NC2=C1 TZCYLJGNWDVJRA-UHFFFAOYSA-N 0.000 claims description 4
- 239000007821 HATU Substances 0.000 claims description 4
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 claims description 4
- 239000012043 crude product Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 230000003647 oxidation Effects 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 claims 1
- 108010051696 Growth Hormone Proteins 0.000 description 6
- 102000018997 Growth Hormone Human genes 0.000 description 5
- 239000000122 growth hormone Substances 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000006340 racemization Effects 0.000 description 3
- KWGRBVOPPLSCSI-WPRPVWTQSA-N (-)-ephedrine Chemical compound CN[C@@H](C)[C@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WPRPVWTQSA-N 0.000 description 2
- PFRGGOIBYLYVKM-UHFFFAOYSA-N 15alpha-hydroxylup-20(29)-en-3-one Natural products CC(=C)C1CCC2(C)CC(O)C3(C)C(CCC4C5(C)CCC(=O)C(C)(C)C5CCC34C)C12 PFRGGOIBYLYVKM-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-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
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- SOKRNBGSNZXYIO-UHFFFAOYSA-N Resinone Natural products CC(=C)C1CCC2(C)C(O)CC3(C)C(CCC4C5(C)CCC(=O)C(C)(C)C5CCC34C)C12 SOKRNBGSNZXYIO-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- BXRNXXXXHLBUKK-UHFFFAOYSA-N piperazine-2,5-dione Chemical compound O=C1CNC(=O)CN1 BXRNXXXXHLBUKK-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
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- 230000001681 protective effect Effects 0.000 description 2
- 230000001603 reducing effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- HBGPNLPABVUVKZ-POTXQNELSA-N (1r,3as,4s,5ar,5br,7r,7ar,11ar,11br,13as,13br)-4,7-dihydroxy-3a,5a,5b,8,8,11a-hexamethyl-1-prop-1-en-2-yl-2,3,4,5,6,7,7a,10,11,11b,12,13,13a,13b-tetradecahydro-1h-cyclopenta[a]chrysen-9-one Chemical compound C([C@@]12C)CC(=O)C(C)(C)[C@@H]1[C@H](O)C[C@]([C@]1(C)C[C@@H]3O)(C)[C@@H]2CC[C@H]1[C@@H]1[C@]3(C)CC[C@H]1C(=C)C HBGPNLPABVUVKZ-POTXQNELSA-N 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 description 1
- 229960001117 clenbuterol Drugs 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 description 1
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- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229960002179 ephedrine Drugs 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
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- 230000004060 metabolic process Effects 0.000 description 1
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- 238000007243 oxidative cyclization reaction Methods 0.000 description 1
- 208000035824 paresthesia Diseases 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
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- 210000000707 wrist Anatomy 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
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- 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)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for preparing HGH (176-191) by continuous flow solid phase synthesis, which uses a continuous flow solid phase synthesis method to prepare fragments Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH, uses a Wang resin as a carrier, and uses a continuous flow solid phase synthesis method to sequentially connect Fmoc-Phe-OH, fmoc-Cys (Trt) -Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH to obtain peptide resin. The method shortens the deprotection and coupling time, reduces the dosage of amino acid and solvent, ensures that the purity of the prepared crude peptide reaches more than 80 percent, and greatly reduces the synthesis cost.
Description
Technical Field
The invention belongs to the technical field of polypeptide drug synthesis, and particularly relates to a method for preparing HGH (176-191) by using a continuous flow solid phase synthesis strategy.
Background
Human growth hormone fragment HGH (176-191) is a protein peptide prepared from amino acid positions 176-191. HGH of HGH (176-191) represents human growth hormone, and has been proved to be a very desirable substance for reducing fat and helping to lose weight. HGH (176-191) works as part of HGH growth hormone peptide by mimicking the way natural HGH regulates human metabolism, but does not have adverse side effects on insulin sensitivity, blood glucose, cell proliferation or muscle growth as whole-segment HGH, and such drugs may also promote anti-aging. In fact, HGH (176-191) is more potent than HGH in stimulating lipolysis, and can inhibit the production of lipids such as fatty acids in the body. HGH (176-191) has a strength 10 times that of normal growth hormone or more, to promote weight loss in vivo. Unlike other common lipid-lowering drugs, the user does not have a great increase in hunger sensation and does not have side effects such as stress caused by clenbuterol, ephedrine and the like. Since HGH (176-191) is only the terminal portion of HGH growth hormone, it does not participate in competitive binding to HGH receptor. Several studies have shown that HGH (176-191) also promotes lean body mass gain, protein synthesis, increases bone density, and improves sleep. Compared with the whole section, the growth hormone fragments are used, the use fragments do not worry about side effects possibly caused by the whole section of the growth hormone such as thyroid gland problem, glucose sensitivity, tingling sensation of joints and wrists and the like, and meanwhile, the growth hormone fragments have strong lipid reducing effect and can increase vascularity.
The peptide sequence of HGH (176-191) is: H-Tyr 176 -Leu 177 -Arg 178 -Ile 179 -Val 180 -Gln 181 -cyclo[Cys 182 -Arg 183 -Ser 184 -Val 185 -Glu 186 -Gly 187 -Ser 188 -Cys 189 ]-Gly 190 -Phe 191 -OH；
Few reports on HGH (176-191) synthesis have been made on the method of grafting amino acids one by one using a conventional solid phase synthesis strategy, and diketopiperazine is easily generated at amino acid position 191 to 190, resulting in the active site falling off from the resin, resulting in an excessively low yield. In addition, the difficult sequence and racemization of amino acid exist in the whole peptide sequence, and the amount of amino acid fed and the reaction time which are generally used cannot reach the total reaction, so that the purity of the crude product is very low, and the purification is too difficult.
In the process of polypeptide coupling, the liquid phase synthesis of dipeptide fragment and the solid phase synthesis of decapeptide fragment are used, and the fragment peptide is connected in the main chain of HGH (176-191) by solid phase synthesis method, so that the method can effectively avoid the production of diketopiperazine and missing peptide, but the problems of overlong coupling time of the fragment peptide and the main chain and overlarge consumption of solvent and amino acid are faced. Based on the method, the invention adopts a continuous flow solid-phase synthesis method to prepare fragments Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH and participates in main chain reaction, and the main chain uses Wang resin as a carrier to carry out coupling according to peptide sequence.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel method for preparing HGH (176-191) by continuous flow solid phase synthesis, which has high synthesis efficiency and less impurity of synthesized products.
The object of the invention is achieved by the following technical scheme. The invention relates to a method for preparing HGH (176-191) by continuous flow solid phase synthesis, which is characterized in that a continuous flow solid phase synthesis method is used for preparing fragments Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH, a main chain takes Wang resin as a carrier, and Fmoc-Phe-OH, fmoc-Cys (Trt) -Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH are sequentially connected by using the continuous flow solid phase synthesis method to obtain peptide resin.
The invention relates to a method for preparing HGH (176-191) by continuous flow solid phase synthesis, which further adopts the preferable technical scheme that:
1. the method for preparing HGH (176-191) comprises the following specific reaction steps:
(1) Swelling 2-CTC resin in advance, and then loading the 2-CTC resin into an adjustable glass reaction column, and adjusting the piston, the liquid flow rate and the ultraviolet wavelength;
(2) The 2-CTC resin is sequentially connected with amino acids Fmoc-Val-OH, fmoc-Ser (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Cys (Trt) -OH, fmoc-Gln (Trt) -OH, fmoc-Val-OH, fmoc-Ile-OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH and Fmoc-Tyr (tBu) -OH with protecting groups through a continuous flow solid phase synthesis strategy to prepare Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Pbf) -Arg (Ser (tBu) -Val-CTC resin;
(3) Performing full protection cutting on the peptide resin prepared in the step (2) to obtain Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH fragments;
(4) Swelling Wang resin in advance, and then loading the swelled Wang resin into an adjustable glass reaction column, and adjusting the piston, the liquid flow rate and the ultraviolet wavelength;
(5) The Wang resin is sequentially connected with amino acid with protecting groups and polypeptide fragments Fmoc-Phe-OH, fmoc-Cys (Trt) -Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Pbf) -Arg (Pbf) -Ser (tBu) -Val-OH through a continuous flow solid phase synthesis strategy to prepare H-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-Glu-Gly-Ser (tBu) -Cys (Trt) -Gly-Phe-Wang resin;
(6) Cutting the polypeptide resin prepared in the step (5) to obtain HGH (176-191) linear peptide;
(7) Cyclizing the linear peptide prepared in the step (6) to obtain a crude HGH (176-191).
2. In the method for preparing HGH (176-191):
the Fmoc removal solution is a piperidine solution with the mass concentration of 15% -30%;
the solvent in the Fmoc removing solution is one or more of N, N-dimethylformamide, N-methylpyrrolidone and tetrahydrofuran.
3. In the method for preparing HGH (176-191): the amino acid coupling solvent is one or more of N, N-dimethylformamide, N-methylpyrrolidone and methylene dichloride;
the condensing agent is any one of DIC/HOBt, DIC/Cl-HOBt, HBTU/DIEA, HATU/DIEA, pyBOP/DIEA, (HBUT+HOBt)/DIEA, and (HAUT+HOAt)/DIEA.
4. In the method for preparing HGH (176-191): the acylation catalyst is 4-dimethylaminopyridine.
5. In the method for preparing HGH (176-191): the molar ratio of the amino acid or fragment with the protecting group, the condensing reagent and the resin is 1.1-3: 1.1-3: 1, the mass ratio of the reaction solvent to the resin is 1-5: 1.
6. in the method for preparing HGH (176-191): the coupling reaction temperature of the amino acid or the fragment with the protecting group is 20-35 ℃; fmoc removal temperature is 15-25 ℃.
7. In the method for preparing HGH (176-191): the resin filling height is 1-10 cm; the flow rate of the Fmoc removal solution is 5-30 ml/min, and the reaction time is 2-10 min; the flow rate of the washing solvent is 5-30 ml/min, and the time is 10-20 min.
8. In the method for preparing HGH (176-191): the reaction pressure is 0-1.0 MPa; the ultraviolet wavelength is 210-400 nm.
9. The preparation method of the HGH (176-191) comprises the following steps:
1. Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH fragment:
adding 2-CTC-resin into dichloromethane, swelling for 10min, loading into an adjustable reaction column, adjusting a piston, washing with pure DMF until a UV baseline is stable and returns to zero, pumping a DMF mixed solution of Fmoc-Val-OH and a condensing agent at a certain flow rate, reacting until the change value of a UV curve is less than 1mAu in one minute, and washing with pure DMF until the UV curve is less than 100mAu; removing Fmoc to reach a maximum value by using a deprotection solution, returning to less than 100mAu, and then coupling amino acids with protecting groups to H-Val-resin one by one according to a fragment peptide sequence;
cleaving the peptide resin using a 1% trifluoroacetic acid/dichloromethane mixed solution or a 20% trifluoroethanol/dichloromethane mixed solution to obtain fully protected polypeptide fragments;
the Fmoc removal solution is 20% piperidine solution, and the solvent is one of DMF, NMP, THF;
the amino acids with protecting groups are: fmoc-Val-OH, fmoc-Ser (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Cys (Trt) -OH, fmoc-Gln (Trt) -OH, fmoc-Val-OH, fmoc-Ile-OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Cys (Trt) -Gly-OH;
2. H-Tyr (tBu) -Leu-Arg (pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (pbf) -Ser (tBu) -Val-Glu (OtBu) -Gly-Ser (tBu) -Cys (Trt) -Gly-Phe-Wang Synthesis:
adding Wang resin into dichloromethane, swelling for 10min, loading into an adjustable reaction column, adjusting a piston, washing with pure DMF until a UV baseline is stable and returns to zero, pumping a DMF mixed solution of Fmoc-Phe-OH, a condensing agent and 4-dimethylaminopyridine at a certain flow rate, reacting until a UV curve changes to a value less than 1mAu in one minute, and washing with pure DMF until the UV curve is less than 100mAu; removing Fmoc to a maximum value of a UV curve by using a deprotection solution, returning to less than 100mAu, washing, coupling amino acid and fragments with protecting groups to H-Phe-resin one by one according to a peptide sequence, and returning to less than 100mAu after removing Fmoc to the maximum value of the UV curve by using the deprotection solution, washing, and collecting samples;
3. H-Tyr-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-OH synthesis: cutting by using TFA, filtering, settling, washing and drying to obtain HGH (176-191) linear peptide;
4. H-Tyr-Leu-Arg-Ile-Val-Gln-cyclo [ Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys ] -Gly-Phe-OH synthesis:
dissolving the prepared linear peptide in acetic acid aqueous solution, and adding iodoethanol or hydrogen peroxide for oxidation to obtain a crude product of HGH (176-191).
Compared with the prior art, the invention has the following innovation points and advantages:
1. the method of the present invention utilizes a continuous flow solid phase synthesis strategy to produce HGH (176-191). Compared with intermittent solid phase synthesis, the continuous flow solid phase synthesis process of HGH (176-191) shortens the coupling time of each step of amino acid from 1.5-3 hours to less than 30 minutes, shortens the Fmoc removal protection time from original 30 minutes to less than 10 minutes, greatly improves the production efficiency of the synthesis procedure, greatly reduces the dosage of amino acid, condensing agent, deprotection solution and solvent, does not need manual feeding during the coupling and Fmoc removal protection of amino acid, effectively avoids solvent volatilization and personnel contact, and ensures safer production process.
2. According to the method, the Fmoc solid-phase synthesis strategy is adopted to realize efficient coupling in the adjustable reaction column, the resin density is maintained through the adjustable piston, and the Fmoc coupling and Fmoc removing efficiency can be detected in real time by utilizing UV/Vis. The method shortens the deprotection and coupling time, reduces the dosage of amino acid and solvent, ensures that the purity of the prepared crude peptide reaches more than 80 percent, and greatly reduces the synthesis cost.
3. When Fmoc-Arg (Pbf) -OH is coupled, the reaction time is too long, racemization of amino acid is easy to cause, the coupling can be completed in a short time by continuous flow solid phase synthesis, the racemization of the amino acid is avoided, and the impurity of the crude polypeptide is reduced.
Drawings
FIG. 1 is a high performance liquid chromatography for preparing a crude HGH (176-191) product in an embodiment;
FIG. 2 is a mass spectrum of a crude HGH (176-191) prepared in example;
FIG. 3 is a UV curve of the coupling reaction.
Detailed Description
The invention discloses a preparation method of HGH (176-191) by continuous flow solid phase synthesis, and examples are only a part of the invention, but not all the examples. The embodiments are only for illustrating the technical features and features of the present invention, and should not be construed as limiting the scope of the present invention, and all equivalent changes or modifications made according to the essence of the present invention are included in the scope of the present invention.
Example 1, a method of preparing HGH (176-191) by continuous flow solid phase Synthesis
The fragments Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH were prepared using a continuous flow solid phase synthesis method, and Fmoc-Phe-OH, fmoc-Cys (Trt) -Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH were sequentially connected using a continuous flow solid phase synthesis method to obtain peptide resins.
The specific reaction steps are as follows:
(1) Swelling 2-CTC resin in advance, and then loading the 2-CTC resin into an adjustable glass reaction column, and adjusting the piston, the liquid flow rate and the ultraviolet wavelength;
(2) The 2-CTC resin is sequentially connected with amino acids Fmoc-Val-OH, fmoc-Ser (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Cys (Trt) -OH, fmoc-Gln (Trt) -OH, fmoc-Val-OH, fmoc-Ile-OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH and Fmoc-Tyr (tBu) -OH with protecting groups through a continuous flow solid phase synthesis strategy to prepare Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Pbf) -Arg (Ser (tBu) -Val-CTC resin;
(3) Performing full protection cutting on the peptide resin prepared in the step (2) to obtain Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH fragments;
(4) Swelling Wang resin in advance, and then loading the swelled Wang resin into an adjustable glass reaction column, and adjusting the piston, the liquid flow rate and the ultraviolet wavelength;
(5) The Wang resin is sequentially connected with amino acid with protecting groups and polypeptide fragments Fmoc-Phe-OH, fmoc-Cys (Trt) -Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Pbf) -Arg (Pbf) -Ser (tBu) -Val-OH through a continuous flow solid phase synthesis strategy to prepare H-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-Glu-Gly-Ser (tBu) -Cys (Trt) -Gly-Phe-Wang resin;
(6) Cutting the polypeptide resin prepared in the step (5) to obtain HGH (176-191) linear peptide;
(7) Cyclizing the linear peptide prepared in the step (6) to obtain a crude HGH (176-191).
In the method for preparing HGH (176-191):
the Fmoc removal solution is a piperidine solution with the mass concentration of 15 percent;
the solvent in the Fmoc removal solution is N, N-dimethylformamide;
the amino acid coupling solvent is N, N-dimethylformamide;
the condensing agent is DIC/HOBt;
the acylation catalyst is 4-dimethylaminopyridine;
the molar ratio of the amino acid or the fragment with the protecting group, the condensing reagent and the resin is 1.1:3:1, the mass ratio of the reaction solvent to the resin is 1:1. the coupling reaction temperature of the amino acid or fragment with the protecting group is 20 ℃; the Fmoc removal temperature was 15 ℃. The filling height of the resin is 1cm; the flow rate of Fmoc removal solution is 5ml/min, and the reaction time is 2min; the washing solvent flow rate was 30ml/min for 10min. The reaction pressure is 1.0MPa; the ultraviolet wavelength was 210nm.
Example 1, a method of preparing HGH (176-191) by continuous flow solid phase Synthesis
In the method for preparing HGH (176-191):
the Fmoc removal solution is a piperidine solution with the mass concentration of 15% -30%;
the solvent in the Fmoc removing solution is one or more of N, N-dimethylformamide, N-methylpyrrolidone and tetrahydrofuran.
The amino acid coupling solvent is one or more of N, N-dimethylformamide, N-methylpyrrolidone and methylene dichloride;
the condensing agent is any one of DIC/HOBt, DIC/Cl-HOBt, HBTU/DIEA, HATU/DIEA, pyBOP/DIEA, (HBUT+HOBt)/DIEA, and (HAUT+HOAt)/DIEA.
The acylation catalyst is 4-dimethylaminopyridine.
The molar ratio of the amino acid or fragment with the protecting group, the condensing reagent and the resin is 1.1-3: 1.1-3: 1, the mass ratio of the reaction solvent to the resin is 1-5: 1. the coupling reaction temperature of the amino acid or the fragment with the protecting group is 20-35 ℃; fmoc removal temperature is 15-25 ℃. The resin filling height is 1-10 cm; the flow rate of the Fmoc removal solution is 5-30 ml/min, and the reaction time is 2-10 min; the flow rate of the washing solvent is 5-30 ml/min, and the time is 10-20 min. The reaction pressure is 0-1.0 MPa; the ultraviolet wavelength is 210-400 nm.
The remainder was the same as in example 1.
Example 2, a method of preparing HGH (176-191) by continuous flow solid phase Synthesis
In the method for preparing HGH (176-191):
the Fmoc removal solution is a piperidine solution with the mass concentration of 30%;
the solvent in the Fmoc removal solution is N-methyl pyrrolidone;
the amino acid coupling solvent is N-methyl pyrrolidone;
condensing agent is DIC/Cl-HOBt or HBTU/DIEA;
the acylation catalyst is 4-dimethylaminopyridine;
the molar ratio of the amino acid or the fragment with the protecting group, the condensing reagent and the resin is 3:1.1:1, the mass ratio of the reaction solvent to the resin is 5:1. the coupling reaction temperature of the amino acid or fragment with the protecting group is 35 ℃; fmoc removal temperature was 25 ℃. The filling height of the resin is 10cm; the flow rate of Fmoc removal solution is 30ml/min, and the reaction time is 10min; the washing solvent flow rate was 5ml/min for 20min. The reaction pressure is 0.5MPa; the ultraviolet wavelength is 400nm.
The remainder was the same as in example 1.
Example 3A method for preparing HGH (176-191) by continuous flow solid phase Synthesis
In the method for preparing HGH (176-191):
the Fmoc removal solution is a piperidine solution with the mass concentration of 25%;
the solvent in the Fmoc removal solution is tetrahydrofuran;
the amino acid coupling solvent is methylene dichloride;
the condensing agent is HATU/DIEA or PyBOP/DIEA or (HBUT+HOBt)/DIEA or (HAUT+HOAt)/DIEA;
the acylation catalyst is 4-dimethylaminopyridine.
The molar ratio of the amino acid or the fragment with the protecting group, the condensing reagent and the resin is 2:2:1, the mass ratio of the reaction solvent to the resin is 3:1. the coupling reaction temperature of the amino acid or fragment with the protecting group is 25 ℃; fmoc removal temperature was 20 ℃. The filling height of the resin is 5cm; the flow rate of Fmoc removal solution is 20ml/min, and the reaction time is 5min; the washing solvent flow rate was 15 ml/min for 15min. The reaction pressure is 0.3MPa; the ultraviolet wavelength was 300nm.
Example 4, a method for preparing HGH (176-191) by continuous flow solid phase synthesis, comprising the following steps:
1. Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH fragment:
adding 2-CTC-resin into dichloromethane, swelling for 10min, loading into an adjustable reaction column, adjusting a piston, washing with pure DMF until a UV baseline is stable and returns to zero, pumping a DMF mixed solution of Fmoc-Val-OH and a condensing agent at a certain flow rate, reacting until the change value of a UV curve is less than 1mAu in one minute, and washing with pure DMF until the UV curve is less than 100mAu; removing Fmoc to reach a maximum value by using a deprotection solution, returning to less than 100mAu, and then coupling amino acids with protecting groups to H-Val-resin one by one according to a fragment peptide sequence;
cleaving the peptide resin using a 1% trifluoroacetic acid/dichloromethane mixed solution or a 20% trifluoroethanol/dichloromethane mixed solution to obtain fully protected polypeptide fragments;
the Fmoc removal solution is 20% piperidine solution, and the solvent is one of DMF, NMP, THF;
the amino acids with protecting groups are: fmoc-Val-OH, fmoc-Ser (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Cys (Trt) -OH, fmoc-Gln (Trt) -OH, fmoc-Val-OH, fmoc-Ile-OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Cys (Trt) -Gly-OH;
2. H-Tyr (tBu) -Leu-Arg (pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (pbf) -Ser (tBu) -Val-Glu (OtBu) -Gly-Ser (tBu) -Cys (Trt) -Gly-Phe-Wang Synthesis:
adding Wang resin into dichloromethane, swelling for 10min, loading into an adjustable reaction column, adjusting a piston, washing with pure DMF until a UV baseline is stable and returns to zero, pumping a DMF mixed solution of Fmoc-Phe-OH, a condensing agent and 4-dimethylaminopyridine at a certain flow rate, reacting until a UV curve changes to a value less than 1mAu in one minute, and washing with pure DMF until the UV curve is less than 100mAu; removing Fmoc to a maximum value of a UV curve by using a deprotection solution, returning to less than 100mAu, washing, coupling amino acid and fragments with protecting groups to H-Phe-resin one by one according to a peptide sequence, and returning to less than 100mAu after removing Fmoc to the maximum value of the UV curve by using the deprotection solution, washing, and collecting samples;
3. H-Tyr-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-OH synthesis: cutting by using TFA, filtering, settling, washing and drying to obtain HGH (176-191) linear peptide;
4. H-Tyr-Leu-Arg-Ile-Val-Gln-cyclo [ Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys ] -Gly-Phe-OH synthesis:
dissolving the prepared linear peptide in acetic acid aqueous solution, and adding iodoethanol or hydrogen peroxide for oxidation to obtain a crude product of HGH (176-191).
Example 5: a method for preparing HGH (176-191) by continuous flow solid phase synthesis comprises the following steps:
2-CTC-resin 18.2 g (substitution = 1.1 mmol/g) was added to the beaker and the resin was swelled with 50 mL DCM for 5min and then added to the adjustable glass reaction column. The tubing was connected and the UV was turned on and the UV wavelength 270 nm was set and the reaction column piston was adjusted to have the bottom of the piston approximately 3mm from the resin surface. The DMF channel was opened and the flow rate was adjusted to 20mL/min, and zeroed after the UV curve was stationary. The amino acid with protecting group is coupled with 2-CTC resin one by one according to peptide sequence, the condensing agent uses DIC/HOBt system, and the dosage of amino acid and condensing agent is 1.5 times of resin. Deprotection and coupling resin heights and reaction pressures were observed in real time using a 20% PIP/DMF solution to give Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-CTC peptide resin 21.53 g. Removing Fmoc protection and coupling, wherein the UV absorption value in one minute is less than 1mAu, and the reaction end point is taken as the coupling end point; taking a UV absorption value less than 100mAu as a post-coupling washing endpoint; UV absorbance values less than 1mAu are post-deprotection wash endpoints. The flow rates of the coupling circulating liquid, the Fmoc-removing protective liquid and the washing liquid are all 20mL/min.
Full protection cleavage was performed using 10 times the amount of peptide resin 20% TFE/DCM and spin-dried on a rotary evaporator to give 10.91 g of decapeptide dry powder.
9.43 g Wang resin (substitution = 0.53 mmol/g) was added to the beaker and the resin was swollen with 50 mL DCM for 5 minutes and added to the adjustable glass reaction column. The pipeline is connected, UV is turned on, ultraviolet wavelength is set to 280nm, and the piston of the reaction column is regulated, so that the distance between the bottom of the piston and the surface of the resin is about 3mm. And opening the DMF channel, regulating the flow rate to be 20mL/min, and returning to zero after the UV curve is stable. The amino acid and fragment with protecting group are coupled with Wang resin one by one according to peptide sequence, and the condensing agent adopts DIC/HOBt system, and the dosage of amino acid and condensing agent is 1.5 times of that of resin. Deprotection and coupling resin heights and reaction pressures were observed in real time using a 20% PIP/DMF solution to give 24.8 g of H-Tyr (tBu) -Leu-Arg (pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (pbf) -Ser (tBu) -Val-Glu (OtBu) -Gly-Ser (tBu) -Cys (Trt) -Gly-Phe-Wang peptide resin. Removing Fmoc protection and coupling, wherein the UV absorption value in one minute is less than 1mAu, and the reaction end point is taken as the coupling end point; taking a UV absorption value less than 100mAu as a post-coupling washing endpoint; UV absorbance values less than 1mAu are post-deprotection wash endpoints. The flow rates of the coupling circulating solution, the Fmoc-removing protective solution and the washing solution are all 20mL/min.
Claims (10)
1. A method for preparing HGH (176-191) by continuous flow solid phase synthesis, which is characterized in that: preparation of fragments Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val using continuous flow solid phase Synthesis
And (3) taking Wang resin as a carrier for the main chain, and sequentially connecting Fmoc-Phe-OH, fmoc-Cys (Trt) -Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Pbf) -Arg (Pbf) -Ser (tBu) -Val-OH by using a continuous flow solid phase synthesis method to obtain the peptide resin.
2. The method for preparing HGH (176-191) according to claim 1, characterized in that the specific reaction steps are as follows:
(1) Swelling 2-CTC resin in advance, and then loading the 2-CTC resin into an adjustable glass reaction column, and adjusting the piston, the liquid flow rate and the ultraviolet wavelength;
(2) The 2-CTC resin is sequentially connected with amino acids Fmoc-Val-OH, fmoc-Ser (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Cys (Trt) -OH, fmoc-Gln (Trt) -OH, fmoc-Val-OH, fmoc-Ile-OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH and Fmoc-Tyr (tBu) -OH with protecting groups through a continuous flow solid phase synthesis strategy to prepare Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Pbf) -Arg (Ser (tBu) -Val-CTC resin;
(3) Performing full protection cutting on the peptide resin prepared in the step (2) to obtain Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH fragments;
(4) Swelling Wang resin in advance, and then loading the swelled Wang resin into an adjustable glass reaction column, and adjusting the piston, the liquid flow rate and the ultraviolet wavelength;
(5) The Wang resin is sequentially connected with amino acid with protecting groups and polypeptide fragments Fmoc-Phe-OH, fmoc-Cys (Trt) -Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Pbf) -Arg (Pbf) -Ser (tBu) -Val-OH through a continuous flow solid phase synthesis strategy to prepare H-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-Glu-Gly-Ser (tBu) -Cys (Trt) -Gly-Phe-Wang resin;
(6) Cutting the polypeptide resin prepared in the step (5) to obtain HGH (176-191) linear peptide;
(7) Cyclizing the linear peptide prepared in the step (6) to obtain a crude HGH (176-191).
3. A method of preparing HGH (176-191) according to claim 2, characterized in that:
the Fmoc removal solution is a piperidine solution with the mass concentration of 15% -30%;
the solvent in the Fmoc removing solution is one or more of N, N-dimethylformamide, N-methylpyrrolidone and tetrahydrofuran.
4. A method of preparing HGH (176-191) according to claim 2, characterized in that: the amino acid coupling solvent is one or more of N, N-dimethylformamide, N-methylpyrrolidone and methylene dichloride;
the condensing agent is any one of DIC/HOBt, DIC/Cl-HOBt, HBTU/DIEA, HATU/DIEA, pyBOP/DIEA, (HBUT+HOBt)/DIEA, and (HAUT+HOAt)/DIEA.
5. A method of preparing HGH (176-191) according to claim 2, characterized in that: the acylation catalyst is 4-dimethylaminopyridine.
6. A method of preparing HGH (176-191) according to claim 2, characterized in that: the molar ratio of the amino acid or fragment with the protecting group, the condensing reagent and the resin is 1.1-3: 1.1-3: 1, the mass ratio of the reaction solvent to the resin is 1-5: 1.
7. a method of preparing HGH (176-191) according to claim 2, characterized in that: the coupling reaction temperature of the amino acid or the fragment with the protecting group is 20-35 ℃; fmoc removal temperature is 15-25 ℃.
8. A method of preparing HGH (176-191) according to claim 2, characterized in that: the resin filling height is 1-10 cm; the flow rate of the Fmoc removal solution is 5-30 ml/min, and the reaction time is 2-10 min; the flow rate of the washing solvent is 5-30 ml/min, and the time is 10-20 min.
9. A method of preparing HGH (176-191) according to claim 2, characterized in that: the reaction pressure is 0-1.0 MPa; the ultraviolet wavelength is 210-400 nm.
10. The method for producing HGH (176-191) according to claim 2, characterized in that the method comprises the steps of:
1. Fmoc-Tyr (tBu) -Leu-Arg (Pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (Pbf) -Ser (tBu) -Val-OH fragment:
adding 2-CTC-resin into dichloromethane, swelling for 10min, loading into an adjustable reaction column, adjusting a piston, washing with pure DMF until a UV baseline is stable and returns to zero, pumping a DMF mixed solution of Fmoc-Val-OH and a condensing agent at a certain flow rate, reacting until the change value of a UV curve is less than 1mAu in one minute, and washing with pure DMF until the UV curve is less than 100mAu; removing Fmoc to reach a maximum value by using a deprotection solution, returning to less than 100mAu, and then coupling amino acids with protecting groups to H-Val-resin one by one according to a fragment peptide sequence;
cleaving the peptide resin using a 1% trifluoroacetic acid/dichloromethane mixed solution or a 20% trifluoroethanol/dichloromethane mixed solution to obtain fully protected polypeptide fragments;
the Fmoc removal solution is 20% piperidine solution, and the solvent is one of DMF, NMP, THF;
the amino acids with protecting groups are: fmoc-Val-OH, fmoc-Ser (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Cys (Trt) -OH, fmoc-Gln (Trt) -OH, fmoc-Val-OH, fmoc-Ile-OH, fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Cys (Trt) -Gly-OH;
2. H-Tyr (tBu) -Leu-Arg (pbf) -Ile-Val-Gln (Trt) -Cys (Trt) -Arg (pbf) -Ser (tBu) -Val-Glu (OtBu) He-Glu (tBu)
Gly-Ser (tBu) -Cys (Trt) -Gly-Phe-Wang Synthesis:
adding Wang resin into dichloromethane, swelling for 10min, loading into an adjustable reaction column, adjusting a piston, washing with pure DMF until a UV baseline is stable and returns to zero, pumping a DMF mixed solution of Fmoc-Phe-OH, a condensing agent and 4-dimethylaminopyridine at a certain flow rate, reacting until a UV curve changes to a value less than 1mAu in one minute, and washing with pure DMF until the UV curve is less than 100mAu; removing Fmoc to a maximum value of a UV curve by using a deprotection solution, returning to less than 100mAu, washing, coupling amino acid and fragments with protecting groups to H-Phe-resin one by one according to a peptide sequence, and returning to less than 100mAu after removing Fmoc to the maximum value of the UV curve by using the deprotection solution, washing, and collecting samples;
3. H-Tyr-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-OH synthesis: cutting by using TFA, filtering, settling, washing and drying to obtain HGH (176-191) linear peptide;
4. H-Tyr-Leu-Arg-Ile-Val-Gln-cyclo [ Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys ] -Gly-Phe-OH synthesis:
dissolving the prepared linear peptide in acetic acid aqueous solution, and adding iodoethanol or hydrogen peroxide for oxidation to obtain a crude product of HGH (176-191).
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