CN112851747B - Method for synthesizing polypeptide modified by quaternary ammonium base derivative - Google Patents
Method for synthesizing polypeptide modified by quaternary ammonium base derivative Download PDFInfo
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- CN112851747B CN112851747B CN201911101870.7A CN201911101870A CN112851747B CN 112851747 B CN112851747 B CN 112851747B CN 201911101870 A CN201911101870 A CN 201911101870A CN 112851747 B CN112851747 B CN 112851747B
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- Prior art keywords
- quaternary ammonium
- ammonium base
- polypeptide
- resin
- solid phase
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 88
- 125000001453 quaternary ammonium group Chemical group 0.000 title claims abstract description 82
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 64
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 9
- 239000011347 resin Substances 0.000 claims abstract description 63
- 229920005989 resin Polymers 0.000 claims abstract description 63
- 238000005859 coupling reaction Methods 0.000 claims abstract description 25
- 239000007790 solid phase Substances 0.000 claims abstract description 20
- 125000003277 amino group Chemical group 0.000 claims abstract description 15
- 230000008878 coupling Effects 0.000 claims abstract description 14
- 238000010168 coupling process Methods 0.000 claims abstract description 14
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 11
- 239000002994 raw material Substances 0.000 claims abstract description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 20
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 14
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 13
- 239000006166 lysate Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 5
- 239000007822 coupling agent Substances 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000004007 reversed phase HPLC Methods 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 2
- XOMRRQXKHMYMOC-OAQYLSRUSA-O [(2r)-3-carboxy-2-hexadecanoyloxypropyl]-trimethylazanium Chemical group CCCCCCCCCCCCCCCC(=O)O[C@H](CC(O)=O)C[N+](C)(C)C XOMRRQXKHMYMOC-OAQYLSRUSA-O 0.000 claims 1
- 230000004048 modification Effects 0.000 abstract description 17
- 238000012986 modification Methods 0.000 abstract description 17
- 230000015572 biosynthetic process Effects 0.000 abstract description 12
- 238000003786 synthesis reaction Methods 0.000 abstract description 12
- 238000003776 cleavage reaction Methods 0.000 abstract description 8
- 230000007017 scission Effects 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 7
- 238000001308 synthesis method Methods 0.000 abstract description 7
- 239000012535 impurity Substances 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract 1
- 239000002585 base Substances 0.000 description 47
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 18
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 14
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 12
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 12
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 12
- 229960003237 betaine Drugs 0.000 description 12
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 description 11
- 239000012071 phase Substances 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- 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
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 8
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 8
- 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
- 238000010189 synthetic method Methods 0.000 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 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000005457 ice water Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- -1 9-fluorenylmethoxycarbonyl Chemical group 0.000 description 5
- 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 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 229960001518 levocarnitine Drugs 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
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 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
- 238000006467 substitution reaction Methods 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
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 239000007821 HATU Substances 0.000 description 3
- 239000012317 TBTU Substances 0.000 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 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000010511 deprotection reaction Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000006011 modification reaction Methods 0.000 description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 3
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 2
- 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 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 description 2
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 2
- AHVYPIQETPWLSZ-UHFFFAOYSA-N N-methyl-pyrrolidine Natural products CN1CC=CC1 AHVYPIQETPWLSZ-UHFFFAOYSA-N 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
- 239000003875 Wang resin Substances 0.000 description 2
- 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 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 150000001875 compounds Chemical class 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
- 238000006116 polymerization reaction Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 235000003441 saturated fatty acids Nutrition 0.000 description 2
- 150000004671 saturated fatty acids Chemical class 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 2
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 1
- 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
- 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 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
- 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
- IXKVYKPPJAWZLH-UHFFFAOYSA-N 7-thiabicyclo[4.1.0]hepta-2,4-diene Chemical compound C1=CC=CC2SC21 IXKVYKPPJAWZLH-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- KXADPELPQCWDHL-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1.COC1=CC=CC=C1 KXADPELPQCWDHL-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit 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
- 239000003153 chemical reaction reagent Substances 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
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 description 1
- 238000010586 diagram Methods 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
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 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
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000007867 post-reaction treatment Methods 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
- 238000004659 sterilization and disinfection Methods 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
Classifications
-
- 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
Landscapes
- 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 a polypeptide containing quaternary ammonium base modification. The synthesis method of the invention prepares polypeptide-solid phase resin with exposed amino through a solid phase synthesis method; coupling quaternary ammonium base or a derivative of the quaternary ammonium base with polypeptide-solid phase resin with exposed amino groups to obtain the quaternary ammonium base modified polypeptide resin; and (3) performing cleavage on the quaternary ammonium base modified polypeptide resin to obtain a 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 industrialized production of the polypeptide containing the quaternary ammonium base modification, and the prepared polypeptide containing the quaternary ammonium base modification 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 polypeptide containing quaternary ammonium base modification.
Background
Quaternary ammonium bases are a class of quaternary ammonium (-N) containing compounds + (CH 3 ) 3 ) Organic base with structure, wherein carboxyl terminal (-COOH) is also present in the molecule such as betaine, L-carnitine, etc., to make the molecule at the same timeThere are two different charge ion characteristics.
The substances with zwitterionic characteristics have the characteristics of surfactants and generally have the functions of washing, emulsifying, sterilizing and inhibiting bacteria in the fields of cosmetics and medicines. The positively charged ends in the zwitterions can be adsorbed on the surface of the negatively charged bacterial cell membranes to change the permeability of the cell walls so as to achieve the effects of inhibition and sterilization.
Under some conditions proteins or polypeptides are prone to polymerization and have low solubility in some organic solvents, which can create difficulties for their use in industrial or medical fields. In 2008, xiao et al found for the first time that chemical modification of proteins or polypeptides with small molecule betaines (betaines) increased protein solubility and prevented polymerization of the polypeptides. By performing beet alkali chemical modification on the N end of the protein, the protein solubility is found to be obviously improved. The research result provides a novel small molecule modification method for improving the enzyme solubility, and provides a novel method for solving the application problem that the low solubility of protein or polypeptide is unfavorable for the industrial or medical field.
Bioconjugate chem.2008,19 (6), pp 1113-1118 reports a method for modifying an N-terminal cysteine polypeptide by thioesterification of betaine, but the raw materials used in this method are not commercially available and are not readily available. US5958886 reports a series of compounds which modify L-carnitine at a polypeptide, but no synthetic method of polypeptide modification of L-carnitine is disclosed therein. EP0514359B1 provides a method for modifying the N end of amino acid by acetyl L-carnitine in a liquid phase, but the synthetic method has longer reaction time and more complex post-reaction treatment, separation and purification.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide an easy to handle method for synthesizing quaternary ammonium base modified polypeptide derivatives on solid phase resins.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a method for synthesizing a polypeptide containing a quaternary ammonium base modification, comprising the steps of:
1) Preparing polypeptide-solid phase resin with exposed amino groups by a solid phase synthesis method;
2) Coupling quaternary ammonium base or a derivative of the quaternary ammonium base with polypeptide-solid phase resin with exposed amino groups to obtain the quaternary ammonium base modified polypeptide resin;
3) And (3) performing cleavage on the quaternary ammonium base modified polypeptide resin to obtain a 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, R 1 H, OH or saturated or unsaturated fatty acids containing 2 to 18 carbon atoms.
Further, the quaternary ammonium derivative in the step 2) is L-carnitine, betaine, palmitoyl carnitine, lauroyl carnitine or acetyl L-carnitine.
Further, the molar ratio of the quaternary ammonium base and the quaternary ammonium base derivative raw material to the polypeptide in the step 2) is (1-8): 1.
further, the solvent of the coupling reaction in the step 2) is any one or a mixture solution of a plurality of DMF, NMP, dichloromethane and DMSO in any proportion.
Further, the coupling agent in 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 hours.
Further, the lysate of the cleavage in the step 3) is a mixture of more than two of TFA, H2O, phOMe and TIS.
Further, the purification in step 3) is reversed phase high performance liquid chromatography.
According to the technical scheme, the synthetic method of the polypeptide containing quaternary ammonium base modification prepares the polypeptide-solid phase resin with exposed amino groups through a solid phase synthetic method; coupling quaternary ammonium base or a derivative of the quaternary ammonium base with polypeptide-solid phase resin with exposed amino groups to obtain the quaternary ammonium base modified polypeptide resin; and (3) performing cleavage on the quaternary ammonium base modified polypeptide resin to obtain a 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 industrialized production of the polypeptide containing the quaternary ammonium base modification, and the prepared polypeptide containing the quaternary ammonium base modification has high purity and low impurity content and has wide application prospects in the industrial or medical fields.
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 synthetic route diagram of the synthetic method of the present invention;
FIG. 2 shows example 1betaine-GIACL-CONH 2 Crude peptide chromatograms;
FIG. 3 shows example 1betaine-GIACL-CONH 2 Refined peptide chromatogram;
FIG. 4 shows example 1betaine-GIACL-CONH 2 A mass spectrogram;
FIG. 5 shows example 2L-carnitine-IIGAC-CONH 2 Crude peptide chromatograms;
FIG. 6 shows example 2L-carnitine-IIGAC-CONH 2 Refined peptide chromatogram;
FIG. 7 shows example 2L-carnitine-IIGAC-CONH 2 A mass spectrogram;
FIG. 8 shows a chromatogram of example 3 Palmitonyl-L-carnitine-LCQATL-COOH;
FIG. 9 shows a mass spectrum of Palmitonyl-L-carnitine-LCQATL-COOH of example 3.
Detailed Description
The invention discloses a method for synthesizing a polypeptide containing quaternary ammonium base modification. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the method and product of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods described herein without departing from the spirit and scope of the invention.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a method for synthesizing a polypeptide containing a quaternary ammonium base modification, comprising the steps of:
1) Preparing polypeptide-solid phase resin with exposed amino groups by a solid phase synthesis method;
2) Coupling quaternary ammonium base or a derivative of the quaternary ammonium base with polypeptide-solid phase resin with exposed amino groups to obtain the quaternary ammonium base modified polypeptide resin;
3) And (3) performing cleavage on the quaternary ammonium base modified polypeptide resin to obtain a 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 exposed 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, R 1 H, OH or saturated or unsaturated fatty acids containing 2 to 18 carbon atoms.
In some embodiments, n=0, r in the quaternary ammonium derivative 1 =h, the structure of which is shown in formula ii, i.e. the quaternary ammonium derivative is betaine;
in some embodiments, n=1, r in the quaternary ammonium derivative 1 =oh, which is l-carnitine, having the structure shown in formula iii, i.e., the quaternary ammonium derivative is l-carnitine;
in some embodiments, n=1, r in the quaternary ammonium derivative 1 ＝OCOCH 3 The structure of the acetyl L-carnitine is shown as a formula IV, namely the quaternary ammonium derivative is acetyl L-carnitine;
in some embodiments, n=1, r in the quaternary ammonium derivative 1 ＝OCO(CH 2 ) 14 CH 3 The structure of the quaternary ammonium derivative is shown in a formula V, namely, the quaternary ammonium derivative is lauroyl carnitine;
in some embodiments, n=1, r in the quaternary ammonium derivative 1 ＝OCO(CH 2 ) 14 CH 3 The structure of the quaternary ammonium derivative is shown in a formula VI, namely, the quaternary ammonium derivative is palmitoyl carnitine;
further, the molar ratio of the quaternary ammonium base and the quaternary ammonium base derivative raw material to the polypeptide in the step 2) is (1-8): 1. in some embodiments, 3:1.
further, the solvent of the coupling reaction in the step 2) is any one or a mixture solution of a plurality of DMF, NMP, dichloromethane and DMSO in any proportion. In some embodiments, the solvent for the coupling reaction is DMSO: nmp=1:1 mixed solution.
Further, the coupling agent for the coupling reaction in step 2) of the synthesis method 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, the coupling agent is dipea+hobt+hbtu when betaine, acetyl l-carnitine, palmitoyl carnitine, or lauroyl carnitine is coupled. The molar ratio of betaine, acetyl-L-carnitine, palmitoyl carnitine or lauroyl carnitine and other quaternary ammonium base derivatives DIPEA, HOBT, HBTU is 1:2.5:1.5:1.5.
in some embodiments, when coupling l-carnitine, the coupling agent is dic+hobt, the molar ratio of l-carnitine to DIC, HOB is 1:1.5:1.5.
further, 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 hours. In some embodiments, the coupling reaction is 3h coupling effective.
Further, the cracking liquid obtained in step 3) of the synthesis method is TFA and H 2 O, phOMe and TIS.
In some embodiments, when the polypeptide is modified with a quaternary ammonium base of higher molecular weight such as palmitoyl carnitine, lauroyl carnitine, etc., the cleavage lysate is TFA: H 2 O =95:5 (v/v) lysate.
In some embodiments, when smaller molecular weight quaternary ammonium bases such as L-carnitine, acetyl L-carnitine, betaine and the like are used for modification, the cracked lysate is TFA: H 2 O:TIS:PhOMe＝90:5:4:1(v/v) lysate.
The crude peptide obtained by cleavage in step 3) of the preparation method of the invention is further purified to obtain refined peptide, and the purification is preferably reversed-phase high performance liquid chromatography.
In some embodiments, the purification is specifically: the reverse phase octadecylsilane is used as a stationary phase, 0.1% acetic acid aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and gradient elution is carried out.
The synthetic method of the polypeptide containing quaternary ammonium base modification prepares polypeptide-solid phase resin with exposed amino groups through a solid phase synthetic method; coupling quaternary ammonium base or a derivative of the quaternary ammonium base with polypeptide-solid phase resin with exposed amino groups to obtain the quaternary ammonium base modified polypeptide resin; and (3) performing cleavage on the quaternary ammonium base modified polypeptide resin to obtain a 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 industrialized production of the polypeptide containing the quaternary ammonium base modification, and the prepared polypeptide containing the quaternary ammonium base modification has high purity and low impurity content and has wide application prospects in the industrial or medical fields.
In order to further understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Abbreviations and English meanings in the specification and examples are as follows:
| abbreviations and English | Meaning of |
| betaine | Betaine (betaine) |
| L-carnitine | L-carnitine |
| Palmitoyl-L-carnitine | palmitoyl-L-carnitine |
| SPPS | Solid phase polypeptide synthesis |
| HOAt | 1-hydroxy-7-azobenzotriazole |
| Fmoc | 9-fluorenylmethoxycarbonyl |
| DIPCDI | Diisopropylcarbodiimide |
| HOBt | 1-hydroxybenzotriazoles |
| HATU | 2- (7-Azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate |
| HBTU | benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate |
| DIPEA | N, N-diisopropylethylamine |
| PyBOP | Benzotriazol-1-yl-oxy-tripyrrolidinyl hexafluorophosphate |
| PyAOP | (3H-1, 2, 3-triazolo [4, 5-b)]Pyridin-3-yloxy) tris-1-pyrrolidinylphosphonium hexafluorophosphate. |
| TBTU | O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid |
| DMF | N, N-dimethylformamide |
| DCM | Dichloromethane (dichloromethane) |
| THF | Tetrahydrofuran (THF) |
| TFE | Trifluoroethanol |
| TFA | Trifluoroacetic acid |
| TA | Benzene sulfide |
| PhOMe | Anisole (anisole) |
| TIS | Triisopropylsilane |
| DBLK | 20% piperidine/DMF (V/V) solution |
| tBu | Tert-butyl group |
| NMP | N-methylpyrrolidone |
Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Example 1: synthesis of betaine-GIACL-CONH 2 (FW: 574.32) as an example, the following is given as examples:
step 1): preparation of polypeptide-solid phase resin with naked amino groups
12.2g of Rink Amide-AM resin with substitution of 0.41mmol/g was taken and put into a reactor, after washing twice with DMF, the resin was swelled with DMF for 30 minutes, fmoc-protecting groups were removed with 20% piperidine/DMF solution (DBLK) twice for 10 minutes, and after deprotection the resin was washed with DMF six times. 5.3g (15 mmol) of Fmoc-Leu-OH, 3.05g (22.5 mmol) of HOBT and 8.55g of HBTU (22.5 mmol) were weighed out and dissolved in 35ml of a mixed solution of NMP=1:1, and after 4.85g (35.5 mmol) of DIPEA was added under ice water bath to activate for 3min, the mixed solution was added to the reaction column and reacted at room temperature for 2 hours. At the end of the reaction, the resin was washed 3 times with 35mL of LDMF, deprotected 5min+10min by addition of 20mL of BLK, washed 6 times with 35mL of LDMF, and coupling of Fmoc-Cys (Trt) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Gly-OH, 15mmol of each amino acid charge, 22.5mmol of HOBT charge, 22.5mmol of HBTU charge, and 35.5mmol of DIPEA charge was continued in sequence according to the peptide sequence. And after the coupling is finished, waiting for the next quaternary ammonium base modification reaction.
Step 2): introduction of betaine into the N-terminus of a polypeptide
3.5g (30 mmol) betaine, 6.1g (45 mmol) HOBT and 17.1g HBTU (45 mmol) were weighed out with 35ml DMSO: NMP=1: 1, adding 9.7g (75 mmol) of DIPEA under ice water bath for 3min for activation, adding into a reactor for reaction for three hours, washing with DMF three times after the reaction is finished, and washing with DCM three times. The resin was contracted with 35mL of methanol and drained to give 16.3g of peptide resin.
Step 3): betaine-polypeptide preparation
16.3g of the resin from step 2) was added to a 250mL three-necked flask, 160mL of preconfigured TFA/H2O/TIS/PhOMe=90:5:4:1 (v/v) was added, the reaction was carried out at room temperature for 2 hours, the resin was filtered under reduced pressure, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates were combined. The filtrate was slowly precipitated by adding 1.6L of ice methyl tert-butyl ether, centrifuged, and washed 3 times with 1.0L of ice 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 peptide
The crude peptide obtained in step 3 was purified by high performance liquid chromatography through a 15cm×25cm preparative column. Reversed-phase octadecylsilane is used as a stationary phase, 0.1% acetic acid aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and the gradient A phase is eluted: 70% -30%, elution preparation flow rate: 70-80ml/min; detection wavelength: 220nm; the desired peak fraction was collected, concentrated and lyophilized to give 2.41g of a pure product having a purity of 99.57%, a yield of 82.5% and a molecular weight FW of 574.32 (FIGS. 3 and 4).
Example 2: synthesis of L-carnitine-IIGAC-CONH 2 (FW:617.8)
Step 1): preparation of polypeptide-solid phase resin with naked amino groups
24.4g of Rink Amide-AM resin with substitution of 0.41mmol/g was taken and put into a reactor, after washing twice with DMF, the resin was swelled with DMF for 30 minutes, fmoc-protecting groups were removed with 20% piperidine/DMF solution (DBLK) twice for 10 minutes, and after deprotection the resin was washed with DMF six times. 17.57g (30 mmol) of Fmoc-Cys (Trt) -OH and 6.10g (45 mmol) of HOBT were weighed and dissolved in 60ml of DMF, and after 5.7g (45 mmol) of DIC was added under ice-water bath to activate for 3min, the mixture was added to the reaction column and reacted at room temperature for 2 hours. After the reaction, the resin was washed 3 times with 70mL of LDMF, deprotected 5min+10min by adding 70mL of BLK, washed 6 times with 70mL of LDMF, and coupling, fmoc-Ala-OH, fmoc-Gly-OH, fmoc-Ile-OH, 30mmol of each amino acid charge, 45mmol of HOBT charge, and 45mmol of DIC charge were continued in this order according to the peptide sequence. And after the coupling is finished, waiting for the next quaternary ammonium base modification reaction.
Step 2): introduction of L-carnitine into the N-terminus of a polypeptide
4.84g (30 mmol) of l-carnitine, 6.10g (45 mmol) of HOBT,60ml DMSO:NMP =1 are weighed out: 1, 5.7g (45 mmol) of DIC was added under ice-water bath to activate for 3min, and the mixture was added to a reaction column and reacted at room temperature for 3 hours. After the reaction was completed, the reaction was washed three times with DMF and three times with DCM. The resin was contracted with 70mL of methanol and drained to give 31.4g of peptide resin.
Step 3): preparation of L-carnitine-polypeptide
31.4g of the resin from step 2 was added to a 500mL three-necked flask, 350mL of preconfigured TFA/H2O/TIS/PhOMe=90:5:4:1 (v/v) was added, the reaction was carried out at room temperature for 2 hours, the resin was filtered under reduced pressure, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates were combined. The filtrate was slowly precipitated in 3.5L of ice methyl tert-butyl ether, centrifuged, and 1.0L of ice methyl tert-butyl ether was washed 3 times, and dried under reduced pressure to give 6.5g of L-carnitine-polypeptide derivative, crude peptide of 75.75% (FIG. 5).
Step 4): preparation of refined peptide
The crude peptide obtained in step 3 was purified by high performance liquid chromatography through a 15cm×25cm preparative column. Reversed-phase octadecylsilane is used as a stationary phase, 0.1% acetic acid aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and the gradient A phase is eluted: 65% -25%, elution preparation flow rate: 70-80ml/min; detection wavelength: 220nm; the desired peak fraction was collected, concentrated and lyophilized to give 5.8g of pure product with a purity of 98.35%, a yield of 93.85% and a molecular weight m= 618.34 (fig. 6 and 7).
Example 3: synthesis of Palmitonyl-L-carnitine-LCQATL-COOH (FW: 1031.37)
Step 1): preparation of polypeptide-solid phase resin with naked amino groups
1.22g of Fmoc-Leu-wang resin with a substitution degree of 0.40mmol/g was taken and added to the reactor, and after washing twice with DMF, the resin was swelled with DMF for 30 minutes, and Fmoc-protecting groups were removed with 20% piperidine/DMF solution (DBLK) twice for 10 minutes, and after deprotection, the resin was washed six times with DMF. 0.596g (1.5 mmol) Fmoc-Thr (tBu) -OH, 0.304g (2.25 mmol) HOBT were weighed and dissolved in 5ml DMF, 0.28g (2.25 mmol) DIC was added under ice water bath to activate for 3min, and the mixture was added to the reaction column and reacted at room temperature for 2 hours. At the end of the reaction, the resin was washed 3 times with 10mL of LDMF, deprotected 5min+10min by addition of 10mL of BLK, washed 6 times with 10mL of LDMF, and coupling of Fmoc-Ala-OH, fmoc-Gln (Trt) -OH, fmoc-Cys (Trt) -OH, fmoc-Leu-OH, 1.5mmol of each amino acid charge, 2.25mmol of HOBT charge, 2.25mmol of DIC charge, was continued in sequence. And after the coupling is finished, waiting for the next quaternary ammonium base modification reaction.
Step 2): introduction of palmitoyl-L-carnitine to the N-terminus of a polypeptide
0.60g (1.5 mmol) of palmitoyl-L-carnitine hydrochloride 0.304g (2.25 mmol) HOBT,5ml DMSO:NMP =1: 1, 0.28g (2.25 mmol) of DIC was added under ice-water bath to activate for 3min, and the mixture was added to a reaction column and reacted at room temperature for 3 hours. After the reaction was completed, the reaction was washed three times with DMF and three times with DCM. The resin was contracted with 10mL of methanol and drained to give 1.93g of peptide resin.
Step 3): palmitoyl-L-carnitine-polypeptide preparation
1.93g of the resin obtained in step 2 was put into a 50mL three-necked flask, 20mL of a preconfigured TFA, H2O=95:5 (v/v), was added, the reaction was carried out at room temperature for 2 hours, the resin was filtered under reduced pressure, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrates were combined. The filtrate was slowly precipitated in 200mL of ice methyl tert-butyl ether, centrifuged, and 50mL of ice methyl tert-butyl ether was washed 3 times and dried under reduced pressure to give 0.526g of palmitoyl-L-carnitine-polypeptide derivative, crude peptide having a purity of 96.56% (FIGS. 8 and 9).
Claims (1)
1. A method for synthesizing a polypeptide modified by a quaternary ammonium base derivative, comprising the steps of:
1) Preparing polypeptide-solid phase resin with exposed amino groups by a solid phase synthesis method; the solid phase synthesis is an Fmoc/tBu solid phase synthesis method, and the carrier resin is Rink Amide-AM resin;
2) Coupling a derivative of quaternary ammonium base containing a quaternary ammonium structure with a polypeptide-solid phase resin with exposed amino groups to obtain a quaternary ammonium base modified polypeptide resin; the derivative of the quaternary ammonium base is palmitoyl-L-carnitine, and the coupling agent for the coupling reaction is DIC and HOBT; the molar ratio of the quaternary ammonium base derivative raw material to the polypeptide is (1-8): 1, a step of; the solvent of the coupling reaction is NMP and DMSO in a ratio of 1:1; the reaction temperature of the coupling reaction is 20-30 ℃ and the reaction time is 1-4 hours;
3) Cracking the quaternary ammonium base modified polypeptide resin to obtain a quaternary ammonium base modified polypeptide crude peptide, and purifying to obtain a quaternary ammonium base modified polypeptide refined peptide, wherein the purification is reversed-phase high performance liquid chromatography purification; the cleaved lysate was 95:5 TFA and H 2 O。
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