CN112851747A - Method for synthesizing quaternary ammonium base modified polypeptide - Google Patents
Method for synthesizing quaternary ammonium base modified polypeptide Download PDFInfo
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- CN112851747A CN112851747A CN201911101870.7A CN201911101870A CN112851747A CN 112851747 A CN112851747 A CN 112851747A CN 201911101870 A CN201911101870 A CN 201911101870A CN 112851747 A CN112851747 A CN 112851747A
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- Prior art keywords
- quaternary ammonium
- resin
- polypeptide
- modified polypeptide
- base modified
- Prior art date
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- Granted
Links
- 125000001453 quaternary ammonium group Chemical group 0.000 title claims abstract description 91
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 89
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 64
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 37
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 9
- 239000011347 resin Substances 0.000 claims abstract description 64
- 229920005989 resin Polymers 0.000 claims abstract description 64
- 239000000908 ammonium hydroxide Substances 0.000 claims abstract description 27
- 238000005859 coupling reaction Methods 0.000 claims abstract description 24
- 239000007790 solid phase Substances 0.000 claims abstract description 18
- 238000001308 synthesis method Methods 0.000 claims abstract description 15
- 230000008878 coupling Effects 0.000 claims abstract description 13
- 238000010168 coupling process Methods 0.000 claims abstract description 13
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 13
- 125000003277 amino group Chemical group 0.000 claims abstract description 9
- 238000005336 cracking Methods 0.000 claims abstract description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 21
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 19
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 16
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 16
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 15
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 15
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 15
- 229960003237 betaine Drugs 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 15
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 claims description 12
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 9
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 8
- RDHQFKQIGNGIED-MRVPVSSYSA-N O-acetyl-L-carnitine Chemical compound CC(=O)O[C@H](CC([O-])=O)C[N+](C)(C)C RDHQFKQIGNGIED-MRVPVSSYSA-N 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 7
- FUJLYHJROOYKRA-QGZVFWFLSA-N O-lauroyl-L-carnitine Chemical compound CCCCCCCCCCCC(=O)O[C@H](CC([O-])=O)C[N+](C)(C)C FUJLYHJROOYKRA-QGZVFWFLSA-N 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 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
- 239000007821 HATU Substances 0.000 claims description 4
- 239000012317 TBTU Substances 0.000 claims description 4
- 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 4
- 239000002904 solvent Substances 0.000 claims description 4
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 claims description 3
- 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 3
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims description 3
- AHVYPIQETPWLSZ-UHFFFAOYSA-N N-methyl-pyrrolidine Natural products CN1CC=CC1 AHVYPIQETPWLSZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000003875 Wang resin Substances 0.000 claims description 3
- 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 description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 3
- 238000004007 reversed phase HPLC Methods 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 150000004671 saturated fatty acids Chemical class 0.000 claims description 3
- 235000021122 unsaturated fatty acids Nutrition 0.000 claims description 3
- 150000004670 unsaturated fatty acids Chemical class 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000010189 synthetic method Methods 0.000 claims description 2
- XOMRRQXKHMYMOC-NRFANRHFSA-N (3s)-3-hexadecanoyloxy-4-(trimethylazaniumyl)butanoate Chemical compound CCCCCCCCCCCCCCCC(=O)O[C@@H](CC([O-])=O)C[N+](C)(C)C XOMRRQXKHMYMOC-NRFANRHFSA-N 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 13
- 238000003786 synthesis reaction Methods 0.000 abstract description 13
- 230000008569 process Effects 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 6
- 239000012535 impurity Substances 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 239000002585 base Substances 0.000 description 29
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 12
- 238000005406 washing Methods 0.000 description 12
- 239000012071 phase Substances 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000000706 filtrate Substances 0.000 description 9
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 9
- XOMRRQXKHMYMOC-OAQYLSRUSA-N O-palmitoyl-L-carnitine Chemical compound CCCCCCCCCCCCCCCC(=O)O[C@H](CC([O-])=O)C[N+](C)(C)C XOMRRQXKHMYMOC-OAQYLSRUSA-N 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- -1 9-fluorenylmethoxycarbonyl group Chemical group 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000010511 deprotection reaction Methods 0.000 description 6
- 239000005457 ice water Substances 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- YDJXDYKQMRNUSA-UHFFFAOYSA-N tri(propan-2-yl)silane Chemical compound CC(C)[SiH](C(C)C)C(C)C YDJXDYKQMRNUSA-UHFFFAOYSA-N 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 230000004913 activation Effects 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000007822 coupling agent Substances 0.000 description 4
- 229960001518 levocarnitine Drugs 0.000 description 4
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 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 description 3
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 3
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000006011 modification reaction Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 230000008961 swelling Effects 0.000 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 description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 2
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 2
- NTFTULBKHJJQAW-HNNXBMFYSA-N 9h-fluoren-9-ylmethyl n-[(2s)-4-methyl-1-oxopentan-2-yl]carbamate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C=O)C3=CC=CC=C3C2=C1 NTFTULBKHJJQAW-HNNXBMFYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101100074988 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) nmp-1 gene Proteins 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 2
- WDGICUODAOGOMO-DHUJRADRSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino)pentanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)CC(=O)NC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 WDGICUODAOGOMO-DHUJRADRSA-N 0.000 description 1
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 description 1
- GJTJQSPLLQWKMB-SECBINFHSA-N (3R)-3-hydroxy-4-oxo-3-[(trimethylazaniumyl)methyl]pentanoate Chemical compound C(C)(=O)[C@](O)(C[N+](C)(C)C)CC([O-])=O GJTJQSPLLQWKMB-SECBINFHSA-N 0.000 description 1
- GAMKNLFIHBMGQT-ZMBIFBSDSA-N (3r)-3-hexadecanoyloxy-4-(trimethylazaniumyl)butanoate;hydrochloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC(=O)O[C@H](CC(O)=O)C[N+](C)(C)C GAMKNLFIHBMGQT-ZMBIFBSDSA-N 0.000 description 1
- BQMPVGMHZKZPBH-BYZBDTJCSA-N (3r)-3-hydroxy-4-(trimethylazaniumyl)butanoate Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O.C[N+](C)(C)C[C@H](O)CC([O-])=O BQMPVGMHZKZPBH-BYZBDTJCSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical class C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 1
- IIVWHGMLFGNMOW-UHFFFAOYSA-N 2-methylpropane Chemical compound C[C](C)C IIVWHGMLFGNMOW-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- WOOWBQQQJXZGIE-UHFFFAOYSA-N n-ethyl-n-propan-2-ylpropan-2-amine Chemical compound CCN(C(C)C)C(C)C.CCN(C(C)C)C(C)C WOOWBQQQJXZGIE-UHFFFAOYSA-N 0.000 description 1
- VWBWQOUWDOULQN-UHFFFAOYSA-N nmp n-methylpyrrolidone Chemical compound CN1CCCC1=O.CN1CCCC1=O VWBWQOUWDOULQN-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 108010004034 stable plasma protein solution Proteins 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- WHRNULOCNSKMGB-UHFFFAOYSA-N tetrahydrofuran thf Chemical compound C1CCOC1.C1CCOC1 WHRNULOCNSKMGB-UHFFFAOYSA-N 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application belongs to the technical field of polypeptide synthesis and discloses a method for synthesizing quaternary ammonium base modified polypeptide. The synthesis method of the invention prepares polypeptide-solid phase resin with naked amino groups by a solid phase synthesis method; coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin; and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide. The synthesis method has the characteristics of high synthesis yield, mild synthesis conditions, simple process, easily obtained raw materials, stable process and the like, is suitable for large-scale industrial production of the quaternary ammonium base modified polypeptide, and the prepared quaternary ammonium base modified polypeptide has high purity and low impurity content and has wide application prospect in the industrial or medical field.
Description
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing a quaternary ammonium base modified polypeptide.
Background
Quaternary ammonium bases are a class of quaternary ammonium (-N) -containing bases+(CH3)3) The organic alkali with the structure, wherein carboxyl terminal (-COOH) is also present in molecules such as betaine, L-carnitine and the like, so that the molecules have the characteristic of two ions with different charges.
Substances with zwitterionic characteristics mostly have the characteristics of surfactants, and generally have the functions of washing, emulsifying, sterilizing and bacteriostasis in the fields of cosmetics and medicines. The positive charge end in the zwitterion can change the permeability of cell walls to achieve the effects of inhibiting and killing bacteria by adsorbing the zwitterion on the surface of a cell membrane of the negatively charged bacteria.
Under some conditions proteins or polypeptides readily form polymerization, and their low solubility in some organic solvents can create difficulties for their use in industrial or medical fields. In 2008, Xiao et al found that chemical modification of protein or polypeptide by small-molecule betaine (betaine) can increase protein solubility and prevent polypeptide polymerization for the first time. The solubility of the protein is obviously improved by carrying out betaine chemical modification on the N end of the protein. The research result provides a brand-new small molecule modification method for improving the enzyme solubility, and provides a new method for solving the application problem that the low solubility of protein or polypeptide is not beneficial to the industrial or medical field.
Bioconjugate chem.2008,19(6), pp 1113-1118 reported a method for modifying N-terminal cysteine polypeptide by thioesterification of betaine, but the raw materials used in the method are not commercialized and are not easily available. US5958886 reports a series of compounds for modifying L-carnitine with polypeptide, but the synthesis method of L-carnitine modified with polypeptide is not disclosed. EP0514359B1 provides a method for modifying the N end of amino acid by acetyl L-carnitine in a liquid phase, but the synthesis method has longer reaction time and more complicated separation and purification after reaction.
Disclosure of Invention
In view of the above, the present invention provides a method for synthesizing quaternary ammonium hydroxide modified polypeptide derivatives on a solid phase resin, which is easy to operate.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for synthesizing a quaternary ammonium base modified polypeptide comprises the following steps:
1) preparing polypeptide-solid phase resin with naked amino groups by a solid phase synthesis method;
2) coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin;
3) and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide.
Further, the solid-phase synthesis in the step 1) is an Fmoc/tBu solid-phase synthesis method, and the carrier resin is any one of Wang resin, Rink Amide-MBHA resin, Rink Amide resin or Rink Amide-AM resin.
Further, the quaternary ammonium derivative in the step 2) has a structure shown in a formula I,
wherein n is 0, 1, R1H, OH or a saturated or unsaturated fatty acid containing 2 to 18 carbon atoms.
Further, in the step 2), the quaternary ammonium derivatives are L-carnitine, betaine, palmitoyl carnitine, lauroyl carnitine and acetyl L-carnitine.
Further, the molar ratio of the raw materials of the quaternary ammonium hydroxide and the quaternary ammonium hydroxide derivatives in the step 2) to the polypeptide is (1-8): 1.
further, the solvent for the coupling reaction in the step 2) is a mixed solution of any one or more of DMF, NMP, dichloromethane and DMSO in any proportion.
Further, a coupling agent for the coupling reaction in the step 2) is DIPEA + A + B or DIC + A, wherein A is HOBT or HOAT, and B is any one of PyBOP, PyAOP, HATU, HBTU or TBTU.
Further, the reaction temperature of the coupling reaction in the step 2) is 20-30 ℃, and the reaction time is 1-4 h.
Further, the lysis solution obtained in the step 3) is a mixture of more than two of TFA, H2O, PhOMe and TIS.
Further, the purification in the step 3) is reverse phase high performance liquid chromatography purification.
According to the technical scheme, the synthesis method of the quaternary ammonium base modified polypeptide prepares polypeptide-solid phase resin with naked amino groups by a solid phase synthesis method; coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin; and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide. The synthesis method has the characteristics of high synthesis yield, mild synthesis conditions, simple process, easily available raw materials, environmental friendliness, stable process and the like, is suitable for large-scale industrial production of the quaternary ammonium base modified polypeptide, and the prepared quaternary ammonium base modified polypeptide has high purity and low impurity content and has wide application prospect in the industrial or medical field.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a synthesis scheme for the synthesis method according to the invention;
FIG. 2 shows example 1 beta-GIACL-CONH2A crude peptide chromatogram;
FIG. 3 shows example 1 beta-GIACL-CONH2A chromatogram of the semen peptide;
FIG. 4 shows example 1 beta-GIACL-CONH2Mass spectrogram;
FIG. 5 shows example 2L-carnitine-IIGAC-CONH2A crude peptide chromatogram;
FIG. 6 shows example 2L-carnitine-IIGAC-CONH2A chromatogram of the semen peptide;
FIG. 7 shows example 2L-carnitine-IIGAC-CONH2Mass spectrogram;
FIG. 8 shows a chromatogram of Palmitoyl-L-carnitine-LCQATL-COOH of example 3;
FIG. 9 shows the mass spectrum of Palmitoyl-L-carnitine-LCQATL-COOH of example 3.
Detailed Description
The invention discloses a method for synthesizing quaternary ammonium base modified polypeptide. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for synthesizing a quaternary ammonium base modified polypeptide comprises the following steps:
1) preparing polypeptide-solid phase resin with naked amino groups by a solid phase synthesis method;
2) coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin;
3) and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide.
The synthetic route of the synthetic method of the invention is shown in figure 1.
Further, the solid-phase synthesis in the step 1) is an Fmoc/tBu solid-phase synthesis method, and the carrier resin is any one of Wang resin, Rink Amide-MBHA resin, Rink Amide resin or Rink Amide-AM resin.
In some embodiments, the polypeptide-solid phase resin with naked amino groups is prepared by Fmoc/tBu solid phase synthesis using Rink Amide-AM resin as a carrier resin.
Further, the quaternary ammonium derivative in the step 2) has a structure shown in a formula I,
wherein n is 0, 1, R1H, OH or a saturated or unsaturated fatty acid containing 2 to 18 carbon atoms.
In some embodiments, n ═ 0, R in the quaternary ammonium derivatives1The structure of the quaternary ammonium derivative is shown as a formula II, namely the quaternary ammonium derivative is betaine;
in some embodiments, n ═ 1, R in the quaternary ammonium derivatives1OH, which is l-carnitine, and the structure of which is shown in formula III, i.e. the quaternary ammonium derivative is l-carnitine;
in some embodiments, n ═ 1, R in the quaternary ammonium derivatives1＝OCOCH3Acetyl L-carnitine with a structure shown in a formula IV, namely the quaternary ammonium derivative is acetyl L-carnitine;
in some embodiments, n ═ 1, R in the quaternary ammonium derivatives1＝OCO(CH2)14CH3The structure of the quaternary ammonium derivative is shown as a formula V, namely the quaternary ammonium derivative is lauroyl carnitine;
in some embodiments, n ═ 1 in the quaternary ammonium derivatives,R1＝OCO(CH2)14CH3the structure of the quaternary ammonium derivative is shown as a formula VI, namely the quaternary ammonium derivative is palmitoyl carnitine;
further, the molar ratio of the raw materials of the quaternary ammonium hydroxide and the quaternary ammonium hydroxide derivatives in the step 2) to the polypeptide is (1-8): 1. in some embodiments, is 3: 1.
further, the solvent for the coupling reaction in the step 2) is a mixed solution of any one or more of DMF, NMP, dichloromethane and DMSO in any proportion. In some embodiments, the solvent of the coupling reaction is DMSO: NMP is a 1:1 mixed solution.
Further, the coupling agent in the coupling reaction in step 2) of the synthesis method of the present invention is DIPEA + a + B or DIC + a, wherein a is HOBT or HOAT, and B is any one of PyBOP, PyAOP, HATU, HBTU or TBTU.
In some embodiments, when betaine, acetyl-l-carnitine, palmitoyl-carnitine, or lauroyl-carnitine is coupled, the coupling agent is DIPEA + HOBT + HBTU. The molar ratio of quaternary ammonium base derivatives such as betaine, acetyl L-carnitine, palmitoyl carnitine or lauroyl carnitine, namely DIPEA, HOBT and HBTU is 1: 2.5: 1.5: 1.5.
in some embodiments, where l-carnitine is coupled, the coupling agent is DIC + HOBT, and the molar ratio of l-carnitine to DIC, HOB is 1: 1.5: 1.5.
furthermore, the reaction temperature of the coupling reaction in the step 2) of the synthesis method is 20-30 ℃, and the reaction time is 1-4 h. In some embodiments, the coupling reaction is most effective for 3 h.
Further, the lysis solution cleaved in step 3) of the synthesis method of the present invention is TFA or H2O, PhOMe and TIS.
In some embodiments, when a polypeptide is modified with a higher molecular weight quaternary ammonium base, such as palmitoyl carnitine, lauroyl carnitine, etc., the cleaved lysate is TFA H2And O:95: 5(v/v) lysate.
In some embodiments, when modified with a lower molecular weight quaternary ammonium base such as L-carnitine, acetyl L-carnitine, betaine, etc., the cleaved lysate is TFA H2(vi) lysates from TIS: PhOMe ═ 90:5:4:1 (v/v).
The crude peptide obtained by the cracking in the step 3) of the preparation method is further purified to obtain the refined peptide, and the purification is preferably reversed-phase high performance liquid chromatography purification.
In some embodiments, the purification is specifically: gradient elution is carried out by taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B.
The synthesis method of the quaternary ammonium base modified polypeptide prepares polypeptide-solid phase resin with naked amino by a solid phase synthesis method; coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin; and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide. The synthesis method has the characteristics of high synthesis yield, mild synthesis conditions, simple process, easily available raw materials, environmental friendliness, stable process and the like, is suitable for large-scale industrial production of the quaternary ammonium hydroxide modified polypeptide, and the prepared quaternary ammonium hydroxide modified polypeptide has high purity and low impurity content and has wide application prospects in the industrial or medical field.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Abbreviations and English meanings in the specification and examples are as follows:
| abbreviations and English | Means of |
| betaine | Betaine |
| L-carnitine | L-carnitine |
| Palmitoyl-L-carnitine | palmitoyl-L-carnitine |
| SPPS | Solid phase polypeptide synthesis |
| HOAt | 1-hydroxy-7-azobenzotriazol |
| Fmoc | 9-fluorenylmethoxycarbonyl group |
| DIPCDI | Diisopropylcarbodiimide |
| HOBt | 1-hydroxybenzotriazoles |
| HATU | 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate |
| HBTU | benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate |
| DIPEA | N, N-diisopropylethylamine |
| PyBOP | Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphates |
| PyAOP | (3H-1,2, 3-triazolo [4,5-b ]]Pyridin-3-yloxy) tris-1-pyrrolidinophosphonium hexafluorophosphate. |
| TBTU | O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate |
| DMF | N, N-dimethylformamide |
| DCM | Methylene dichloride |
| THF | Tetrahydrofuran (THF) |
| TFE | Trifluoroethanol |
| TFA | Trifluoroacetic acid |
| TA | Phenylmethyl sulfide |
| PhOMe | Phenylmethyl ether |
| TIS | Tri-isopropyl silane |
| DBLK | 20% piperidine/DMF (V/V) solution |
| tBu | Tert-butyl radical |
| NMP | N-methyl pyrrolidone |
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1: synthesis of beta-GIACL-CONH2(FW:574.32) for example, the examples are as follows:
step 1): preparation of polypeptide-solid phase resin with naked amino group
Taking 12.2g of Rink Amide-AM resin with the substitution degree of 0.41mmol/g, adding the Rink Amide-AM resin into a reactor, adding DMF to wash twice, then swelling the Rink Amide-AM resin with DMF for 30 minutes, removing Fmoc protective groups twice with 20% piperidine/DMF solution (DBLK), each time for 10 minutes, and washing six times with DMF after deprotection. 5.3g (15mmol) of Fmoc-Leu-OH, 3.05g (22.5mmol) of HOBT and 8.55g of HBTU (22.5mmol) were dissolved in 35ml of a mixed solution of NMP 1:1, and after activation with 4.85g (35.5mmol) of DIPEA in an ice-water bath for 3min, the mixture was loaded on a reaction column and reacted at room temperature for 2 hours. After the reaction is finished, washing the resin with 35mLDMF for 3 times, adding 20mLDBLK for deprotection for 5min +10min, washing the resin with 35mLDMF for 6 times, and sequentially coupling Fmoc-Cys (Trt) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH and Fmoc-Gly-OH according to peptide sequence, wherein each amino acid is fed with 15mmol, HOBT is fed with 22.5mmol, HBTU is fed with 22.5mmol and DIPEA is fed with 35.5 mmol. After the coupling is finished, waiting for the next quaternary ammonium base modification reaction.
Step 2): introduction of betaine to the nitrogen terminus of a polypeptide
3.5g (30mmol) betaine, 6.1g (45mmol) HOBT and 17.1g HBTU (45mmol) were weighed out in 35ml DMSO: NMP ═ 1:1, adding 9.7g (75mmol) of DIPEA under the condition of ice water bath for activating for 3min, adding the mixture into a reactor for reacting for three hours, washing the mixture with DMF for three times after the reaction is finished, and washing the mixture with DCM for three times. The resin was shrunk with 35mL of methanol and suction dried to give 16.3g of peptide resin.
Step 3): betaine-polypeptide preparation
16.3g of the resin obtained in step 2) was put into a 250mL three-necked flask, 160mL of the prepared TFA H2O TIS PhOMe 90:5:4:1(v/v) was added, and the mixture was reacted at room temperature for 2 hours, and the resin was filtered under reduced pressure to collect the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 1.6L of glacial methyl tert-butyl ether for precipitation, centrifuged, washed 3 times with 1.0L of glacial methyl tert-butyl ether, and dried under reduced pressure to give 2.92g of betaine-polypeptide derivative, with a crude peptide purity of 85.24% (FIG. 2).
Step 4): preparation of refined peptides
The crude peptide obtained in step 3 was purified by high performance liquid chromatography through a 15cm × 25cm preparative column. Taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution as a mobile phase A, taking acetonitrile as a mobile phase B, and eluting a gradient phase A: 70% -30%, elution preparation flow rate: 70-80 ml/min; detection wavelength: 220 nm; the desired peak fraction was collected, concentrated and lyophilized to obtain 2.41g of pure product with purity of 99.57%, yield of 82.5% and molecular weight FW:574.32 (FIGS. 3 and 4).
Example 2: synthesis of L-carnitine-IIGAC-CONH2(FW:617.8)
Step 1): preparation of polypeptide-solid phase resin with naked amino group
Taking 24.4g of Rink Amide-AM resin with the substitution degree of 0.41mmol/g, adding the Rink Amide-AM resin into a reactor, adding DMF to wash twice, then swelling the Rink Amide-AM resin with DMF for 30 minutes, removing Fmoc protective groups twice with 20% piperidine/DMF solution (DBLK), each time for 10 minutes, and washing six times with DMF after deprotection. 17.57g (30mmol) of Fmoc-Cys (Trt) -OH and 6.10g (45mmol) of HOBT were dissolved in 60ml of DMF, and after 5.7g (45mmol) of DIC was added in an ice-water bath to activate the mixture for 3min, the mixture was put into a reaction column and reacted at room temperature for 2 hours. And after the reaction is finished, washing the resin by using 70mLDMF for 3 times, adding 70mLDBLK for deprotection for 5min +10min, washing the resin by using 70mLDMF for 6 times, and sequentially coupling, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH and Fmoc-Ile-OH according to the peptide sequence, wherein each amino acid is fed by 30mmol, HOBT is fed by 45mmol, and DIC is fed by 45 mmol. After the coupling is finished, waiting for the next quaternary ammonium base modification reaction.
Step 2): introduction of L-carnitine into the nitrogen end of polypeptide
Weighing 4.84g (30mmol) of L-carnitine, 6.10g (45mmol) of HOBT, 60ml of DMSO, NMP 1:1, adding 5.7g (45mmol) of DIC under ice water bath for activation for 3min, adding the mixture into a reaction column, and reacting at room temperature for 3 h. After the reaction was complete, it was washed three times with DMF and three times with DCM. The resin was shrunk with 70mL of methanol and suction dried to give 31.4g of peptide resin.
Step 3): preparation of L-carnitine-polypeptide
31.4g of the resin obtained in step 3 was put into a 500mL three-necked flask, 350mL of previously prepared TFA, H2O, TIS, PhOMe, 90:5:4:1(v/v) was added, and the mixture was reacted at room temperature for 2 hours, followed by filtration of the resin under reduced pressure to collect the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 3.5L of glacial methyl tert-butyl ether for precipitation, centrifuged, washed 3 times with 1.0L of glacial methyl tert-butyl ether and dried under reduced pressure to give 6.5g of L-carnitine-polypeptide derivative in a crude peptide purity of 75.75% (FIG. 5).
Step 4): preparation of refined peptides
The crude peptide obtained in step 4 was purified by high performance liquid chromatography through a 15cm × 25cm preparative column. Taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution as a mobile phase A, taking acetonitrile as a mobile phase B, and eluting a gradient phase A: 65% -25%, elution preparation flow rate: 70-80 ml/min; detection wavelength: 220 nm; the desired peak fraction was collected, concentrated and lyophilized to obtain 5.8g of a pure product having a purity of 98.35%, a yield of 93.85% and a molecular weight M of 618.34 (fig. 6 and 7).
Example 3: synthesis of Palmitoyl-L-carbonitine-LCQATL-COOH (FW:1031.37)
Step 1): preparation of polypeptide-solid phase resin with naked amino group
Taking 1.22g of Fmoc-Leu-wang resin with the substitution degree of 0.40mmol/g, adding the Fmoc-Leu-wang resin into a reactor, adding DMF to wash twice, then swelling the mixture with DMF for 30 minutes, removing Fmoc protective groups twice with 20% piperidine/DMF solution (DBLK) for 10 minutes each time, and washing the mixture with DMF for six times after deprotection. 0.596g (1.5mmol) of Fmoc-Thr (tBu) -OH and 0.304g (2.25mmol) of HOBT were dissolved in 5ml of DMF, and 0.28g (2.25mmol) of DIC was added thereto in an ice-water bath for 3min of activation, and then the mixture was put into a reaction column and reacted at room temperature for 2 hours. After the reaction is finished, washing the resin by using 10mLDMF for 3 times, adding 10mLDBLK for deprotection for 5min +10min, washing the resin by using 10mLDMF for 6 times, and sequentially coupling Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Cys (Trt) -OH and Fmoc-Leu-OH according to the peptide sequence, wherein each amino acid is fed by 1.5mmol, HOBT is fed by 2.25mmol and DIC is fed by 2.25 mmol. After the coupling is finished, waiting for the next quaternary ammonium base modification reaction.
Step 2): introduction of palmitoyl-L-carnitine into the nitrogen terminal of a polypeptide
0.60g (1.5.mmol) palmitoyl-L-carnitine hydrochloride 0.304g (2.25mmol) HOBT, 5ml DMSO NMP ═ 1:1, adding 0.28g (2.25mmol) of DIC under ice water bath for activation for 3min, adding the mixture into a reaction column, and reacting at room temperature for 3 h. After the reaction was complete, it was washed three times with DMF and three times with DCM. The resin was shrunk with 10mL of methanol and suction dried to give 1.93g of peptide resin.
Step 3): preparation of palmitoyl-L-carnitine-polypeptide
1.93g of the resin obtained in step 3 was put into a 50mL three-necked flask, and prepared TFA, H2O (95: 5(v/v)20mL, was added thereto, and the mixture was reacted at room temperature for 2 hours, and the resin was filtered under reduced pressure to collect the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 200mL of glacial methyl tert-butyl ether for precipitation, centrifuged, washed 3 times with 50mL of glacial methyl tert-butyl ether, and dried under reduced pressure to give 0.526g of palmitoyl-L-carnitine-polypeptide derivative, crude peptide purity 96.56% (FIGS. 8 and 9).
Claims (10)
1. A method for synthesizing a quaternary ammonium base modified polypeptide comprises the following steps:
1) preparing polypeptide-solid phase resin with naked amino groups by a solid phase synthesis method;
2) coupling quaternary ammonium hydroxide containing a quaternary ammonium structure or a derivative of the quaternary ammonium hydroxide with polypeptide-solid phase resin with naked amino to obtain quaternary ammonium hydroxide modified polypeptide resin;
3) and (3) cracking the quaternary ammonium base modified polypeptide resin to obtain the quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain the quaternary ammonium base modified polypeptide refined peptide.
2. The method of claim 1, wherein the solid phase synthesis in step 1) is Fmoc/tBu solid phase synthesis method, and the carrier resin is any one of Wang resin, Rink Amide-MBHA resin, Rink Amide resin, or Rink Amide-AM resin.
3. The method of claim 1, wherein the quaternary ammonium derivative of step 2) has a structure of formula I,
wherein n is 0, 1, R1H, OH or a saturated or unsaturated fatty acid containing 2 to 18 carbon atoms.
4. The method of claim 3, wherein the quaternary ammonium derivative of step 2) is L-carnitine, betaine, palmitoyl carnitine, lauroyl carnitine, acetyl L-carnitine.
5. The method of claim 1, wherein the molar ratio of the starting quaternary ammonium hydroxide and quaternary ammonium hydroxide derivative in step 2) to the polypeptide is (1-8): 1.
6. the synthesis method according to claim 1, wherein the solvent for the coupling reaction in step 2) is a mixed solution of any one or more of DMF, NMP, dichloromethane and DMSO in any proportion.
7. The synthesis method of claim 1, wherein the coupling reagent in the coupling reaction in step 2) is DIPEA + A + B or DIC + A, wherein A is HOBT or HOAT, and B is any one of PyBOP, PyAOP, HATU, HBTU or TBTU.
8. The synthesis method of claim 1, wherein the coupling reaction in step 2) is carried out at a reaction temperature of 20-30 ℃ for 1-4 h.
9. The method of claim 1, wherein the cleaved lysate of step 3) is a mixture of two or more of TFA, H2O, PhOMe, and TIS.
10. The synthetic method of claim 1 wherein the purification of step 3) is by reverse phase high performance liquid chromatography.
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