CN117486976B - Synthesis method of self-assembled polypeptide RAKA 16 - Google Patents
Synthesis method of self-assembled polypeptide RAKA 16 Download PDFInfo
- Publication number
- CN117486976B CN117486976B CN202410004984.4A CN202410004984A CN117486976B CN 117486976 B CN117486976 B CN 117486976B CN 202410004984 A CN202410004984 A CN 202410004984A CN 117486976 B CN117486976 B CN 117486976B
- Authority
- CN
- China
- Prior art keywords
- ala
- fmoc
- otbu
- resin
- full
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 101
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 23
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 15
- 238000001308 synthesis method Methods 0.000 title description 4
- 239000011347 resin Substances 0.000 claims abstract description 103
- 229920005989 resin Polymers 0.000 claims abstract description 103
- 239000012634 fragment Substances 0.000 claims abstract description 51
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 45
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 26
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000005336 cracking Methods 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 14
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 13
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 12
- 125000006239 protecting group Chemical group 0.000 claims abstract description 12
- 239000011259 mixed solution Substances 0.000 claims abstract description 9
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007787 solid Substances 0.000 claims abstract description 7
- 238000006640 acetylation reaction Methods 0.000 claims abstract description 4
- 230000021736 acetylation Effects 0.000 claims abstract description 3
- 239000008176 lyophilized powder Substances 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 54
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical group C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 claims description 38
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 35
- 238000005406 washing Methods 0.000 claims description 30
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 28
- 238000005859 coupling reaction Methods 0.000 claims description 28
- 230000008878 coupling Effects 0.000 claims description 25
- 238000010168 coupling process Methods 0.000 claims description 25
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 24
- 238000001914 filtration Methods 0.000 claims description 22
- 238000002360 preparation method Methods 0.000 claims description 20
- 238000003776 cleavage reaction Methods 0.000 claims description 19
- 238000004108 freeze drying Methods 0.000 claims description 19
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 claims description 16
- 230000007017 scission Effects 0.000 claims description 16
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 15
- 150000001413 amino acids Chemical class 0.000 claims description 14
- 239000006166 lysate Substances 0.000 claims description 13
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 claims description 12
- 238000009833 condensation Methods 0.000 claims description 12
- 230000005494 condensation Effects 0.000 claims description 12
- 150000007530 organic bases Chemical class 0.000 claims description 11
- 239000012043 crude product Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 claims description 9
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 claims description 8
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 claims description 7
- 238000006467 substitution reaction Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 4
- 239000012535 impurity Substances 0.000 abstract description 5
- 238000000746 purification Methods 0.000 abstract description 5
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 20
- 238000003786 synthesis reaction Methods 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 8
- -1 fmoc-Ala-OH Chemical compound 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 239000012488 sample solution Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000005303 weighing Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000005086 pumping Methods 0.000 description 6
- 238000000197 pyrolysis Methods 0.000 description 6
- 239000012295 chemical reaction liquid Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000007858 starting material Substances 0.000 description 5
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 4
- LTYMSROWYAPPGB-UHFFFAOYSA-N diphenyl sulfide Chemical compound C=1C=CC=CC=1SC1=CC=CC=C1 LTYMSROWYAPPGB-UHFFFAOYSA-N 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000006340 racemization Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- VVQIIIAZJXTLRE-QMMMGPOBSA-N (2s)-2-amino-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound CC(C)(C)OC(=O)NCCCC[C@H](N)C(O)=O VVQIIIAZJXTLRE-QMMMGPOBSA-N 0.000 description 1
- RVLOMLVNNBWRSR-KNIFDHDWSA-N (2s)-2-aminopropanoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound C[C@H](N)C(O)=O.NCCCC[C@H](N)C(O)=O RVLOMLVNNBWRSR-KNIFDHDWSA-N 0.000 description 1
- 238000010146 3D printing Methods 0.000 description 1
- XAEWTDMGFGHWFK-IMJSIDKUSA-N Ala-Asp Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O XAEWTDMGFGHWFK-IMJSIDKUSA-N 0.000 description 1
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229940030225 antihemorrhagics Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000002874 hemostatic agent Substances 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a solid phase synthesis method of self-assembled polypeptide RAKA 16. The method comprises the following steps: preparing X1 full-protection fragments and X2 full-protection fragments; sequentially connecting the X1 full-protection segment and the X2 full-protection segment to Rink Resin at intervals; removing Fmoc protecting group by deprotection reagent, performing N-terminal acetylation on mixed solution of acetic anhydride and pyridine, cracking, settling to obtain solid crude peptide, purifying the crude peptide by HPLC, and lyophilizing to obtain lyophilized powder of refined peptide. The invention synthesizes RAKA 16 by selecting the segment X1 and the segment X2 full-protection peptide, simplifies the operation steps, shortens the reaction period, can effectively control the generation of racemized impurities and missing impurities, reduces the purification difficulty, and has better application prospect.
Description
Technical Field
The invention belongs to the field of organic synthesis, and particularly relates to a solid-phase synthesis method of self-assembled polypeptide RAKA 16.
Background
The self-assembled polypeptide can be self-assembled into an assembly body with specific morphology and structure by utilizing non-covalent bond forces such as hydrogen bond, hydrophobic effect, pi-pi stacking effect and the like, and the polypeptide has good biocompatibility and controllable degradation performance, so that the self-assembled polypeptide is widely applied to the research fields of 3D printing, cell storage, tissue engineering scaffolds and the like. Among these, RAKA 16 has been developed as a medical product such as hemostatic agents and hydrogel dressings.
The peptide sequence of RAKA 16 is:
Ac- Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH
the synthesis of self-assembled polypeptide RAKA 16 generally employs a stepwise coupling approach. The amino acid residues from 16 to 1 are sequentially connected to the amino acid resin by deprotection and coupling. Because of the influence of the secondary structure of the polypeptide, the steric hindrance of the reaction site becomes large when the polypeptide is connected to the 9 th Arg and the 1 st Arg, the coupling reaction is difficult, more missing impurities are easy to generate, the synthesis yield is influenced, and the purification difficulty is improved.
In summary, the existing method for synthesizing RAKA 16 has a long synthesis period, and is easy to introduce some deletion peptides and racemization peptides which are difficult to remove, and affects the yield.
Disclosure of Invention
The invention provides a synthesis method of self-assembled polypeptide RAKA 16, and aims to solve the problems that the existing method for synthesizing RAKA 16 is long in synthesis period, some deletion peptides and racemization peptides which are difficult to remove are easily introduced, and the yield is influenced.
The technical scheme is as follows:
the invention provides a solid phase synthesis method of self-assembled polypeptide RAKA 16, which comprises the following steps:
s1, preparing an X1 full-protection fragment, wherein the X1 full-protection fragment is Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH;
s2, preparing an X2 full-protection fragment, wherein the X2 full-protection fragment is Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH;
s3, removing Fmoc protecting groups of the Rink Resin by using a deprotection reagent, and sequentially coupling X1 full-protection fragments and X2 full-protection fragments on the Rink Resin at intervals by using a condensation reagent to obtain X1-X2-X1-X2-Rink Resin, namely Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin;
s4, removing Fmoc protecting groups from X1-X2-X1-X2-Rink Resin by using a deprotection reagent, and performing N-terminal acetylation by using a mixed solution of acetic anhydride and pyridine to obtain Ac-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin;
s5 cleavage of Ac-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink RFiltering and settling the esin, cracking reaction liquid to obtain solid crude peptide, repeatedly washing and centrifuging the solid crude peptide with diethyl ether for 2-5 times to obtain washed crude peptide, and drying the crude peptide until weight is no longer reduced to obtain Ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH 2 Crude products;
s6, purifying the crude product by HPLC, and freeze-drying to obtain Ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH 2 Lyophilized powder of the refined peptide.
Further, the substitution degree of the Rink Resin is 0.2-0.8 mmol/g.
Further, in the step S4, the molar ratio of X1-X2-X1-X2-Rink Resin with Fmoc protecting groups removed to acetic anhydride and pyridine is 1: (5-20): (5-20).
Further, before purifying the crude product in the step S5 by HPLC, the crude product needs to be dissolved by N-methyl pyrrolidone or DMF until the concentration of the crude product is 0.5-1 mmol/L.
Preferably, the preparation steps of the X1 full protection fragment are as follows:
1a, preparation of Fmoc-Ala-CTC Resin: fmoc-Ala-OH and organic base are added into the swelled 2-CTC Rsein to obtain Fmoc-Ala-CTC Resin;
1b, fmoc-Ala-CTC Resin adopts a gradual coupling mode, firstly uses a deprotection reagent to remove Fmoc protecting groups, and then uses a condensation reagent to sequentially couple amino acid raw materials Fmoc-Asp (OtBu) -OH, fmoc-Ala-OH and Fmoc-Arg (Pbf) -OH to obtain Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin;
1c, splitting Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin by adopting a full protection splitting mode to obtain an X1 full protection fragment, namely Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH;
the preparation steps of the X2 full-protection fragment are as follows:
2a, adding Fmoc-Ala-OH and organic base into the swelled 2-CTC Rsein to obtain Fmoc-Ala-CTC Resin;
2b, removing Fmoc protecting groups by using a deprotection reagent firstly in a stepwise coupling mode, and then sequentially coupling amino acid raw materials Fmoc-Lys (Boc) -OH, fmoc-Ala-OH and Fmoc-Glu (OtBu) -OH by using a condensation reagent to obtain Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin;
2c, adopting a full protection cleavage mode to cleave Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin to obtain X2 full protection fragments, namely Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH.
Preferably, the organic base in the steps 1a and 2a is DIPEA, and the molar ratio of the organic base to 2-CTC Rsein and Fmoc-Ala-OH is (2-10): 1: (1-5).
Preferably, the fully-protected cracking reagent is a dichloromethane solution of TFE, and the volume ratio of TFE to dichloromethane is (10-25): (75-90); the amount of the full-protection cleavage reagent used was 1g of peptide resin per 10ml of the full-protection cleavage reagent.
Preferably, the condensing agent is a mixed solution of DIC and HOBt with equal molar ratio or a mixed solution of PyBOP, DIEA and HOBt with equal molar ratio; or a mixture of HBTU, DIEA and HOBt in equimolar ratio;
in the step S3, the molar ratio of the condensing reagent, rink Resin, X1 full-protection fragment or X2 full-protection fragment is (4.8-12): 1: (2-5);
in the steps 1b and 2b, the molar ratio of the condensation reagent, fmoc-Ala-CTC Resin and the amino acid raw material is (4.8-12): 1: (2-5).
Preferably, the deprotection reagent is a mixed solution of piperidine and DMF, and the volume ratio of the piperidine to the DMF is: dmf=1: 4, a step of; the amount of deprotection reagent used is 1g of peptide resin/(10-20) ml of deprotection reagent.
Preferably, the cracked pyrolysis liquid is trifluoroacetic acid solution, wherein the volume ratio of trifluoroacetic acid is 85% -95%; the cracking time is 2-4 hours, and the cracking temperature is 25-45 ℃; the amount of lysate was 1g of peptide resin per 10ml of lysate.
The invention has the following advantages:
the invention synthesizes fully protected fragments Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH, and then sequentially couples the fragments on Rink Resin by using a condensation reagent in a stepwise coupling mode to obtain Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin. Removing Fmoc groups, performing acetylation reaction, shrinking peptide resin, cracking the peptide resin, and purifying crude peptide to obtain the RAKA 16 refined peptide. The conventional stepwise coupling synthesis period is 14 days, the total yield is about 10%, the synthesis period is shortened by about 8 days, the yield is improved, and the total yield is more than 20%.
The invention synthesizes RAKA 16 by a fragment method, which avoids the situation of difficult coupling of medium-length peptide due to the influence of secondary structure, shortens the synthesis period, simplifies the synthesis operation and improves the synthesis efficiency. Meanwhile, the generation of deletion peptide and racemization peptide is effectively reduced, so that the purification difficulty is reduced, and the method has wide application prospect.
Drawings
FIG. 1 is an HPLC plot of crude peptides;
FIG. 2 is an HPLC chromatogram of the refined peptide.
Detailed Description
The technical scheme of the invention is further described below with reference to the attached drawings and examples. In this example, the chinese meaning corresponding to the english abbreviation is shown in table 1 below.
Table 1 reagents and apparatus
The invention provides a solid-phase synthesis method of self-assembled polypeptide RAKA 16, which comprises the steps of firstly preparing X1 and X2 full-protection fragments, preparing RAKA 16 peptide resin by using a solid-phase polypeptide synthesis method, and using one or a combination of DMF, DCM and DMSO as a solvent. Cleavage of the peptide resin of RAKA 16 to obtain crude RAKA 16, purification and freeze-drying to obtain refined peptide Ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH of RAKA 16 2 。
Example 1
1. Preparing Fmoc-Ala-CTC Resin;
and weighing 0.032mol of 2-CTC Resin, swelling with DCM for 30min, wherein the volume of DCM is required to satisfy the requirement that 1g of Resin corresponds to 10-20ml of DCM. The molar ratio of the organic base to the 2-CTC Rsein and Fmoc-Ala-OH is (2-10): 1: (1-5), in this example, 0.032mol Fmoc-Ala-OH,0.064mol DIPEA and 500ml DMF were added. The amount of DMF dissolved and eluted was 1g of resin corresponding to 10-30ml. The reaction was carried out at 30℃for 3h. Filtering and washing to obtain Fmoc-Ala-CTC Resin.
2. Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin were prepared;
2.1 preparation of Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin;
to Fmoc-Ala-CTC Resin was added 500ml of 20% piperidine/DMF (v/v) solution for deprotection. Reacting for 20min, taking a small amount of resin, detecting with ninhydrin, detecting positive, washing, and suction filtering to obtain NH 2 -Ala-CTC Resin。
The amount of deprotection reagent used in this example was about 1g resin for 10-20ml of deprotection reagent.
In this example ninhydrin detection was: and (3) sequentially adding a small amount of resin into the reagent a, the absolute ethanol solution (w/v) of 5% ninhydrin, b, the absolute ethanol solution (w/v) of phenol, c and 1-2 drops of pyridine respectively, shaking uniformly, and heating for 5min at 105 ℃. If the solution and the resin are blue or deep blue, the detection is positive; if the solution and resin were bluish or no color change, the test was negative.
According to the characteristics of RAKA 16 peptide sequences, the X1 full-protection fragment and the X2 full-protection fragment are ingeniously designed with the purposes of shortening the coupling times and avoiding Arg coupling. Coupling amino acid raw materials and condensation reagents, wherein the amino acid raw materials comprise Fmoc-Asp (OtBu) -OH, fmoc-Ala-OH, fmoc-Arg (Pbf) -OH, fmoc-Glu (OtBu) -OH and Fmoc-Lys (Boc) -OH, and the condensation reagents comprise DIC and HOBt, NH 2 -Ala-CTC Resin, amino acid starting material, DIC, HOBt molar ratio 1: (2-5): (2.4 to 6): (2.4-6). The coupling temperature is 20-40 ℃. Preferably at about 30 ℃.
The specific coupling steps are as follows:
weighing 0.064mol Fmoc-Asp (OtBu) -OH,0.096mol HOBt into NH 2 To Ala-CTC Resin, 500ml DMF was added and stirred well, followed by 0.096mmol DIC. At 30 DEG CAnd (3) reacting for 2 hours, taking a small amount of resin, detecting by ninhydrin, detecting to be negative, and pumping out the reaction solution. Washing and suction filtering to obtain Fmoc-Asp (OtBu) -Ala-CTC Resin.
Fmoc-Ala-OH, fmoc-Arg (Pbf) -OH were sequentially ligated using the same procedure as described above.
Specifically, deprotection was performed by adding 500ml of 20% piperidine/DMF (v/v) solution to Fmoc-Asp (OtBu) -Ala-CTC Resin. Reacting for 20min, taking a small amount of resin, detecting with ninhydrin, detecting positive, washing, and suction filtering to obtain NH 2 -Asp(OtBu)-Ala-CTC Resin。
Weighing 0.048mol Fmoc-Ala-OH,0.096mol HOBt into NH 2 To the Asp (OtBu) -Ala-CTC Resin was added 500ml DMF and stirred well and 0.096mmol DIC was added. Reacting for 2 hours at 30 ℃, taking a small amount of resin, detecting with ninhydrin, detecting to be negative, and pumping out the reaction liquid. Washing and suction filtering to obtain Fmoc-Ala-Asp (OtBu) -Ala-CTC Resin.
To NH 2 To the Ala-Asp (OtBu) -Ala-CTC Resin was added 500ml of 20% piperidine/DMF (v/v) solution for deprotection. Reacting for 20min, taking a small amount of resin, detecting with ninhydrin, detecting positive, washing, and suction filtering to obtain NH 2 -Ala-Asp(OtBu)-Ala-CTC Resin。
Then 0.048mol Fmoc-Arg (Pbf) -OH and 0.096mol HOBt were weighed into Fmoc-Ala-Asp (OtBu) -Ala-CTC Resin, 500ml DMF was added and stirred well, and then 0.096mmol DIC was added. Reacting for 2 hours at 30 ℃, taking a small amount of resin, detecting with ninhydrin, detecting to be negative, and pumping out the reaction liquid. Washing and suction filtering to obtain Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin.
2.2 preparation of Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin;
to Fmoc-Ala-CTC Resin was added 500ml of 20% piperidine/DMF (v/v) solution for deprotection. Reacting for 20min, taking a small amount of resin, detecting with ninhydrin, detecting positive, washing, and suction filtering to obtain NH 2 -Ala-CTC Resin。
Weighing 0.048mol Fmoc-Lys (Boc) -OH,0.096mol HOBt into NH 2 To Ala-CTC Resin, 500ml of DMF was added and stirred well, followed by 0.096mmol of DIC. Reacting at 30deg.C for 2 hr, taking a small amount of resin, detecting with ninhydrin, detecting to show negative, and drainingAnd (3) liquid. Washing and suction filtering to obtain Fmoc-Lys (Boc) -Ala-CTC Resin.
Fmoc-Ala-OH, fmoc-Glu (OtBu) -OH were sequentially ligated using the same procedure as described above.
Specifically, deprotection was performed by adding 500ml of 20% piperidine/DMF (v/v) solution to Fmoc-Lys (Boc) -Ala-CTC Resin. Reacting for 20min, taking a small amount of resin, detecting with ninhydrin, detecting positive, washing, and suction filtering to obtain NH 2 -Lys(Boc)-Ala-CTC Resin。
Weighing 0.048mol Fmoc-Ala-OH,0.096mol HOBt into NH 2 To the Lys (Boc) -Ala-CTC Resin, 500ml DMF was added and stirred well before 0.096mmol DIC was added. Reacting for 2 hours at 30 ℃, taking a small amount of resin, detecting with ninhydrin, detecting to be negative, and pumping out the reaction liquid. Washing and suction filtering to obtain Fmoc-Ala-Lys (Boc) -Ala-CTC Resin.
Deprotection was performed by adding 500ml of 20% piperidine/DMF (v/v) solution to Fmoc-Ala-Lys (Boc) -Ala-CTC Resin. Reacting for 20min, taking a small amount of resin, detecting with ninhydrin, detecting positive, washing, and suction filtering to obtain NH 2 -Ala-Lys(Boc)-Ala-CTC Resin。
Then 0.048mol of Fmoc-Glu (OtBu) -OH and 0.096mol of HOBt are weighed into NH 2 To Ala-Lys (Boc) -Ala-CTC Resin, 500ml DMF was added and stirred well before 0.096mmol DIC was added. Reacting for 2 hours at 30 ℃, taking a small amount of resin, detecting with ninhydrin, detecting to be negative, and pumping out the reaction liquid. Washing and suction filtering to obtain Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin.
3. Preparation of full protection fragments
Splitting Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin according to a splitting ratio of 1g/10ml, wherein the full-protection splitting reagent is a dichloromethane solution of TFE, and the volume ratio of TFE to dichloromethane is 10-25: 75-90, preferably the volume ratio of the full-protection pyrolysis liquid is TFE: dcm=1: 9. cracking for 3h, wherein the cracking temperature is about 30 ℃. The cleavage reaction solution was filtered through a sand core funnel, leaving the filtrate. The filtrate was rotary evaporated to give a white or off-white solid. And then placing the solid obtained by rotary evaporation into a vacuum drying oven for drying under reduced pressure until the weight is no longer reduced, and finally obtaining the full-protection segment: x1 full protection fragment Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH and X2 full protection fragment Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH.
4. Preparation of RAKA 16 peptide resins
Weighing 0.001mol of Rink Resin, swelling with 40ml of DCM for 30min, wherein the adding proportion of DCM is required to satisfy the requirement that 1g of Resin corresponds to 10-20ml of DCM. The substitution degree of the Rink Resin is 0.2-0.8 mmol/g. The substitution degree of the Rink Resin is preferably 0.3-0.6 mmol/g.
40ml of 20% piperidine/DMF (v/v) solution was added to 0.001mol rink Resin for deprotection. Reacting for 20min, taking a small amount of resin, detecting with ninhydrin, detecting positive, washing, and suction filtering to obtain NH 2 -Rink Resin。
NH 2 -Rink Resin, X1 full protection fragment or X2 full protection fragment, DIC and HOBt in a molar ratio of 1: (2-5): (2.4 to 6): (2.4-6).
In this example, 0.004mol X2 of Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH was weighed, 0.0048mol HOBt was put into Rink Resin, 40ml DMF was added and stirred well, and then 0.0048mol DIC was added. And (3) reacting for 4 hours, taking a small amount of resin, detecting by ninhydrin, detecting to be negative, and pumping out the reaction solution. Washing and suction filtering to obtain Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin.
The same procedure as described above was used to sequentially ligate X1 full protection fragment Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH, X2 full protection fragment Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH, and X1 full protection fragment Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH, followed by washing and drying to obtain Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin.
A proper amount of 20% piperidine/DMF (v/v) solution was added to the reaction column for deprotection. Reacting for 20min, taking a small amount of resin, detecting with ninhydrin, detecting positive, washing, and suction filtering to obtain NH 2 -Arg(Pbf)-Ala-Asp(OtBu)-Ala-Glu（OtBu）-Ala-Lys（Boc)-Ala-Arg(Pbf)-Ala-Asp(OtBu)-Ala-Glu（OtBu）-Ala-Lys（Boc)-Ala-Rink Resin。
A mixture of 0.02mol of acetic anhydride, 0.02mol of pyridine and 40ml of DMF was added to the reaction column. The reaction was carried out for 60min. The peptide resin was washed 6 times with DMF, the washing waste was removed, and 40ml of anhydrous methanol was added to shrink the peptide resin 3 times, and the shrink waste was removed. Transferring the peptide resin into a vacuum drying oven for reduced pressure drying until the weight is no longer reduced, and obtaining the RAKA 16 peptide resin: ac-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin.
5. Preparation of RAKA 16 refined peptide
And (3) adding RAKA 16 peptide resin into the lysate to carry out a cleavage reaction. The amount of lysate was 1g of peptide resin per 10ml of lysate. The pyrolysis liquid is high-concentration trifluoroacetic acid solution with the concentration of 85% -95%, and other components of the pyrolysis liquid are corresponding side chain protecting group capturing reagents. The specific trifluoroacetic acid solution is a mixed solution of trifluoroacetic acid, phenylsulfide, EDT and water, wherein the volume ratio of the trifluoroacetic acid is 85% -95%, the other three reagents are the same, and the sum of the volume ratios of the four reagents is 100%; in this example, the preferred lysate ratio is TFA: phenyl sulfide: EDT: water=91: 3:3:3 (v/v), cleaved at 30℃for 3h. Settling by methyl tertiary butyl ether, washing and centrifuging to obtain the RAKA 16 crude peptide. The crude peptide was 68.597% pure.
Dissolving the crude peptide with N-methylpyrrolidone or DMF to 0.5mmol/L to 1mmol/L, further purifying by HPLC under the conditions that phase A is a purified water solution of 0.1% trifluoroacetic acid and phase B is acetonitrile, gradient eluting for 40min, collecting sample solution with purity of more than 90%, and freeze-drying. Before freeze-drying, pre-freezing the sample solution, freeze-drying in a freeze dryer for more than 48 hours, and freeze-drying to obtain RAKA 16 refined peptide: ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH 2 。
HPLC analysis was performed on a small sample of the crude peptide from example 1, resulting in FIG. 1, which was 68.597% pure, which was superior. HPLC analysis is performed on the refined peptide sample to obtain the sample with the purity of 95.480 percent in figure 2, the purity is better, and the impurities attached to the main peak are almost absent, so that the purity is still further improved.
Example 2
1. Preparing Fmoc-Ala-CTC Resin;
0.032mol of 2-CTC Resin was weighed and swollen with DCM for 30min. The molar ratio of the organic base to the 2-CTC Rsein and Fmoc-Ala-OH is (2-10): 1: (1-5), in this example, 0.16mol Fmoc-Ala-OH,0.32mol DIPEA and 500ml DMF were added. The reaction was carried out at 30℃for 3h. Filtering and washing to obtain Fmoc-Ala-CTC Resin.
2. Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin were prepared;
unlike example 1, the condensing agent includes DIC and HOBt, NH 2 -Ala-CTC Resin, amino acid starting material, DIC, HOBt molar ratio 1:5:6:6. the temperature of the coupling was 38 ℃.
Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin were obtained.
3. Preparation of full protection fragments
Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin were cleaved at a cleavage ratio of 1g/10ml using a full-protection cleavage reagent in methylene chloride solution of TFE in a volume ratio of TFE to methylene chloride of 25:75, cracking for 3h, wherein the cracking temperature is about 30 ℃. Finally obtaining the full protection segment: x1 full protection fragment Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH and X2 full protection fragment Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH.
4. Preparation of RAKA 16 peptide resins
The difference from example 1 is that the substitution degree of the Rink Resin is preferably 0.5mmol/g. NH (NH) 2 -Rink Resin, X1 full protection fragment or X2 full protection fragment, DIC and HOBt in a molar ratio of 1:5:6:6.
obtaining RAKA 16 peptide resin: ac-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin.
5. Preparation of RAKA 16 refined peptide
The difference from example 1 is that the lysate is a high concentration trifluoroacetic acid solution with a concentration of 95%, the cleavage time is 2 hours, and the cleavage temperature is 25 ℃. Settling by methyl tertiary butyl ether, washing and centrifuging to obtain the RAKA 16 crude peptide. The crude peptide was 69.125% pure.
Dissolving the crude peptide with N-methylpyrrolidone or DMF to 1mmol/L, further purifying by HPLC under the condition that phase A is a purified water solution of 0.1% trifluoroacetic acid, phase B is acetonitrile, gradient eluting for 40min, collecting sample solution with purity of more than 90%, and freeze-drying. Before freeze-drying, pre-freezing the sample solution, freeze-drying in a freeze dryer for more than 48 hours, and freeze-drying to obtain RAKA 16 refined peptide: ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH 2 。
Example 3
1. Preparing Fmoc-Ala-CTC Resin;
0.032mol of 2-CTC Resin was weighed and swollen with DCM for 30min. The molar ratio of the organic base to the 2-CTC Rsein and Fmoc-Ala-OH is (2-10): 1: (1-5), in this example, 0.096mol Fmoc-Ala-OH,0.16mol DIPEA and 500ml DMF were added. The reaction was carried out at 30℃for 3h. Filtering and washing to obtain Fmoc-Ala-CTC Resin.
2. Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin were prepared;
the difference from example 1 is that the condensing agent comprises PyBOP and DIEA and HOBt, NH 2 -Ala-CTC Resin, amino acid starting material, pyBOP, DIC, HOBt in a molar ratio of 1:2:2.4:2.4:2.4. the temperature of the coupling was 25 ℃.
Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin were obtained.
3. Preparation of full protection fragments
Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin and Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin were cleaved at a cleavage ratio of 1g/10ml using a full-protection cleavage reagent in methylene chloride solution of TFE in a volume ratio of TFE to methylene chloride of 20:80, cracking for 3h, wherein the cracking temperature is about 30 ℃. Finally obtaining the full protection segment: x1 full protection fragment Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH and X2 full protection fragment Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH.
4. Preparation of RAKA 16 peptide resins
The difference from example 1 is that the substitution degree of the Rink Resin is preferably 0.6mmol/g. NH (NH) 2 -Rink Resin, X1 full protection fragment or X2 full protection fragment, DIC and HOBt in a molar ratio of 1:3:4:4.
obtaining RAKA 16 peptide resin: ac-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin.
5. Preparation of RAKA 16 refined peptide
The difference from example 1 is that the lysate is a high concentration trifluoroacetic acid solution with a concentration of 85%, the cleavage time is 4 hours, and the cleavage temperature is 45 ℃. Settling by methyl tertiary butyl ether, washing and centrifuging to obtain the RAKA 16 crude peptide. The crude peptide was 58.667% pure.
Dissolving the crude peptide with N-methylpyrrolidone or DMF to 0.5mmol/L, further purifying by HPLC under the condition that phase A is a purified water solution of 0.1% trifluoroacetic acid, phase B is acetonitrile, gradient eluting for 40min, collecting sample solution with purity of more than 90%, and freeze-drying. Before freeze-drying, pre-freezing the sample solution, freeze-drying in a freeze dryer for more than 48 hours, and freeze-drying to obtain RAKA 16 refined peptide: ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH 2 。
Comparative example 1
The difference between this comparative example and example 1 is: example 1A RAKA peptide resin was synthesized by combining stepwise coupling and fragmentation. The comparative example adopts a stepwise coupling mode to synthesize the RAKA peptide resin in the whole process.
1. Preparation of RAKA 16 peptide resins
Weighing 0.001mol of Rink Resin, swelling with 40ml of DCM for 30min, wherein the adding proportion of DCM is required to satisfy the requirement that 1g of Resin corresponds to 10-20ml of DCM. The substitution degree of the Rink Resin is preferably 0.3-0.6 mmol/g.
40ml of 20% piperidine/DMF (v/v) solution was added to 0.001mol rink Resin for deprotection. After the reaction is finished, washing and suction filtering to obtain NH 2 -Rink Resin。
NH 2 -Rink Resin, amino acid starting material, DIC and HOBt in a molar ratio of 1: (2-5): (2.4 to 6): (2.4-6). Amino acid starting materials include Fmoc-Ala-OH, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Lys (Boc) -OH and Fmoc-Glu (OtBu) -OH.
In this comparative example, 0.004mol Fmoc-Ala-OH and 0.005mol HOBt were weighed into NH 2 To Rink Resin, 40ml of DMF was added and stirred well, followed by 0.005mol of DIC. The reaction was carried out for 4 hours. Washing and suction filtering to obtain Fmoc-Ala-Rink Resin.
Fmoc-Lys (Boc) -OH, fmoc-Ala-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Asp (OtBu) -OH, fmoc-Ala-OH, fmoc-Arg (Pbf) -OH, fmoc-Ala-OH, fmoc-Lys (Boc) -OH, fmoc-Ala-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, fmoc-Asp (OtBu) -OH, fmoc-Ala-OH, fmoc-Arg (Pbf) -OH were then sequentially connected by the same method as described above. Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin was obtained. Fmoc-Arg (Pbf) -OH coupling reaction of two sites is difficult, repeated feeding is carried out, and the reaction time is prolonged. And the ninhydrin detection is still positive when the coupling is finished, but compared with the ninhydrin detection of deprotection of the previous site, the ninhydrin detection has obvious change, and most sites are judged to have completed the coupling reaction, so that the next reaction is continued.
A proper amount of 20% piperidine/DMF (v/v) solution was added to the reaction column for deprotection. After the reaction is finished, washing and suction filtering to obtain NH 2 -Arg(Pbf)-Ala-Asp(OtBu)-Ala-Glu（OtBu）-Ala-Lys（Boc)-Ala-Arg(Pbf)-Ala-Asp(OtBu)-Ala-Glu（OtBu）-Ala-Lys（Boc)-Ala-Rink Resin。
A mixture of 0.02mol of acetic anhydride, 0.02mol of pyridine and 40ml of DMF was added to the reaction column. The reaction was carried out for 60min. The peptide resin was washed 6 times with DMF, the washing waste was removed, and 40ml of anhydrous methanol was added to shrink the peptide resin 3 times, and the shrink waste was removed. Transferring the peptide resin into a vacuum drying oven for reduced pressure drying until the weight is no longer reduced, and obtaining the RAKA 16 peptide resin: ac-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin.
2. Preparation of crude RAKA 16 peptides
And (3) adding the RAKA 16 peptide resin obtained in step (1) into a lysate to carry out a cleavage reaction. The amount of lysate was 1g of peptide resin per 10ml of lysate. The pyrolysis liquid is high-concentration trifluoroacetic acid solution with the concentration of 85% -95%, the trifluoroacetic acid solution is mixed solution of trifluoroacetic acid, phenylsulfide, EDT and water, and in the comparative example, the preferable ratio of the pyrolysis liquid is TFA: phenyl sulfide: EDT: water=91: 3:3:3 (v/v). Cracking for 3h at 30 ℃. Settling by methyl tertiary butyl ether, washing and centrifuging to obtain the RAKA 16 crude peptide. The crude peptide was 45.083 in purity.
3. Preparation of RAKA refined peptide
Dissolving the crude peptide with N-methylpyrrolidone or DMF to 0.5mmol/L to 1mmol/L, further purifying by HPLC under the conditions that phase A is a purified water solution of 0.1% trifluoroacetic acid and phase B is acetonitrile, gradient eluting for 40min, collecting sample solution with purity of more than 90%, and freeze-drying. Before freeze-drying, pre-freezing the sample solution, freeze-drying in a freeze dryer for more than 48 hours, and freeze-drying to obtain RAKA 16 refined peptide: ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH 2 。
The RAKA 16 refined peptides of examples 1-3 and comparative example 1 were compared and the data obtained are shown in Table 2.
Table 2 shows comparative data
As can be seen from the data in Table 2, the conventional stepwise coupling (comparative example 1) had a synthesis period of 14 days and a total yield of about 10%, and the present invention shortened the synthesis period by about 8 days, increased the purities of the crude and refined peptides and increased the total yield by about 20%.
In conclusion, the RAKA 16 is synthesized by a fragment method, so that the situation of difficult coupling of the medium-length peptide due to the influence of a secondary structure is avoided, the synthesis period is shortened, the synthesis operation is simplified, and the synthesis efficiency is improved. Meanwhile, the problem that more missing impurities are easy to generate when the 9 th Arg and the 1 st Arg are connected is skillfully avoided, and the generation of missing peptide and racemized peptide is effectively reduced, so that the purification difficulty is reduced, and the method has a wide application prospect.
Claims (10)
1. A solid phase synthesis method of self-assembled polypeptide RAKA 16, comprising the steps of:
s1, preparing an X1 full-protection fragment, wherein the X1 full-protection fragment is Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH;
s2, preparing an X2 full-protection fragment, wherein the X2 full-protection fragment is Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH;
s3, removing Fmoc protecting groups of the Rink Resin by using a deprotection reagent, and sequentially coupling X1 full-protection fragments and X2 full-protection fragments on the Rink Resin at intervals by using a condensation reagent to obtain X1-X2-X1-X2-Rink Resin, namely Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin;
s4, removing Fmoc protecting groups from X1-X2-X1-X2-Rink Resin by using a deprotection reagent, and performing N-terminal acetylation by using a mixed solution of acetic anhydride and pyridine to obtain Ac-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin;
s5, cracking Ac-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Arg (Pbf) -Ala-Asp (OtBu) -Ala-Glu (OtBu) -Ala-Lys (Boc) -Ala-Rink Resin, filtering the cracking reaction solution, settling to obtain a solid crude peptide, repeatedly washing and centrifuging the solid crude peptide with diethyl ether for 2-5 times to obtain a washed crude peptide, and drying the crude peptide until weight is not reduced to obtain Ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Lys-Ala-Glu-Ala-NH 2 Crude products;
s6, purifying the crude product by HPLC, and freeze-drying to obtain Ac-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-Arg-Ala-Asp-Ala-Glu-Ala-Lys-Ala-NH 2 Lyophilized powder of the refined peptide.
2. The method for solid phase synthesis of RAKA 16 according to claim 1, wherein: the substitution degree of the Rink Resin is 0.2-0.8 mmol/g.
3. The method for solid phase synthesis of RAKA 16 according to claim 1, wherein: the molar ratio of X1-X2-X1-X2-Rink Resin to acetic anhydride and pyridine for removing Fmoc protecting groups in the step S4 is 1: (5-20): (5-20).
4. The method for solid phase synthesis of RAKA 16 according to claim 1, wherein: before the crude product in the step S5 is purified by HPLC, the crude product is dissolved by N-methyl pyrrolidone or DMF until the concentration of the crude product is 0.5-1 mmol/L.
5. The method for solid phase synthesis of RAKA 16 according to claim 1, wherein:
the preparation steps of the X1 full-protection fragment are as follows:
1a, preparation of Fmoc-Ala-CTC Resin: fmoc-Ala-OH and organic base are added into the swelled 2-CTC Rsein to obtain Fmoc-Ala-CTC Resin;
1b, fmoc-Ala-CTC Resin adopts a gradual coupling mode, firstly uses a deprotection reagent to remove Fmoc protecting groups, and then uses a condensation reagent to sequentially couple amino acid raw materials Fmoc-Asp (OtBu) -OH, fmoc-Ala-OH and Fmoc-Arg (Pbf) -OH to obtain Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin;
1c, splitting Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-CTC Resin by adopting a full protection splitting mode to obtain an X1 full protection fragment, namely Fmoc-Arg (Pbf) -Ala-Asp (OtBu) -Ala-OH;
the preparation steps of the X2 full-protection fragment are as follows:
2a, adding Fmoc-Ala-OH and organic base into the swelled 2-CTC Rsein to obtain Fmoc-Ala-CTC Resin;
2b, removing Fmoc protecting groups by using a deprotection reagent firstly in a stepwise coupling mode, and then sequentially coupling amino acid raw materials Fmoc-Lys (Boc) -OH, fmoc-Ala-OH and Fmoc-Glu (OtBu) -OH by using a condensation reagent to obtain Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin;
2c, adopting a full protection cleavage mode to cleave Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-CTC Resin to obtain X2 full protection fragments, namely Fmoc-Glu (OtBu) -Ala-Lys (Boc) -Ala-OH.
6. The method for solid phase synthesis of RAKA 16 according to claim 5, wherein: the organic base in the steps 1a and 2a is DIPEA, and the molar ratio of the organic base to 2-CTC Rsein and Fmoc-Ala-OH is (2-10): 1: (1-5).
7. The method for solid phase synthesis of RAKA 16 according to claim 5, wherein: the full-protection cracking reagent is a dichloromethane solution of TFE, and the volume ratio of TFE to dichloromethane is (10-25): (75-90); the amount of the full-protection cleavage reagent used was 1g of peptide resin per 10ml of the full-protection cleavage reagent.
8. The method of solid phase synthesis of RAKA 16 according to claim 1 or 5, wherein: the condensation reagent is mixed liquid of DIC and HOBt with equal molar ratio or mixed liquid of PyBOP, DIEA and HOBt with equal molar ratio; or a mixture of HBTU, DIEA and HOBt in equimolar ratio;
in the step S3, the molar ratio of the condensing reagent, rink Resin, X1 full-protection fragment or X2 full-protection fragment is (4.8-12): 1: (2-5);
in the steps 1b and 2b, the molar ratio of the condensation reagent, fmoc-Ala-CTC Resin and the amino acid raw material is (4.8-12): 1: (2-5).
9. The method of solid phase synthesis of RAKA 16 according to claim 1 or 5, wherein: the deprotection reagent is a mixed solution of piperidine and DMF, and the volume ratio of the piperidine to the DMF is: dmf=1: 4, a step of; the amount of deprotection reagent used is 1g of peptide resin/(10-20) ml of deprotection reagent.
10. The method for solid phase synthesis of RAKA 16 according to claim 1, wherein: the cracked cracking liquid is trifluoroacetic acid solution, wherein the volume ratio of trifluoroacetic acid is 85% -95%; the cracking time is 2-4 hours, and the cracking temperature is 25-45 ℃; the amount of lysate was 1g of peptide resin per 10ml of lysate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410004984.4A CN117486976B (en) | 2024-01-03 | 2024-01-03 | Synthesis method of self-assembled polypeptide RAKA 16 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410004984.4A CN117486976B (en) | 2024-01-03 | 2024-01-03 | Synthesis method of self-assembled polypeptide RAKA 16 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117486976A CN117486976A (en) | 2024-02-02 |
| CN117486976B true CN117486976B (en) | 2024-04-09 |
Family
ID=89674859
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410004984.4A Active CN117486976B (en) | 2024-01-03 | 2024-01-03 | Synthesis method of self-assembled polypeptide RAKA 16 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117486976B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201730203A (en) * | 2015-12-04 | 2017-09-01 | 廣州南沙龍沙有限公司 | Method for preparation of RADA-16 |
| CN110396124A (en) * | 2019-08-21 | 2019-11-01 | 上海交通大学 | Self assembly polypeptide and its purposes as hemostat |
| WO2023222057A1 (en) * | 2022-05-19 | 2023-11-23 | 江苏奥赛康药业有限公司 | Method for preparing self-assembling peptide rada16 by means of solid phase convergent synthesis |
-
2024
- 2024-01-03 CN CN202410004984.4A patent/CN117486976B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201730203A (en) * | 2015-12-04 | 2017-09-01 | 廣州南沙龍沙有限公司 | Method for preparation of RADA-16 |
| CN110396124A (en) * | 2019-08-21 | 2019-11-01 | 上海交通大学 | Self assembly polypeptide and its purposes as hemostat |
| WO2023222057A1 (en) * | 2022-05-19 | 2023-11-23 | 江苏奥赛康药业有限公司 | Method for preparing self-assembling peptide rada16 by means of solid phase convergent synthesis |
Non-Patent Citations (2)
| Title |
|---|
| RADA-16: A Tough Peptide – Strategies for Synthesis and Purification;Marta Paradís-Bas等;Eur. J. Org. Chem.;20131231;第5871-5878页 * |
| 多肽LGEAKAL的参数和合成路线|三字母为Leu-Gly-Glu-Ala-Lys-Ala-Leu|;专肽生物VX;https://www.allpeptide.com/article/2376.html;20230327;第1-4页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117486976A (en) | 2024-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3398960B1 (en) | Method for preparing semaglutide | |
| CN106699871B (en) | Preparation method of liraglutide | |
| JP4405594B2 (en) | Improved solid phase peptide synthesis and reagents for use in such synthesis | |
| KR101297942B1 (en) | Synthesis of glucagon-like peptide | |
| CN113880935B (en) | Preparation method of Somaloutide full-protection peptide resin and preparation method of Somaloutide | |
| CN113423723A (en) | Preparation method of somaglutide | |
| CN112679602B (en) | Solid phase synthesis method of cable Ma Lutai | |
| CN117486976B (en) | Synthesis method of self-assembled polypeptide RAKA 16 | |
| CN110642936B (en) | Method for preparing teriparatide | |
| CN112175067B (en) | Preparation method of teduglutide | |
| CN115038711B (en) | Synthesis method of atosiban | |
| CN111732632B (en) | Synthesis method of linaclotide | |
| CN114478708A (en) | Solid phase synthesis method of ganirelix fragment | |
| CN110256532B (en) | RGD cyclopeptide synthesis method | |
| CN110845600B (en) | Method for preparing liraglutide | |
| CN113045641A (en) | Preparation method of Somalutide | |
| CN111018962A (en) | Method for preparing teduglutide based on solid phase step-by-step method | |
| CN111018963B (en) | Preparation method of glucagon | |
| CN110981939A (en) | Preparation method of polycaprolactam | |
| CN111499719B (en) | Method for synthesizing pramlintide | |
| CN114920804B (en) | Cetrorelix synthesis process capable of being directly used for pilot scale amplification | |
| CN112521482B (en) | Preparation method for synthesizing nesiritide by solid-liquid combination | |
| CN114456236A (en) | Preparation method of degarelix acetylated impurities | |
| CN108239147B (en) | Process for preparing thymosin alpha1 derivatives | |
| US20230406900A1 (en) | Process for preparing glucagon-like peptide-1 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |