CN112500453A - Solid-phase synthesis method of fragment of antibacterial peptide Iseganan - Google Patents
Solid-phase synthesis method of fragment of antibacterial peptide Iseganan Download PDFInfo
- Publication number
- CN112500453A CN112500453A CN202011457209.2A CN202011457209A CN112500453A CN 112500453 A CN112500453 A CN 112500453A CN 202011457209 A CN202011457209 A CN 202011457209A CN 112500453 A CN112500453 A CN 112500453A
- Authority
- CN
- China
- Prior art keywords
- fmoc
- resin
- iseganan
- reagent
- amino acid
- 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
- GUCYBPFJNGVFEB-XELKFLSISA-N iseganan Chemical compound C([C@H]1C(=O)N[C@H]2CSSC[C@H](NC(=O)[C@H](CC=3C=CC=CC=3)NC(=O)[C@H](CCCNC(N)=N)NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@H](C(=O)N[C@@H](CSSC[C@@H](C(N1)=O)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)C(C)C)C1=CC=C(O)C=C1 GUCYBPFJNGVFEB-XELKFLSISA-N 0.000 title claims abstract description 54
- 108010078256 antimicrobial peptide IB-367 Proteins 0.000 title claims abstract description 50
- 229950000488 iseganan Drugs 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 39
- 238000010532 solid phase synthesis reaction Methods 0.000 title claims abstract description 16
- 239000012634 fragment Substances 0.000 title claims abstract description 10
- 239000003910 polypeptide antibiotic agent Substances 0.000 title description 4
- 229920005989 resin Polymers 0.000 claims abstract description 112
- 239000011347 resin Substances 0.000 claims abstract description 112
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 34
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 22
- 125000006239 protecting group Chemical group 0.000 claims abstract description 20
- 229920003180 amino resin Polymers 0.000 claims abstract description 17
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims abstract description 16
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 15
- 229910052740 iodine Inorganic materials 0.000 claims abstract description 15
- 239000011630 iodine Substances 0.000 claims abstract description 15
- 239000000047 product Substances 0.000 claims abstract description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000005520 cutting process Methods 0.000 claims abstract description 9
- 230000003647 oxidation Effects 0.000 claims abstract description 9
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 9
- 230000009089 cytolysis Effects 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims abstract description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 claims abstract description 6
- 238000004108 freeze drying Methods 0.000 claims abstract description 5
- 239000012043 crude product Substances 0.000 claims abstract description 4
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims abstract description 4
- 230000001376 precipitating effect Effects 0.000 claims abstract description 4
- 238000005336 cracking Methods 0.000 claims abstract 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 72
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- 239000000243 solution Substances 0.000 claims description 40
- 239000003153 chemical reaction reagent Substances 0.000 claims description 39
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 238000005406 washing Methods 0.000 claims description 19
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 18
- 150000001413 amino acids Chemical group 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 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 16
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 16
- 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 15
- -1 Fmoc- (7-12) -OH Chemical compound 0.000 claims description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- 239000007822 coupling agent Substances 0.000 claims description 12
- 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 9
- 239000002131 composite material Substances 0.000 claims description 8
- 238000006467 substitution reaction Methods 0.000 claims description 8
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
- 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
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- RGTVXXNMOGHRAY-WDSKDSINSA-N Cys-Arg Chemical compound SC[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RGTVXXNMOGHRAY-WDSKDSINSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 239000006166 lysate Substances 0.000 claims description 4
- 230000001590 oxidative effect Effects 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000004007 reversed phase HPLC Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 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 description 3
- 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 3
- 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 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
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 3
- 239000007821 HATU Substances 0.000 claims description 3
- 239000012317 TBTU Substances 0.000 claims description 3
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 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 2
- HXOYWJCDYVODON-UHFFFAOYSA-N 4-[4-(hydroxymethyl)-3-methoxyphenoxy]butanoic acid Chemical compound COC1=CC(OCCCC(O)=O)=CC=C1CO HXOYWJCDYVODON-UHFFFAOYSA-N 0.000 claims description 2
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 claims description 2
- RDRBIXSNGAYLPT-UHFFFAOYSA-N CC1=CC=C(COC2=CC3=C(C=C2)C(NC(=O)OCC2C4=C(C=CC=C4)C4=C2C=CC=C4)C2=C(O3)C=CC=C2)C=C1 Chemical compound CC1=CC=C(COC2=CC3=C(C=C2)C(NC(=O)OCC2C4=C(C=CC=C4)C4=C2C=CC=C4)C2=C(O3)C=CC=C2)C=C1 RDRBIXSNGAYLPT-UHFFFAOYSA-N 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- 238000010511 deprotection reaction Methods 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- QMMFVYPAHWMCMS-UHFFFAOYSA-N Dimethyl sulfide Chemical compound CSC QMMFVYPAHWMCMS-UHFFFAOYSA-N 0.000 claims 3
- 239000007790 solid phase Substances 0.000 abstract description 11
- 238000009776 industrial production Methods 0.000 abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 24
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 12
- 235000001014 amino acid Nutrition 0.000 description 8
- 238000010907 mechanical stirring Methods 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 6
- 238000005086 pumping Methods 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 5
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 4
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000010355 oscillation Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 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 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- IIVWHGMLFGNMOW-UHFFFAOYSA-N 2-methylpropane Chemical compound C[C](C)C IIVWHGMLFGNMOW-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710176384 Peptide 1 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- DIZZIOFQEYSTPV-UHFFFAOYSA-N [I].CO Chemical compound [I].CO DIZZIOFQEYSTPV-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012045 crude solution Substances 0.000 description 1
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 description 1
- 229910000071 diazene Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004719 natural immunity Effects 0.000 description 1
- 230000003448 neutrophilic effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a solid-phase synthesis method of fragments of Iseganan, which comprises the following steps: adopting Fmoc/tBu solid phase synthesis, and connecting Fmoc-amino acid to carboxyl resin according to the amino acid sequence of the Iseganan fragment 7-12; iodine oxidation to form a first pair of disulfide bonds; carrying out full protection and cracking to obtain Fmoc- (7-12) -OH; connecting Fmoc-amino acid and the obtained Fmoc- (7-12) -OH to amino resin according to the sequence of the eosin by adopting Fmoc/tBu solid phase synthesis; iodine oxidation to form a second pair of disulfide bonds; removing Fmoc protecting groups to obtain fully-protected heptadecapeptide peptide resin; cutting by using a lysis solution, simultaneously removing all side chain protecting groups, and precipitating the cutting solution by using glacial ethyl ether to obtain a crude product of Iseganan; and filtering, separating, purifying and freeze-drying the crude Iseganan solution to obtain Iseganan trifluoroacetate or Iseganan acetate. The heptadecapeptide Iseganan is synthesized by adopting a pure solid phase mode, and the oxidation of two pairs of disulfide bonds is completed on resin, so that the positioning accuracy of the disulfide bonds is improved, the purity of the product is improved, and the large-scale industrial production is facilitated.
Description
Technical Field
The invention relates to preparation of a small molecular polypeptide compound, in particular to a preparation method of Iseganan (including Iseganan acetate, trifluoroacetate and free peptide).
Background
Iseganan (Iseganan) is an analog of porcine neutrophilic granulocyte peptide-1, and can bind to extracellular components of bacteria such as lipopolysaccharide or lipoteichoic acid, etc., to cause swelling, rupture, and kill bacteria by destroying cell membrane. It has inhibitory effect on gram-positive and gram-negative bacteria, fungi, and yeast. The molecular formula of Iseganan is C78H126N30O18S4Molecular weight is 1900.2852, and the amino acid sequence is as follows:
iseganan is a small molecular polypeptide which is widely existed in animals, plants, insects and human bodies, is encoded by genes and has a specific spatial structure, and the polypeptide is a component of natural immunity of organisms. The antibacterial peptide has the advantages of broad-spectrum efficient antimicrobial activity, good water solubility, thermal stability and the like. As a novel medicine, the antibacterial peptide has wide development and application prospects in the process of treating and preventing diseases of human beings.
In the prior art, various methods for synthesizing Iseganan have been reported, for example, patent CN102336813A reports a solid phase synthesis lineA method in which a peptide is then cyclized in a liquid phase. Synthesizing linear peptide in solid phase, removing sulfydryl protecting groups Trt on cysteine at 5 and 14 positions when cutting off the protective linear peptide on the solid phase, and oxidizing with hydrogen peroxide to form a first pair of disulfide bonds (Cys)5-14) (ii) a Then removing the sulfhydryl protecting group Acm of the cysteine at the 7 and 12 positions in the iodine methanol solution, and oxidizing to form a second pair of disulfide bonds. After the second pair of disulfide bonds are formed, the product is dissolved in a large amount of iodine solution, so that the method is difficult to carry out post-treatment, has a large amount of waste liquid, easily causes iodine residue and standard exceeding in the product, and is difficult to apply to large-scale production. WO2003/062266 discloses a method for synthesizing Iseganan fragments, wherein after two fragments 1-8 and 9-17 are synthesized in a solid phase manner, the two fragments are coupled in a liquid phase, protective groups are removed, and two pairs of disulfide bonds are formed through oxidation to obtain Iseganan, and the method is difficult to form selectively (Cys)5-14，Cys7-12) Two pairs of disulfide bonds.
In view of this, it is necessary to provide a synthesis method of Iseganan with higher selectivity of disulfide bond formation to improve industrial applicability.
Disclosure of Invention
The invention provides a preparation method of Iseganan, which has the advantages of low cost, simple steps, high yield and high purity, aiming at the problems related in the background technology.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a solid-phase synthesis method of fragment of Iseganan comprises the following steps:
(1) adopting Fmoc/tBu solid phase synthesis, and connecting Fmoc-amino acid to carboxyl resin by using a coupling agent according to the amino acid sequence of the Iseganan fragment 7-12 to obtain fully-protected hexapeptide resin; the Fmoc-amino acids attached to the carboxyl resin are in order: Fmoc-Cys (Trt) -OH, Fmoc-Phe-OH, Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH and Fmoc-Cys (Trt) -OH; the full-protection hexapeptide peptide resin is oxidized by iodine to form a first pair of disulfide bonds, and then is subjected to full-protection cleavage to obtain Fmoc-c (Cys-Arg (pbf) -Gly-Arg (pbf) -Phe-Cys) -OH which is marked as Fmoc- (7-12) -OH;
(2) adopting Fmoc/tBu solid phase synthesis, connecting Fmoc-amino acid and Fmoc- (7-12) -OH obtained in the step (1) to the swelled amino resin by using a coupling agent according to an amino acid sequence of eosin to obtain a fully-protected heptadecapeptide resin; the order of attachment to the amino resin is in the order: Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Cys (Trt) -OH, Fmoc-Val-OH, Fmoc- (7-12) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Leu-OH, Fmoc-Gly-OH and Fmoc-Arg (pbf) -OH; the fully-protected heptadecapeptide resin is oxidized by iodine to form a second pair of disulfide bonds, and Fmoc protecting groups are removed to obtain the fully-protected heptadecapeptide resin with two pairs of disulfide bonds;
(3) cutting the fully-protected heptadecapeptide resin obtained in the step (2) by using a lysis solution, removing all side chain protecting groups simultaneously, and precipitating the cutting solution by using glacial ethyl ether to obtain a crude product of Iseganan;
(4) and (4) filtering, separating, purifying and freeze-drying the crude Iseganan solution obtained in the step (3) to obtain Iseganan trifluoroacetate or Iseganan acetate.
In the scheme of the invention, the carboxyl resin in the step (1) can be selected from any one of the following: 2-chlorotrityllchloride resin (2-CTC resin), HMPB resin, HAL resin, or Rink-Ac resin; in a preferable scheme, the substitution value of the resin is 0.1-1.5 mmol/g, and the particle size of the resin is 100-400 meshes.
In a preferred embodiment of the present invention, the reagent used in the fully protected lysis in step (1) is any one of the following: 20-50% TFE/DCM or 1-5% TFA/DCM.
In a preferred embodiment of the present invention, the amino resin in step (2) may be selected from any one of Rink Amide-AM resin, Sieber Amide resin, PAL Amide resin, Knorr-2-Chlorotrityl resin, or Rink Amide-MBHA resin; in a preferable scheme, the substitution value of the resin is 0.1-1.5 mmol/g, and the particle size of the resin is 100-400 meshes.
In the scheme of the invention, the coupling agent in the steps (1) and (2) can be selected from a plurality of composite coupling agents containing HOBt (1-hydroxybenzotriazole) or HOAt (1-hydroxy-7-azabenzotriazole); preferably said composite coupling agent further comprises DIC (N, N' -diisopropylcarbodiimide); more preferably, the composite coupling agent is selected from any one of the following eight mixtures:
mixing the X reagent, the HATU reagent and the Y reagent according to a molar ratio of 1:1 (1-5);
mixing an X reagent, an HBTU and a Y reagent according to a molar ratio of 1:1 (1-5);
mixing the X reagent, the HCTU and the Y reagent according to a molar ratio of 1:1 (1-5);
mixing the X reagent and the Y reagent DIC according to the molar ratio of 1 (1-5);
mixing the X reagent with EDC & HCl according to a molar ratio of 1 (1-5);
mixing the X reagent, the PyAOP and the Y reagent according to a molar ratio of 1:1 (1-5);
mixing the X reagent, the PyBOP and the Y reagent according to a molar ratio of 1:1 (1-5);
mixing the X reagent, the TBTU and the Y reagent according to a molar ratio of 1:1 (1-5);
wherein, the X reagent is HOBt or HOAt, and the Y reagent is DIC.
In a preferred embodiment of the present invention, the step (1) of attaching the 7 th to 12 th amino acids in the Iseganan amino acid sequence to the swollen carboxyl resin comprises the following steps:
(1.1) respectively weighing the swelled carboxyl resin, Fmoc-amino acid, HOBt and DIC according to the molar ratio of 1 (1.1-20) to 1.1-20 to 1.1-100;
(1.2) dissolving the Fmoc-amino acid and HOBt in DMF, adding DIC to obtain a uniform solution, adding the uniform solution into the swollen carboxyl resin, and connecting the Fmoc-amino acid;
(1.3) removing the Fmoc protecting group from the carboxyl resin treated in the step (1.2) by using 1-50% v/v of piperidine DMF solution for 1-10 times, wherein each time lasts for 1-100 min;
(1.4) repeating the coupling procedure from step (1.2) to step (1.3) using the corresponding Fmoc-amino acids to obtain the fully protected hexapeptide resin, i.e., Fmoc-Cys (Trt) -Arg (pbf) -Gly-Arg (pbf) -Phe-Cys (Trt) -carboxy resin.
In a preferred embodiment of the present invention, the step (2) of attaching Fmoc-amino acid and Fmoc- (7-12) -OH obtained in the step (1) to the swollen amino resin according to the amino acid sequence of Iseganan comprises the steps of:
(2.1) respectively weighing the swelled amino resin, Fmoc-amino acid or Fmoc- (7-12) -OH, HOBt and DIC according to the molar ratio of 1 (1.1-20) to 1.1-20 to 1.1-100;
(2.2) dissolving the Fmoc-amino acid or Fmoc- (7-12) -OH and HOBt in DMF, adding DIC to mix into a uniform solution, adding the uniform solution into the swollen amino resin, and connecting the Fmoc-amino acid or Fmoc- (7-12) -OH;
(2.3) removing the Fmoc protecting group from the amino resin treated in the step (2.2) by using 1-50% v/v of piperidine DMF solution for 1-10 times, wherein each time lasts for 1-100 min;
(2.4) repeating the coupling process from step (2.2) to step (2.3) using corresponding Fmoc-amino acids or Fmoc- (7-12) -OH to obtain a fully protected heptadecapeptide resin, i.e., H-Arg (pbf) -Gly-Gly-Leu-Cys (Trt) -Tyr (tBu) -Cys (Trt) -Arg (pbf) -Gly-Arg (pbf) -Phe-Cys (Trt) -Val-Gly-Arg (pbf) -amino resin.
In a preferred embodiment of the present invention, the iodine oxidation method in steps (1) and (2) comprises: dissolving iodine in DMF, adding the solution into a reaction system containing the peptide resin, wherein the molar ratio of the peptide resin to the iodine is 1 (3-50), and oxidizing to generate a disulfide bond; the reaction temperature is 0-50 ℃; the reaction time is 0.1-10.0 h, preferably 0.5-4 h; after the reaction is completed, the resin is washed for 1-20 times by DMF.
In the preferable scheme of the invention, the solid phase synthesis reaction temperature in the steps (1) and (2) is 0-50 ℃, and the reaction time is 0.1-10.0 h.
In the preferable scheme of the invention, the Fmoc protecting group removing method in the step (2) is to repeat the Fmoc protecting group removing for 1-10 times by using 1-50% v/v piperidine DMF solution, the deprotection time for each time is 1-100 min, and then DMF is used for washing the resin for 1-20 times.
In a preferred scheme of the invention, the lysis solution for cutting the peptide resin in the steps (1) and (3) is a composite lysis solution obtained by mixing trifluoroacetic acid, thioanisole, dithioglycol and water according to a volume ratio of 90:5:3: 2; the cleavage in the step (1) and the step (3) is that 1g of peptide resin is added into 1-100 mL of lysate, the lysate is added into the peptide resin, and the peptide resin is mechanically stirred and reacts for 1-24 hours at the temperature of 0-50 ℃; and then filtering the reaction solution to-50-0 ℃ of ethyl acetate for precipitation, centrifuging, collecting the precipitate, washing the precipitate with ethyl acetate for 1-20 times, and drying under reduced pressure at 0-50 ℃ to constant weight to obtain the crude peptide of Iseganan.
In the preferable scheme of the invention, the filtration in the step (4) is to filter the crude solution of the Iseganan with a 0.1-10.0 μm microporous filter membrane; the separation and purification is to use a reversed-phase high performance liquid chromatography to prepare a chromatographic column for separation and purification, wherein a used mobile phase A is pure acetonitrile or methanol, and a phase B is 0.01-10% v/v trifluoroacetic acid aqueous solution or phosphoric acid aqueous solution; detecting the wavelength at 220nm, collecting the effluent liquid in different time periods by adopting a gradient elution mode, selecting a product with qualified purity by using a detection result of HPLC, and combining and freeze-drying the product with qualified purity to obtain the Iseganan trifluoroacetate or Iseganan acetate.
Compared with the prior art, the method for preparing the Iseganan has the following beneficial effects:
the preparation method of Iseganan adopts a fragment synthesis mode, firstly solid-phase synthesis is carried out on 7-12 fragments of Iseganan, namely oxidation is carried out to generate a first pair of disulfide bonds (Cys)7-12) Then, the synthesized fragment and other amino acids in the Iseganan sequence are utilized to synthesize a full-protection heptadecapeptide resin in a solid phase manner, and the full-protection heptadecapeptide resin is oxidized for the second time to generate a second pair of disulfide bonds (Cys)5-14). The method adopts a pure solid phase mode, completes the oxidation of two pairs of disulfide bonds on the resin, improves the positioning accuracy of the disulfide bonds, improves the purity of the product, and is beneficial to large-scale industrial production. In addition, the method has the advantages of simple and stable process, mild condition, easy control, small influence on human bodies and environment, high product yield and purity, and contribution to large-scale industrial production.
Drawings
FIG. 1 is a process flow diagram for the synthesis of key intermediate F- (7-12) -OH of Iseganan, described in example 1.
FIG. 2 is a process flow diagram of the Iseganan full-sequence synthesis described in example 1.
FIG. 3 is a high performance liquid chromatogram of Iseganan synthesized in example 1.
FIG. 4 is a mass spectrum of Iseganan synthesized by the method of example 1.
Detailed Description
The method of the invention and its core ideas can be better understood from the following examples. The specific material ratios, process conditions and results described in the examples are illustrative of the invention and should not, nor should they limit the invention as detailed in the claims. For those skilled in the art to which the present invention pertains, simple deductions or substitutions made without departing from the concept of the present invention, changes made in the specific embodiments and application ranges, such as changing the condensing agent during the condensation of amino acids, adjusting the ratio of the condensing agent, adjusting the reaction time or temperature, should be considered as the protection scope of the present invention.
Abbreviations used in the specification or claims have the following meanings:
fmoc: 9-fluorenylmethoxycarbonyl group
Linker: linker
DCM: methylene dichloride
DMF: n, N-dimethylformamide
Piperidine: piperidine/piperidine derivatives
HOAt: n-hydroxy-7-azobenzotriazol
HOBt: 1-hydroxybenzotriazoles
HATU: 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate
HCTU: 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate
TBTU: O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate
NMM: n-methylmorpholine
PyBOP: benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphates
PyAOP: (3H-1,2, 3-triazolo [4,5-b ] pyridin-3-yloxy) tri-1-pyrrolidinophosphonium hexafluorophosphate
DIEA: n, N-diisopropylethylamine
DIC: n, N' -diisopropylcarbodiimide
EDC. HCl: 1-Ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC. HCL)
tBu: tert-butyl radical
Pbf: 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
Arg: arginine
Gly: glycine
Val: valine
Cys: cysteine
Phe: phenylalanine
Tyr: tyrosine
Leu: leucine
MeOH: methanol
TFE trifluoroethanol
I2: iodine
TFA: trifluoroacetic acid
EDT (electro-thermal transfer coating): ethanedithiol
Thioanisole: phenylmethyl sulfide
RP-HPLC: reversed phase-high performance liquid chromatography
Example 1
A method for preparing an antimicrobial peptide Iseganan, comprising the steps of:
I. intermediate Fmoc- (7-12) -OH was synthesized as shown in FIG. 1, as follows:
(1) preparation of Fmoc-Cys (Trt) -CTC resin
5.0g of CTC resin (substitution value 1.46mmol/g) was weighed into a jacketed 250mL polypeptide solid phase synthesizer and 50mL of DCM was added and swollen for 30 min. After the resin is completely swelled, DCM is pumped out, 4.703g (8.03mmol) of Fmoc-Cys (Trt) -OH is dissolved by 40mL of DCM, the mixture is added into a reaction system after ultrasonic oscillation and dissolution, 3.980mL (24.09mmol) of DIEA is dropwise added, mechanical stirring reaction is carried out for 1h, the reaction temperature is controlled to be 30 ℃ by circulating water at constant temperature, 4mL of MeOH is added, mechanical stirring reaction is carried out for 40min, 40mL of DCM is used for washing for 2 times, MeOH and DCM are used for washing for 2 times alternately, 40mL of MeOH is used for washing for 3 times, and Fmoc-Cys (Trt) -CTC resin is obtained by drying under reduced pressure at 30 ℃ until the constant weight of the resin, the substitution degree of the resin is 0.638mmol/g when the weight gain method is measured, and the.
(2) Preparation of Fmoc-c (Cys-Arg (pbf) -Gly-Arg (pbf) -Phe-Cys) -CTC resin
50mL of DMF was taken to swell the peptide resin for 30min, after the resin was completely swollen, DMF was pumped off, the Fmoc protecting group was removed 2 times with 40mL of 20% (v/v) Piperidine/DMF solution for 5min and 15min, respectively, and then washed 5 times with 40mL of DMF, and DMF was pumped off. 3.708g (9.57mmol) of Fmoc-Phe-OH and 1.551g (11.484mmol) of HOBt are dissolved in 40mL of DMF, the mixture is subjected to ultrasonic oscillation and dissolution, ice bath is carried out for 10min, 1.775mL (11.484mmol) of DIC is added, the mixture is added into a solid phase reactor after activation for 5min, mechanical stirring is carried out for reaction for 2h, and the reaction temperature is controlled to be 30 ℃ by circulating water at constant temperature. And (3) when the ninhydrin test is negative, namely the resin is colorless and transparent, and the solution is yellow, pumping out the reaction solution, washing with 40mL of DMF for 4 times, and pumping out the DMF to obtain the Fmoc-Phe-Cys (Trt) -CTC resin. Repeating the above operation according to the 7-12 amino acid sequence of Iseganan, weighing amino acid and condensing agent according to the amount in Table 1, adding 40mL DMF to dissolve Fmoc-amino acid and HOBt, ice-cooling for 10min, adding DIC, then Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH and Fmoc-Cys (Trt) -OH are connected in sequence, 6 times of iodine oxidation disulfide bond is carried out, mechanical stirring reaction is carried out for 2h, controlling the reaction temperature to be 30 ℃ by constant-temperature circulating water, washing the resin with DMF 10 times after the reaction is finished, finally washing the resin with 40mL of MeOH three times, then alternately washing the resin with 40mL of DCM and MeOH 2 times, then washing the resin with 40mL of MeOH 2 times, pumping out the MeOH, and drying the resin at 30 ℃ under reduced pressure until the weight of the resin is constant to obtain the Fmoc-c (Cys-Arg (pbf) -Gly-Arg (pbf) -Phe-Cys) -CTC resin.
TABLE 1 amounts of each amino acid and condensing agent
(3) Preparation of Fmoc-c (Cys-Arg (pbf) -Gly-Arg (pbf) -Phe-Cys) -OH.
And (3) placing the peptide resin obtained in the step (2) in 20% TFE/DCM for reacting for 1h, collecting reaction liquid, concentrating, precipitating with water, and drying in vacuum to obtain Fmoc-c (Cys-Arg (pbf) -Gly-Arg (pbf) -Phe-Cys) -OH (namely Fmoc- (7-12) -OH).
Synthesis of the full sequence, in color gurnan, as shown in fig. 2, the procedure is as follows:
(4) preparation of Fmoc-Arg (Pbf) -AM-resin
1.0g (0.62mmol) of Fmoc-Rink-Amide AM resin (substitution value 0.62mmol/g) was weighed into a jacketed 50mL polypeptide solid phase synthesizer, and 10mL of DMF was added to swell for 30 min. After the resin had swelled completely, DMF was pumped off, the Fmoc protecting group was removed 2 times with 8mL of 20% (v/v) Piperidine/DMF solution for 5min and 15min, respectively, and washed 5 times with 8mL of DMF, and DMF was pumped off. Dissolving 1.207g (1.86mmol) of Fmoc-Arg (Pbf) -OH and 0.302g (2.232mmol) of HOBt in 8mL of DMF, dissolving by ultrasonic oscillation, carrying out ice bath for 10min, adding 0.345mL (2.232mmol) of DIC, activating for 5min, adding the mixed solution into a solid phase reactor, carrying out mechanical stirring reaction for 2h, and controlling the reaction temperature to be 30 ℃ by using constant temperature circulating water. When the ninhydrin test is negative, i.e. the resin is colorless and transparent and the solution is yellow, the reaction solution is pumped out, and is washed 4 times with 8mL of DMF, and the DMF is pumped out to obtain Fmoc-Arg (Pbf) -AM-resin.
(5)
Solid phase synthesis of H-Arg (pbf) -Gly-Gly-Leu-Cys (Trt) -Tyr (tBu) -Cys (Trt) -Arg (pbf) -Gly-Arg (pbf) -Phe-Cys (Trt) -Val-Gly-Arg (pbf) -AM-resin.
After removing Fmoc protecting group from Fmoc-Arg (Pbf) -AM-resin prepared above, 0.553g (1.86mmol) of Fmoc-Gly-OH and 0.302g (2.232mmol) of HOBt were dissolved in 8mL of DMF, and after ultrasonic oscillation dissolution, ice-bath was carried out for 10min, 0.345mL (2.232mmol) of DIC was added, after 5min of activation, the mixed solution was added to a solid phase reactor, and mechanical stirring was carried out for 2h, and the reaction temperature was controlled at 30 ℃ with circulating water at constant temperature. And (3) when the ninhydrin test is negative, pumping out the reaction solution, washing the resin with 8mL of DMF, and pumping out the DMF to obtain the Fmoc-Gly-Arg (Pbf) -AM-resin. Repeating the above operation according to the sequence of the amino acid of Geinan, weighing the amino acid and the condensing agent according to the amount shown in Table 2, adding 8mL of DMF to dissolve Fmoc-amino acid and HOBt, carrying out ice bath for 10min, adding DIC, and then sequentially connecting Fmoc-Val-OH, Fmoc-Cys (trt) -OH, Fmoc-Val-OH, F- (7-12) -OH obtained in step (3), Fmoc-Tyr (tBu) -OH, Fmoc-Cys (trt) -OH, Fmoc-Leu-OH, Fmoc-Gly-OH, and Fmoc-Arg (pbf) -OH obtained in step (3), performing mechanical stirring reaction for 2h while controlling the reaction temperature to be 30 ℃ with constant temperature circulating water, completing the washing of the resin with DMF for 10 times, removing the Fmoc protecting group with 8mL of 20% (v/v) Piperidine/DMF for 2 times, the time is 5min and 15min respectively, then washing 5 times with 8mL DMF, and extracting DMF. And finally, washing the second pair of disulfide bond peptide resin by 8mL of MeOH for three times, then respectively washing the third pair of disulfide bond peptide resin by 8mL of DCM and MeOH for 2 times, then washing the third pair of disulfide bond peptide resin by 8mL of MeOH for 2 times, pumping the MeOH, and drying the third pair of disulfide bond peptide resin at the temperature of 30 ℃ under reduced pressure to the constant weight of the resin, thus obtaining the second pair of disulfide bond peptide resin.
TABLE 2 amounts of each amino acid and condensing agent
25mL of the cleavage solution (trifluoroacetic acid, thioanisole, dithioglycol and water mixed at a volume ratio of 90:5:3: 2) was added to the prepared H-Arg (pbf) -Gly-Gly-Leu-Cys (Trt) -Tyr (tBu) -Cys (Trt) -Arg (pbf) -Gly-Arg (pbf) -Phe-Cys (Trt) -Val-Gly-Arg (pbf) -AM-resin, and the mixture was heated in a water bath at 30 ℃ and reacted for 2 hours with mechanical stirring. Then the reaction solution is filtered to 1800mL of glacial ethyl ether at the temperature of-20 ℃ for precipitation, the precipitate is collected after centrifugation, the precipitate is washed by ethyl ether for 3 times, and the precipitate is dried under reduced pressure at the temperature of 30 ℃ to constant weight, so that 1.102g of crude peptide of Iseganan is obtained, and the yield is 93.5%.
(7) HPLC method for preparing pure Iseganan
0.410g of crude Iseganan was weighed, dissolved in 20mL of 10% methanol/water, and pumped under the following HPLC purification conditions: column: unips 10-300: 30 x 250 mm; mobile phase A: 100% methanol; mobile phase B: 0.1% (v/v) TFA/H2O; flow rate: 40 mL/min; detection wavelength: 220 nm;elution gradient: 40% -55%; time: and (3) 30 min. Samples with HPLC purity of more than 99.0% were collected, combined and lyophilized to give 0.199g of pure product with a yield of 48.5%. The high performance liquid chromatogram of the product is shown in FIG. 3, and the mass spectrum is shown in FIG. 4.
Claims (10)
1. A solid-phase synthesis method of fragment of Iseganan comprises the following steps:
(1) adopting Fmoc/tBu solid phase synthesis, and connecting Fmoc-amino acid to the swelled carboxyl resin by using a coupling agent according to the amino acid sequence of the Iseganan fragment 7-12 to obtain fully-protected hexapeptide resin; the Fmoc-amino acids attached to the carboxyl resin are in order: Fmoc-Cys (Trt) -OH, Fmoc-Phe-OH, Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH and Fmoc-Cys (Trt) -OH; the full-protection hexapeptide peptide resin is oxidized by iodine to form a first pair of disulfide bonds, and then is subjected to full-protection cleavage to obtain Fmoc-c (Cys-Arg (pbf) -Gly-Arg (pbf) -Phe-Cys) -OH which is marked as Fmoc- (7-12) -OH;
(2) adopting Fmoc/tBu solid phase synthesis, connecting Fmoc-amino acid and Fmoc- (7-12) -OH obtained in the step (1) to the swelled amino resin by using a coupling agent according to an amino acid sequence of eosin to obtain a fully-protected heptadecapeptide resin; the order of attachment to the amino resin is in the order: Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Cys (Trt) -OH, Fmoc-Val-OH, Fmoc- (7-12) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Leu-OH, Fmoc-Gly-OH and Fmoc-Arg (pbf) -OH; the fully-protected heptadecapeptide resin is oxidized by iodine to form a second pair of disulfide bonds, and Fmoc protecting groups are removed to obtain the fully-protected heptadecapeptide resin with two pairs of disulfide bonds;
(3) cutting the fully-protected heptadecapeptide resin obtained in the step (2) by using a lysis solution, removing all side chain protecting groups simultaneously, and precipitating the cutting solution by using glacial ethyl ether to obtain a crude product of Iseganan;
(4) and (4) filtering, separating, purifying and freeze-drying the crude Iseganan solution obtained in the step (3) to obtain Iseganan trifluoroacetate or Iseganan acetate.
2. The method of claim 1, wherein: the carboxyl resin in the step (1) is selected from any one of the following: 2-chlorotrityllchloride resin (2-CTC resin), HMPB resin, HAL resin, or Rink-Ac resin; in the preferred scheme, the substitution value of the resin is 0.1-1.5 mmol/g, and the particle size of the resin is 100-400 meshes; the amino resin in the step (2) is selected from any one of the following: rink Amide-AM resin, Sieber Amide resin, PAL Amide resin, Knorr-2-Chlorotrityl resin or Rink Amide-MBHA resin; in a preferable scheme, the substitution value of the resin is 0.1-1.5 mmol/g, and the particle size of the resin is 100-400 meshes.
3. The method of claim 1, wherein: the reagent used in the full-protection cracking in the step (1) is any one of the following reagents: 20-50% TFE/DCM or 1-5% TFA/DCM.
4. The method of claim 1, wherein: the coupling agent in the steps (1) and (2) is selected from a plurality of composite coupling agents containing HOBt (1-hydroxy-benzotriazole) or HOAt (1-hydroxy-7-azabenzotriazole); preferably said composite coupling agent further comprises DIC (N, N' -diisopropylcarbodiimide); more preferably, the composite coupling agent is selected from any one of the following eight mixtures:
mixing the X reagent, the HATU reagent and the Y reagent according to a molar ratio of 1:1 (1-5);
mixing an X reagent, an HBTU and a Y reagent according to a molar ratio of 1:1 (1-5);
mixing the X reagent, the HCTU and the Y reagent according to a molar ratio of 1:1 (1-5);
mixing the X reagent and the Y reagent DIC according to the molar ratio of 1 (1-5);
mixing the X reagent with EDC & HCl according to a molar ratio of 1 (1-5);
mixing the X reagent, the PyAOP and the Y reagent according to a molar ratio of 1:1 (1-5);
mixing the X reagent, the PyBOP and the Y reagent according to a molar ratio of 1:1 (1-5);
mixing the X reagent, the TBTU and the Y reagent according to a molar ratio of 1:1 (1-5);
wherein, the X reagent is HOBt or HOAt, and the Y reagent is DIC.
5. The method of claim 1, wherein: connecting Fmoc-amino acids to the swollen carboxyl resin according to the amino acid sequence of the Iseganan fragment 7-12 in step (1), comprising the following steps:
(1.1) respectively weighing the swelled carboxyl resin, Fmoc-amino acid, HOBt and DIC according to the molar ratio of 1 (1.1-20) to 1.1-20 to 1.1-100;
(1.2) dissolving the Fmoc-amino acid and HOBt in DMF, adding DIC to obtain a uniform solution, adding the uniform solution into the swollen carboxyl resin, and connecting the Fmoc-amino acid;
(1.3) removing the Fmoc protecting group from the carboxyl resin treated in the step (1.2) by using 1-50% v/v of piperidine DMF solution for 1-10 times, wherein each time lasts for 1-100 min;
(1.4) repeating the coupling procedure from step (1.2) to step (1.3) using the corresponding Fmoc-amino acids to obtain the fully protected hexapeptide resin, i.e., Fmoc-Cys (Trt) -Arg (pbf) -Gly-Arg (pbf) -Phe-Cys (Trt) -carboxy resin.
6. The method of claim 1, wherein: attaching Fmoc-amino acid and Fmoc- (7-12) -OH obtained in the step (1) to the swollen amino resin according to the amino acid sequence of eosin in (2), comprising the following steps:
(2.1) respectively weighing the swelled amino resin, Fmoc-amino acid or Fmoc- (7-12) -OH, HOBt and DIC according to the molar ratio of 1 (1.1-20) to 1.1-20 to 1.1-100;
(2.2) dissolving the Fmoc-amino acid or Fmoc- (7-12) -OH and HOBt in DMF, adding DIC to mix into a uniform solution, adding the uniform solution into the swollen amino resin, and connecting the Fmoc-amino acid or Fmoc- (7-12) -OH;
(2.3) removing the Fmoc protecting group from the amino resin treated in the step (2.2) by using 1-50% v/v of piperidine DMF solution for 1-10 times, wherein each time lasts for 1-100 min;
(2.4) repeating the coupling process from step (2.2) to step (2.3) using corresponding Fmoc-amino acids or Fmoc- (7-12) -OH to obtain a fully protected heptadecapeptide resin, i.e., H-Arg (pbf) -Gly-Gly-Leu-Cys (Trt) -Tyr (tBu) -Cys (Trt) -Arg (pbf) -Gly-Arg (pbf) -Phe-Cys (Trt) -Val-Gly-Arg (pbf) -amino resin.
7. The method of claim 1, wherein: the iodine oxidation method in the steps (1) and (2) comprises the following steps: dissolving iodine in DMF, adding the solution into a reaction system containing the peptide resin, wherein the molar ratio of the peptide resin to the iodine is 1 (3-50), and oxidizing to generate a disulfide bond; the reaction temperature is 0-50 ℃; the reaction time is 0.1-10.0 h, preferably 0.5-4 h; washing the resin with DMF for 1-20 times after the reaction is completed; the method for removing the Fmoc protecting group in the step (2) is to use 1-50% v/v piperidine DMF solution to remove the Fmoc protecting group for 1-10 times, the deprotection time for each time is 1-100 min, and then DMF is used for washing the resin for 1-20 times.
8. The method of claim 1, wherein: the solid-phase synthesis reaction temperature in the steps (1) and (2) is 0-50 ℃, and the reaction time is 0.1-10.0 h.
9. The method of claim 1, wherein: the lysis solution used for cutting the peptide resin in the steps (1) and (3) is a composite lysis solution obtained by mixing trifluoroacetic acid, dimethyl sulfide, dithioglycol and water according to the volume ratio of 90:5:3: 2; the cleavage in the step (1) and the step (3) is that 1g of peptide resin is added into 1-100 mL of lysate, the lysate is added into the peptide resin, and the peptide resin is mechanically stirred and reacts for 1-24 hours at the temperature of 0-50 ℃; and then filtering the reaction solution to-50-0 ℃ of ethyl acetate for precipitation, centrifuging, collecting the precipitate, washing the precipitate with ethyl acetate for 1-20 times, and drying under reduced pressure at 0-50 ℃ to constant weight to obtain the crude peptide of Iseganan.
10. The method of claim 1, wherein: filtering the solution of the crude product of the Iseganan by using a 0.1-10.0 mu m microporous filter membrane; the separation and purification is to use a reversed-phase high performance liquid chromatography to prepare a chromatographic column for separation and purification, wherein a used mobile phase A is pure acetonitrile or methanol, and a phase B is 0.01-10% v/v trifluoroacetic acid aqueous solution or phosphoric acid aqueous solution; detecting the wavelength at 220nm, collecting the effluent liquid in different time periods by adopting a gradient elution mode, selecting a product with qualified purity by using a detection result of HPLC, and combining and freeze-drying the product with qualified purity to obtain the Iseganan trifluoroacetate or Iseganan acetate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011457209.2A CN112500453A (en) | 2020-12-13 | 2020-12-13 | Solid-phase synthesis method of fragment of antibacterial peptide Iseganan |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011457209.2A CN112500453A (en) | 2020-12-13 | 2020-12-13 | Solid-phase synthesis method of fragment of antibacterial peptide Iseganan |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112500453A true CN112500453A (en) | 2021-03-16 |
Family
ID=74973648
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011457209.2A Pending CN112500453A (en) | 2020-12-13 | 2020-12-13 | Solid-phase synthesis method of fragment of antibacterial peptide Iseganan |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112500453A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003062266A2 (en) * | 2002-01-22 | 2003-07-31 | Intrabiotics Pharmaceuticals, Inc. | Hybrid synthetic method for antimicrobial peptides |
| CN103012563A (en) * | 2012-12-13 | 2013-04-03 | 南京工业大学 | Solid-phase synthesis method of antibacterial peptide Iseganan |
| CN104371008A (en) * | 2014-10-15 | 2015-02-25 | 兰州大学 | Method for preparing terlipressin by virtue of fragment condensation |
| CN104610433A (en) * | 2015-02-13 | 2015-05-13 | 泰州施美康多肽药物技术有限公司 | Preparation method of cetrorelix |
| CN110903352A (en) * | 2019-12-27 | 2020-03-24 | 中肽生化有限公司 | Preparation method of cetrorelix |
-
2020
- 2020-12-13 CN CN202011457209.2A patent/CN112500453A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003062266A2 (en) * | 2002-01-22 | 2003-07-31 | Intrabiotics Pharmaceuticals, Inc. | Hybrid synthetic method for antimicrobial peptides |
| CN103012563A (en) * | 2012-12-13 | 2013-04-03 | 南京工业大学 | Solid-phase synthesis method of antibacterial peptide Iseganan |
| CN104371008A (en) * | 2014-10-15 | 2015-02-25 | 兰州大学 | Method for preparing terlipressin by virtue of fragment condensation |
| CN104610433A (en) * | 2015-02-13 | 2015-05-13 | 泰州施美康多肽药物技术有限公司 | Preparation method of cetrorelix |
| CN110903352A (en) * | 2019-12-27 | 2020-03-24 | 中肽生化有限公司 | Preparation method of cetrorelix |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11518794B2 (en) | Synthesis method for liraglutide with low racemate impurity | |
| AU723268B2 (en) | Improved solid-phase peptide synthesis and agent for use in such synthesis | |
| EP2757107B1 (en) | Method for solid phase synthesis of liraglutide | |
| CN102875665B (en) | Method for synthesizing liraglutide | |
| WO2017114382A1 (en) | Method for synthesizing c-terminal modified peptide | |
| CN103497245B (en) | Method for synthesizing thymalfasin | |
| CN103012563A (en) | Solid-phase synthesis method of antibacterial peptide Iseganan | |
| CN106554391B (en) | Method for synthesizing marine biological peptide Xen2174 | |
| CN110698553A (en) | Preparation method of conus anti-wrinkle agent | |
| CN104045706A (en) | Synthetic method of liraglutide | |
| US20170260247A1 (en) | Method For Synthesizing Degarelix | |
| CN101357938A (en) | Method for synthesizing Exenatide from solid phase polypeptide | |
| CN104788546A (en) | Preparation method of linear peptides containing 24 amino acid residues | |
| CN107022021A (en) | A kind of solid-phase synthesis of Liraglutide | |
| CN105461800A (en) | Method for preparing polypeptide of KPV (Lysine-Proline-Valine) peptide and analogue of KPV peptide | |
| CN113748125A (en) | Glucagon-like peptide-1 (GLP-1) receptor agonists and analogs thereof | |
| US20220033440A1 (en) | An improved process for the preparation of plecanatide | |
| CN109306366B (en) | Method for synthesizing PT141 | |
| CN107778351B (en) | Method for synthesizing octreotide by all-solid-phase method | |
| CN110054679B (en) | Method for synthesizing Abalopratide | |
| CN104428314A (en) | Solid phase peptide synthesis of insulin using side chain anchored lysine | |
| CN112500453A (en) | Solid-phase synthesis method of fragment of antibacterial peptide Iseganan | |
| CN110330560B (en) | Synthetic method of lanreotide acetate | |
| CN112500454A (en) | Preparation method of Iseganan | |
| CN109912709A (en) | A kind of preparation method of the inhibitors of acid-sensing ion channels |
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 |