CN112279905A - Preparation process of conus anti-wrinkle agent - Google Patents
Preparation process of conus anti-wrinkle agent Download PDFInfo
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- CN112279905A CN112279905A CN202011079899.2A CN202011079899A CN112279905A CN 112279905 A CN112279905 A CN 112279905A CN 202011079899 A CN202011079899 A CN 202011079899A CN 112279905 A CN112279905 A CN 112279905A
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- conus
- conotoxin
- wrinkle
- disulfide bonds
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- 230000001153 anti-wrinkle effect Effects 0.000 title claims abstract description 76
- 241000237970 Conus <genus> Species 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 70
- 125000006239 protecting group Chemical group 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- 108050003126 conotoxin Proteins 0.000 claims abstract description 37
- 239000002243 precursor Substances 0.000 claims abstract description 25
- 238000005859 coupling reaction Methods 0.000 claims abstract description 21
- 230000008878 coupling Effects 0.000 claims abstract description 18
- 238000010168 coupling process Methods 0.000 claims abstract description 18
- 239000002994 raw material Substances 0.000 claims abstract description 15
- 150000001413 amino acids Chemical class 0.000 claims abstract description 12
- RDRBIXSNGAYLPT-UHFFFAOYSA-N CC1=CC=C(COC2=CC3=C(C=C2)C(NC(=O)OCC2C4=C(C=CC=C4)C4=C2C=CC=C4)C2=C(O3)C=CC=C2)C=C1 Chemical compound CC1=CC=C(COC2=CC3=C(C=C2)C(NC(=O)OCC2C4=C(C=CC=C4)C4=C2C=CC=C4)C2=C(O3)C=CC=C2)C=C1 RDRBIXSNGAYLPT-UHFFFAOYSA-N 0.000 claims abstract description 11
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims abstract description 10
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 9
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- 230000003647 oxidation Effects 0.000 claims abstract description 9
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 9
- 238000006467 substitution reaction Methods 0.000 claims abstract description 9
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims abstract description 6
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 230000008961 swelling Effects 0.000 claims abstract description 6
- 238000004090 dissolution Methods 0.000 claims abstract description 3
- 238000005336 cracking Methods 0.000 claims abstract 3
- 230000001590 oxidative effect Effects 0.000 claims abstract 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 16
- 238000007363 ring formation reaction Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 12
- 239000011347 resin Substances 0.000 claims description 12
- 229920005989 resin Polymers 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 11
- 239000006166 lysate Substances 0.000 claims description 11
- HPTFPWMPQFBSHP-IBGZPJMESA-N (2s)-1-[2-(9h-fluoren-9-ylmethoxycarbonylamino)acetyl]pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)CNC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 HPTFPWMPQFBSHP-IBGZPJMESA-N 0.000 claims description 8
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 8
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 claims description 7
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 claims description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 claims description 5
- ZDUMTHLUTJOUML-IBGZPJMESA-N (2r)-3-(tert-butyldisulfanyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CSSC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 ZDUMTHLUTJOUML-IBGZPJMESA-N 0.000 claims description 4
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 claims description 4
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000007821 HATU Substances 0.000 claims description 3
- 238000013375 chromatographic separation Methods 0.000 claims description 2
- 230000009089 cytolysis Effects 0.000 claims description 2
- 239000007800 oxidant agent Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 5
- 238000010532 solid phase synthesis reaction Methods 0.000 abstract description 4
- 108010016626 Dipeptides Proteins 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract 1
- 150000003839 salts Chemical class 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 36
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 30
- 239000007788 liquid Substances 0.000 description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 18
- 238000005406 washing Methods 0.000 description 18
- 238000001035 drying Methods 0.000 description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- 238000003756 stirring Methods 0.000 description 12
- 238000005303 weighing Methods 0.000 description 12
- 239000007864 aqueous solution Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 238000005086 pumping Methods 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000000746 purification Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000012317 TBTU Substances 0.000 description 6
- 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 6
- 239000000706 filtrate Substances 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 238000002390 rotary evaporation Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 241001638933 Cochlicella barbara Species 0.000 description 4
- 101800001442 Peptide pr Proteins 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 244000205754 Colocasia esculenta Species 0.000 description 3
- 235000006481 Colocasia esculenta Nutrition 0.000 description 3
- 241000237858 Gastropoda Species 0.000 description 3
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 3
- 230000006835 compression Effects 0.000 description 3
- 238000007906 compression Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000000967 suction filtration Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 2
- 238000007086 side reaction Methods 0.000 description 2
- 238000007792 addition Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- 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
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a preparation process of conus anti-wrinkle essence, which comprises the following steps: 1) selecting Sieber Amide Resin with substitution degree of 0.3-0.6mmol/g, and removing Fmoc after swelling; 2) sequentially coupling the amino acid sequence of the conotoxin as shown in SEQ ID NO.1 to the last amino acid H-Pyr-OH; 3) cracking conus anti-wrinkle linear peptide from Sieber Amide Resin, and cyclizing the first pair of disulfide bonds with 25% mercaptoethanol/DMF solution; 4) cracking and removing all side chain protecting groups to obtain conotoxin precursor peptide containing a pair of disulfide bonds, and naturally oxidizing after dissolution to obtain crude conotoxin peptide forming three pairs of disulfide bonds; 5) separating, purifying, transferring salt and freeze-drying to obtain the conus anti-wrinkle essence product. The technology adopts Fmoc solid phase synthesis strategy, utilizes various condensing agent systems, dipeptide raw materials, positioning oxidation and natural oxidation to obtain the conotoxin, has the characteristics of high reaction operation efficiency, low cost, high yield and the like, and is suitable for large-scale production.
Description
Technical Field
The invention relates to a preparation process for polypeptide synthesis, in particular to a preparation process for conotoxin.
Background
The conus anti-wrinkle agent is a highly folded peptide, is commonly used in the field of cosmetics, can play an anti-wrinkle effect, and has high stability and strong penetrating power due to the unique spatial structure. The conotoxin is composed of 22 amino acids, and the specific sequence is as follows: pyr1-Gly2-Cys3-Cys4-Asn5-Gly6-Pro7-Lys8-Gly9-Cys10-Ser11-Ser12-Lys13-Trp14-Cys15-Arg16-Asp17-His18-Ala19-Arg20-Cys21-Cys22-NH2(Disulfide bond: Cys3-Cys15, Cys4-Cys21, Cys10-Cys22), Casno.:936616-33-0, molecular formula: C92H139N35O28S, molecular weight: 2375.76.
because conus anti-wrinkle essence has three pairs of disulfide bonds, according to the traditional solid-phase synthesis and oxidation thinking, the positioned cyclization is realized by utilizing the differentiation of different side chain protecting groups of three pairs of Cys, and the defects of complicated steps, more side reactions, low yield, low operability, incapability of large-scale production and the like are brought. And three pairs of disulfide bonds are formed by natural oxidation, several forms of isomers can appear, the purification difficulty can be improved, and the final yield of the product is greatly reduced.
Therefore, on the premise, the synthesis scheme and the oxidation process of the conus anti-wrinkle element are improved and optimized, so that the aims of increasing the yield of the conus anti-wrinkle element again and realizing large-scale production are fulfilled.
Disclosure of Invention
The invention provides a preparation process of conotoxin, which aims to shorten the synthesis time, improve the purity of conotoxin straight-chain peptide and well solve the problems of more side reactions, low yield and poor operability caused by cyclization reaction of three pairs of disulfide bonds.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation process of conus anti-wrinkle essence comprises the following steps:
1) selecting Sieber Amide Resin with substitution degree of 0.3-0.6mmol/g, and removing Fmoc after swelling;
2) coupling conus anti-wrinkle straight-chain peptide resin: sequentially coupling the C end to the N end of the amino acid sequence of the conus anti-wrinkle element shown as SEQ ID NO.1 to the last amino acid H-Pyr-OH of the N end to obtain the conus anti-wrinkle element straight-chain peptide resin; wherein, the amino acids used at the 3 rd, 4 th, 15 th and 21 st positions of the N terminal are Fmoc-Cys (Trt) -OH, the amino acids used at the 10 th and 22 th positions are Fmoc-Cys (StBu) -OH, the reaction raw materials used at the 6 th and 7 th positions are Fmoc-Gly-Pro-OH, and the reaction raw materials used at the 11 th and 12 th positions are Fmoc-Ser (tBu) -OH; the side chain protecting groups of Asn, Lys, Trp, Arg, Asp and His are respectively Trt, Boc, Pbf, OtBu and Trt;
3) reacting TFA/DCM solution with conotoxin straight chain peptide resin, filtering to obtain conotoxin straight chain peptide with side chain protecting group, and cyclizing a first pair of disulfide bonds by mercaptoethanol/DMF to obtain a precursor peptide of conotoxin anti-wrinkle peptide with side chain protecting group containing a pair of disulfide bonds;
4) after the first cyclization is completed, removing all side chain protecting groups on the precursor peptide of the conotoxin anti-wrinkle peptide with side chain protecting groups containing a pair of disulfide bonds by using a lysis solution to obtain the conotoxin anti-wrinkle precursor peptide containing a pair of disulfide bonds, adjusting the PH of the solution to 8-9 by using dilute ammonia water after dissolution, and performing cyclization of a second pair of disulfide bonds and a third pair of disulfide bonds under natural conditions to obtain a crude conotoxin peptide;
5) and (4) performing semi-preparative chromatographic separation and freeze-drying to obtain the conotoxin product.
Preferably, the coupling system is formed by combining any one of HOBT, HOAT and Cl-HOBT with DIC, or by combining any one of HBTU, HATU, PyBOP or PyAOP with DIEA, or by combining any one of HBTU, HATU, PyBOP or PyAOP with any one of HOBT, HOAT and Cl-HOBT with DIEA.
Preferably, in the step 1), the substitution degree of the Sieber Amide Resin is 0.34-0.45 mmol/g.
Preferably, in the selection of side chain protecting groups of Cys, the 10 th and 22 th Cys side chain protecting groups can be selected to be StBu, and the rest Cys side chain protecting groups are Trt; or the protecting groups of the Cys side chains at the 4 th position and the 21 st position are StBu, and the protecting groups of the other Cys side chains are Trt; or the Cys side chain protecting groups at the 3 rd and 15 th positions are StBu, and the rest Cys side chain protecting groups are Trt.
Preferably, in step 2), when Fmoc-Gly-Pro-OH is used as a reaction raw material at the 6 th and 7 th positions and Fmoc-Ser (tBu) -OH is used as a reaction raw material at the 11 th and 12 th positions, the reaction raw material is 2 times the mole number of the Sieber Amide Resin.
Preferably, in step 3), the ratio of TFA to DCM in the TFA/DCM solution is 2% -8% by volume.
Preferably, in step 3), the ratio of TFA to DCM in the TFA/DCM solution is 3% by volume.
Preferably, in step 3), the volume ratio of mercaptoethanol to DMF in the mercaptoethanol/DMF solution is 15% -35%, and the oxidizing agent and the solvent used for the cyclization of the first pair of disulfide bonds is H2O2DCM, in which H2O2The volume ratio of the active component to DCM is 0.5% -1.5%.
Preferably, in the step 4), the volume ratio of each component of the lysate for cleaving the conus anti-wrinkle side chain protecting group precursor peptide containing a pair of disulfide bonds is TFA: ethanedithiol: phenol: triisopropylsilane: water ═ 85-90): (5-8): (3-4): (2-3): (1-3).
Preferably, in the step 4), the second and third pairs of disulfide bonds are cyclized by using an air oxidation method, and the reaction time is 48-72 h.
Compared with the prior art, the invention has the beneficial effects that:
the process of the invention uses Fmoc solid phase method, selectively oxidizes the mixture to form a pair of disulfide bonds, then forms the remaining two pairs of disulfide bonds by natural oxidation, selects dipeptide fragments to carry out coupling, has the characteristics of high yield, strong operability and the like, is suitable for large-scale production, has considerable economic and practical values, and has wide application prospect in the field of cosmetics.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows:
a preparation process of conus anti-wrinkle agent with amino acid sequence shown as SEQ ID NO.1
The method comprises the following specific steps:
1) weighing 100g of Sieber Amide Resin with the substitution degree of 0.40mmol/g into a reaction column, adding a proper amount of DCM for swelling for 15min, washing for 3 times by DMF after pumping, pumping for drying, adding a proper amount of decapping solution for reacting for 5min, pumping for drying the decapping solution, washing for one time by DMF, then adding a proper amount of decapping solution for reacting for 10min, pumping for drying the decapping solution, washing for 6 times by DMF, and pumping for drying, wherein the decapping solution is prepared by DBLK and DMF according to the volume ratio of 1: 4 is configured.
2) Weighing 34.52g of Fmoc-Cys (StBu) -OH and 25.69g of TBTU, completely dissolving in a proper amount of DMF, slowly adding 26.44ml of DIEA under an ice bath condition, slowly stirring, slowly adding a uniformly stirred mixed solution of Fmoc-Cys (StBu) -OH and TBTU/DIEA into a reaction column, introducing nitrogen at room temperature for reaction for 1H, then carrying out Kaiser detection, draining after the coupling reaction is finished, washing with DMF for 3 times, and sequentially coupling according to a conotoxin anti-wrinkle sequence until the last amino acid H-Pyr-OH is coupled; the resin was weighed 324.55g by washing 3 times with DMF, DCM and MeOH in sequence and draining.
Wherein, the Cys side chain protecting groups at the 10 th and 22 th positions are StBu, and the rest Cys side chain protecting groups are Trt; the amino acid used at the 6 th and 7 th positions was Fmoc-Gly-Pro-OH, the weight of the Fmoc-Gly-Pro-OH was 31.55g, the reaction material used at the 11 th and 12 th positions was Fmoc-Ser (tBu) -OH, and the weight was 42.14g, wherein the coupling system consisted of 41.63g of PyBop and 26.44ml of DIEA.
3) And (3) filling the conotoxin straight-chain peptide resin after coupling is finished into a reactor, and configuring a TFA/DCM volume ratio of 5: 3500ml of 95 lysate, slow stirring, reaction for 2h, suction filtration to obtain filtrate, adding DIEA to adjust the pH value to alkalinity, and then carrying out rotary evaporation and concentration on the filtrate to obtain 215.42g of taro snail anti-wrinkle linear peptide oily liquid with side chain protecting groups.
4) Preparing 400ml of mercaptoethanol/DMF solution with the volume ratio of 25% for cyclization of a first pair of disulfide bonds, judging the reaction degree through a high performance liquid chromatograph and a mass spectrometer after the reaction is carried out for 30min, adding water to separate out the conus anti-wrinkle tape side chain protecting group precursor peptide containing a pair of disulfide bonds after the reaction is finished, filtering to obtain a conus anti-wrinkle tape side chain protecting group precursor peptide solid matter containing a pair of disulfide bonds, and drying and weighing 191.89 g.
5) TFA in volume ratio: ethanedithiol: phenol: triisopropylsilane: water 87: 5: 3: 3: 2, preparing 1000ml of lysate, slowly adding the lysate into the conus anti-wrinkle element precursor peptide with side chain protecting groups containing a pair of disulfide bonds under the ice bath condition, stirring while adding, removing the ice bath after 30min, continuing to react for 1.5h, adding 8000ml of glacial ethyl ether to precipitate the conus anti-wrinkle element precursor peptide containing a pair of disulfide bonds, filtering the separated crude polypeptide, washing for 3 times by using a proper amount of glacial ethyl ether to obtain the solid conus anti-wrinkle element precursor peptide containing a pair of disulfide bonds, drying, and weighing 107.32 g.
6) Dissolving the conus anti-wrinkle peptide precursor containing a pair of disulfide bonds into 3000ml of pure water, adjusting the pH of a peptide solution to 8 by using dilute ammonia water, slowly stirring, carrying out cyclization of a second pair of disulfide bonds and a third pair of disulfide bonds under natural conditions, tracking and judging by using a high performance liquid chromatograph and a mass spectrometer every 3 hours, detecting that the reaction is finished after 63 hours, obtaining the crude conus anti-wrinkle peptide product, and adding dilute acetic acid to adjust the pH value of the aqueous solution of the crude conus anti-wrinkle peptide product to be 5-6.
7) Filtering the crude product water solution of the conus anti-wrinkle essence by using a 0.45 mu m filter membrane, and purifying the improved crude peptide by using a high performance liquid chromatography instrument: the method comprises the steps of passing through a DAC-HB50 dynamic axial compression column, enabling a mobile phase A to be a trifluoroacetic acid aqueous solution with the mass percentage concentration of 0.05%, enabling a mobile phase B to be a trifluoroacetic acid acetonitrile solution with the mass percentage concentration of 0.05%, carrying out gradient elution separation and purification, detecting a sample by an ultraviolet detector, collecting a peptide solution of a target peak in a segmented mode, carrying out high-efficiency liquid phase purification to obtain 850ml of conus anti-wrinkle element trifluoroacetic acid liquid with the purity of more than 98%, carrying out rotary evaporation concentration to obtain 300ml of liquid, balancing a chromatographic column with deionized water, loading the sample with the sample amount of 300ml of conus anti-wrinkle element trifluoroacetic acid liquid with the purity of more than 98%, eluting for 50min under a 2% acetic acid aqueous solution system, carrying out rotary concentration on the collected target product to 350ml, carrying out pre-freeze-drying and freeze-drying treatment, and finally obtaining the conus anti-wrinkle.
Example two:
a preparation process of conus anti-wrinkle agent with amino acid sequence shown as SEQ ID NO.1
The method comprises the following specific steps:
1) weighing 100g of Sieber Amide Resin with the substitution degree of 0.40mmol/g into a reaction column, adding a proper amount of DCM for swelling for 15min, washing for 3 times by DMF after pumping, pumping for drying, adding a proper amount of decapping solution for reacting for 5min, pumping for drying the decapping solution, washing for one time by DMF, then adding a proper amount of decapping solution for reacting for 10min, pumping for drying the decapping solution, washing for 6 times by DMF, and pumping for drying, wherein the decapping solution is prepared by DBLK and DMF according to the volume ratio of 1: 4 is configured.
2) Weighing 48.86g of Fmoc-Cys (Trt) -OH and 25.69g of TBTU, completely dissolving in a proper amount of DMF, slowly adding 26.44ml of DIEA under an ice bath condition, slowly stirring, slowly adding the uniformly stirred mixed solution of Fmoc-Cys (Trt) -OH and TBTU/DIEA into a reaction column, introducing nitrogen at room temperature for reaction for 1H, carrying out Kaiser detection, draining after the coupling reaction is finished, washing with DMF for 3 times in an anti-wrinkle manner, and sequentially coupling according to the sequence of conotoxin until the last amino acid H-Pyr-OH is coupled. The resin was weighed 335.27g by washing 3 times with DMF, DCM and MeOH in sequence and draining.
Wherein, the protecting groups of the Cys side chains at the 4 th position and the 21 st position are StBu, and the protecting groups of the other Cys side chains are Trt; 31.55g of Fmoc-Gly-Pro-OH was used as a reaction material at the 6 th and 7 th positions, and 42.14g of Fmoc-Ser (tBu) -OH was used as a reaction material at the 11 th and 12 th positions, wherein the coupling system consisted of 41.63g of PyBop and 26.44ml of DIEA.
3) And (3) filling the conotoxin straight-chain peptide resin after coupling is finished into a reactor, and configuring a TFA/DCM volume ratio of 5: 3500ml of 95 lysate, slow stirring, reaction for 2h, suction filtration to obtain filtrate, adding DIEA to adjust the pH value to alkalinity, and then carrying out rotary evaporation and concentration on the filtrate to obtain 209.39g of taro snail anti-wrinkle linear peptide oily liquid with side chain protecting groups.
4) Preparing 400ml of mercaptoethanol/DMF solution with the volume ratio of 25% for cyclization of a first pair of disulfide bonds, judging the reaction degree through a high performance liquid chromatograph and a mass spectrometer after the reaction is carried out for 30min, adding water to separate out the conus anti-wrinkle tape side chain protecting group precursor peptide containing a pair of disulfide bonds after the reaction is finished, filtering to obtain a conus anti-wrinkle tape side chain protecting group precursor peptide solid matter containing a pair of disulfide bonds, and drying and weighing 198.22 g.
5) TFA in volume ratio: ethanedithiol: phenol: triisopropylsilane: water 87: 5: 3: 3: 2, preparing 1000ml of lysate, slowly adding the lysate into the conus anti-wrinkle element precursor peptide with side chain protecting groups containing a pair of disulfide bonds under the ice bath condition, stirring while adding, removing the ice bath after 30min, continuing to react for 1.5h, adding 8000ml of glacial ethyl ether to precipitate the conus anti-wrinkle element precursor peptide containing a pair of disulfide bonds, filtering the separated crude polypeptide, washing for 3 times by using a proper amount of glacial ethyl ether to obtain the solid conus anti-wrinkle element precursor peptide containing a pair of disulfide bonds, drying, and weighing 113.28 g.
6) Dissolving the conus anti-wrinkle peptide precursor containing a pair of disulfide bonds into 3000ml of pure water, adjusting the pH of a peptide solution to 8 by using dilute ammonia water, slowly stirring, carrying out cyclization of a second pair of disulfide bonds and a third pair of disulfide bonds under natural conditions, tracking and judging by using a high performance liquid chromatograph and a mass spectrometer every 3 hours, detecting that the reaction is finished after 66 hours, obtaining the crude conus anti-wrinkle peptide product, and adding dilute acetic acid to adjust the pH value of the aqueous solution of the crude conus anti-wrinkle peptide product to be 5-6.
7) Filtering the aqueous solution of crude conotoxin peptides with a 0.45-micrometer filter membrane, and purifying the improved crude peptides with a high performance liquid chromatography: and (2) performing gradient elution, separation and purification by passing through a DAC-HB50 dynamic axial compression column, wherein a mobile phase A is a trifluoroacetic acid aqueous solution with the mass percentage concentration of 0.05%, and a mobile phase B is a trifluoroacetic acid acetonitrile solution with the mass percentage concentration of 0.05%, detecting a sample by adopting an ultraviolet detector, and collecting a peptide solution of a target peak in a segmented manner. 650ml of conotoxin trifluoroacetic acid liquid with the purity of more than 98 percent is obtained by high performance liquid purification, and 200ml of liquid is obtained by rotary evaporation and concentration. The chromatographic column is balanced by deionized water and then is loaded, 200ml of cone snail anti-wrinkle element trifluoroacetic acid liquid with the purity of more than 98 percent is eluted for 50min in a 2 percent acetic acid aqueous solution system, the collected target product is rotationally concentrated to 280ml, pre-freeze drying and freeze drying are carried out, and finally the weight of the cone snail anti-wrinkle element is 25.72g, and the yield is 27.06 percent.
Example three:
a preparation process of conus anti-wrinkle agent with amino acid sequence shown as SEQ ID NO.1
The method comprises the following specific steps:
1) weighing Sieber Amide Resin (100g) with the substitution degree of 0.40mmol/g into a reaction column, adding a proper amount of DCM for swelling for 15min, washing with DMF for 3 times after draining, adding a proper amount of decapping solution for reaction for 5min, draining the decapping solution, washing with DMF once, then adding a proper amount of decapping solution for reaction for 10min, draining the decapping solution, washing with DMF for 6 times, and draining, wherein the decapping solution is prepared by DBLK and DMF according to the volume ratio of 1: 4 is configured.
2) Weighing 48.86g of Fmoc-Cys (Trt) -OH and 25.69g of TBTU, completely dissolving in a proper amount of DMF, slowly adding 26.44ml of DIEA under an ice bath condition, slowly stirring, slowly adding the uniformly stirred mixed solution of Fmoc-Cys (Trt) -OH and TBTU/DIEA into a reaction column, introducing nitrogen at room temperature for reaction for 1H, carrying out Kaiser detection, draining after the coupling reaction is finished, washing with DMF for 3 times in an anti-wrinkle manner, and sequentially coupling according to the sequence of conotoxin until the last amino acid H-Pyr-OH is coupled. The resin was weighed 322.98g by washing 3 times with DMF, DCM and MeOH in sequence and draining.
Wherein, the Cys side chain protecting groups at the 3 rd and 15 th positions are StBu, and the rest Cys side chain protecting groups are Trt; 31.55g of Fmoc-Gly-Pro-OH is used as a reaction raw material at the 6 th position and the 7 th position, and 42.14g of Fmoc-Ser (tBu) -OH is used as a reaction raw material at the 11 th position and the 12 th position, wherein a coupling system consists of 41.63g of PyBop and 26.44ml of DIEA.
3) And (3) filling the conotoxin straight-chain peptide resin after coupling is finished into a reactor, and configuring a TFA/DCM volume ratio of 5: 3500ml of 95 lysate, slow stirring, reaction for 2h, suction filtration to obtain filtrate, adding DIEA to adjust the pH value to alkalinity, and then carrying out rotary evaporation and concentration on the filtrate to obtain 192.86g of taro snail anti-wrinkle linear peptide oily liquid with side chain protecting groups.
4) Preparing 400ml of mercaptoethanol/DMF solution with the volume ratio of 25% for cyclization of a first pair of disulfide bonds, judging the reaction degree through a high performance liquid chromatograph and a mass spectrometer after the reaction is carried out for 30min, adding water to separate out the conus anti-wrinkle tape side chain protecting group precursor peptide containing a pair of disulfide bonds after the reaction is finished, filtering to obtain a conus anti-wrinkle tape side chain protecting group precursor peptide solid matter containing a pair of disulfide bonds, and drying and weighing 179.92 g.
5) TFA in volume ratio: ethanedithiol: phenol: triisopropylsilane: water 87: 5: 3: 3: 2, preparing 1000ml of lysate, slowly adding the lysate into the conus anti-wrinkle element precursor peptide with side chain protecting groups containing a pair of disulfide bonds under the ice bath condition, stirring while adding, removing the ice bath after 30min, continuing to react for 1.5h, adding 8000ml of glacial ethyl ether to precipitate the conus anti-wrinkle element precursor peptide containing a pair of disulfide bonds, filtering the separated crude polypeptide, washing for 3 times by using a proper amount of glacial ethyl ether to obtain the solid conus anti-wrinkle element precursor peptide containing a pair of disulfide bonds, drying, and weighing 107.68 g.
6) Dissolving the conus anti-wrinkle peptide precursor containing a pair of disulfide bonds into 3000ml of pure water, adjusting the pH of a peptide solution to 8 by using dilute ammonia water, slowly stirring, carrying out cyclization of a second pair of disulfide bonds and a third pair of disulfide bonds under natural conditions, tracking and judging by using a high performance liquid chromatograph and a mass spectrometer every 3h, detecting that the reaction is finished after 69h, obtaining the conus anti-wrinkle peptide crude product, and adding dilute acetic acid to adjust the pH value of the conus anti-wrinkle peptide crude product aqueous solution to 5-6.
7) Filtering the crude product water solution of the conus anti-wrinkle essence by using a 0.45 mu m filter membrane, and purifying the improved crude peptide by using a high performance liquid chromatography instrument: and (2) performing gradient elution, separation and purification by passing through a DAC-HB50 dynamic axial compression column, wherein a mobile phase A is a trifluoroacetic acid aqueous solution with the mass percentage concentration of 0.05%, and a mobile phase B is a trifluoroacetic acid acetonitrile solution with the mass percentage concentration of 0.05%, detecting a sample by adopting an ultraviolet detector, and collecting a peptide solution of a target peak in a segmented manner. 700ml of conus anti-wrinkle element trifluoroacetic acid liquid with the purity of more than 98 percent is obtained by high performance liquid purification, and 280ml of liquid is obtained after rotary evaporation and concentration. The chromatographic column is balanced by deionized water and then is loaded, the loading amount is 280ml of cone snail anti-wrinkle element trifluoroacetic acid liquid with the purity of more than 98 percent, elution is carried out for 50min under a 2 percent acetic acid aqueous solution system, the collected target product is rotationally concentrated to 330ml, pre-freeze-drying and freeze-drying treatment are carried out, and finally the weight of the cone snail anti-wrinkle element is 25.55g, and the yield is 26.89 percent.
In conclusion, the invention synthesizes the conus anti-wrinkle essence by using an Fmoc solid phase method, selects dipeptide fragments as raw materials for coupling, and then positions and oxidizes a pair of disulfide bonds first and then completes the formation of the remaining two pairs of disulfide bonds by natural oxidation, thereby effectively avoiding the formation of isomers, promoting the reaction to be carried out towards a specified line, improving the production efficiency and the product yield, having strong operability, having considerable economic and practical values and being suitable for large-scale production.
The comparison table of the names of the raw materials in the present invention and the names of the raw materials is shown in table 1.
TABLE 1 comparison table of raw material name abbreviations and raw material names
The sequence structures of corresponding substances in different stages in the process of preparing the conus anti-wrinkle agent are as follows:
conus anti-wrinkle straight-chain peptide:
conus anti-wrinkle peptide precursor with side chain protecting group containing a pair of disulfide bonds:
conus anti-wrinkle precursor peptide containing a pair of disulfide bonds:
conotoxin anti-wrinkle agent:
the foregoing is merely exemplary and illustrative of the present invention and various modifications, additions and substitutions may be made by those skilled in the art to the specific embodiments described without departing from the scope of the invention as defined in the following claims.
Claims (10)
1. A preparation process of conotoxin is characterized by comprising the following steps:
1) selecting Sieber Amide Resin with substitution degree of 0.3-0.6mmol/g, and removing Fmoc after swelling;
2) coupling conus anti-wrinkle straight-chain peptide resin: sequentially coupling the C end to the N end of the amino acid sequence of the conus anti-wrinkle element shown as SEQ ID NO.1 to the last amino acid H-Pyr-OH of the N end to obtain the conus anti-wrinkle element straight-chain peptide resin; wherein, the amino acids used at the 3 rd, 4 th, 15 th and 21 st positions of the N terminal are Fmoc-Cys (Trt) -OH, the amino acids used at the 10 th and 22 th positions are Fmoc-Cys (StBu) -OH, the reaction raw materials used at the 6 th and 7 th positions are Fmoc-Gly-Pro-OH, and the reaction raw materials used at the 11 th and 12 th positions are Fmoc-Ser (tBu) -OH; the side chain protecting groups of Asn, Lys, Trp, Arg, Asp and His are respectively Trt, Boc, Pbf, OtBu and Trt;
3) reacting TFA/DCM solution with conotoxin straight chain peptide resin, filtering to obtain conotoxin straight chain peptide with side chain protecting group, and cyclizing a first pair of disulfide bonds by mercaptoethanol/DMF to obtain a precursor peptide of conotoxin anti-wrinkle peptide with side chain protecting group containing a pair of disulfide bonds;
4) after the first cyclization is completed, removing all side chain protecting groups on the conus anti-wrinkle element precursor peptide with side chain protecting groups containing a pair of disulfide bonds by using a lysis solution to obtain the conus anti-wrinkle element precursor peptide containing a pair of disulfide bonds, adjusting the pH of the solution to 8-9 by using dilute ammonia water after dissolution, and performing cyclization of a second pair of disulfide bonds and a third pair of disulfide bonds under natural conditions to obtain a crude conus anti-wrinkle element peptide;
5) and (4) performing semi-preparative chromatographic separation and freeze-drying to obtain the conus anti-wrinkle agent.
2. The preparation process of conotoxin according to claim 1, wherein the preparation process comprises the following steps: the coupling system is formed by combining DIC with any one of HOBT, HOAT and Cl-HOBT, or by combining DIEA with any one of HBTU, HATU, PyBOP or PyAOP, or by combining DIEA with any one of HOBT, HOAT and Cl-HOBT.
3. The preparation process of conotoxin according to claim 1, wherein the preparation process comprises the following steps: in the step 1), the substitution degree of the Sieber Amide Resin is 0.34-0.45 mmol/g.
4. A process for preparing a conotoxin as claimed in claim 2, wherein the process comprises the steps of: in the selection of side chain protecting groups of Cys, the 10 th and 22 th Cys side chain protecting groups can be selected as StBu, and the rest Cys side chain protecting groups are Trt; or the protecting groups of the Cys side chains at the 4 th position and the 21 st position are StBu, and the protecting groups of the other Cys side chains are Trt; or the Cys side chain protecting groups at the 3 rd and 15 th positions are StBu, and the rest Cys side chain protecting groups are Trt.
5. A process for preparing a conotoxin as claimed in claim 3, wherein the process comprises the following steps: in step 2), when Fmoc-Gly-Pro-OH was used as the reaction material at the 6 th and 7 th positions and Fmoc-Ser (tBu) -OH was used as the reaction material at the 11 th and 12 th positions, the reaction material was 2 times the molar number of the Sieber Amide Resin.
6. The preparation process of conotoxin according to claim 1, wherein the preparation process comprises the following steps: in step 3), the ratio of TFA to DCM in the TFA/DCM solution is 2% -8% by volume.
7. A process for preparing a conotoxin as claimed in claim 6, wherein the process comprises the steps of: in step 3), the ratio of TFA to DCM in TFA/DCM solution was 3% by volume.
8. The preparation process of conotoxin according to claim 1, wherein the preparation process comprises the following steps: in the step 3), the volume ratio of mercaptoethanol to DMF in the mercaptoethanol/DMF solution is 15-35%, and the oxidant and solvent used for the cyclization of the first pair of disulfide bonds is H2O2DCM, in which H2O2The volume ratio of the active component to DCM is 0.5% -1.5%.
9. The preparation process of conotoxin according to claim 1, wherein the preparation process comprises the following steps: in the step 4), the volume ratio of each component of the lysate for cracking the precursor peptide of the conus anti-wrinkle peptide with the side chain protecting group containing a pair of disulfide bonds is TFA: ethanedithiol: phenol: triisopropylsilane: water ═ 85-90): (5-8): (3-4): (2-3): (1-3).
10. The preparation process of conotoxin according to claim 1, wherein the preparation process comprises the following steps: in the step 4), the air oxidation method is used for cyclizing the second and third pairs of disulfide bonds, and the reaction time is 48-72 h.
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