CN114031680B - Sodamide sodium salt and preparation method and application thereof - Google Patents
Sodamide sodium salt and preparation method and application thereof Download PDFInfo
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- CN114031680B CN114031680B CN202111020743.1A CN202111020743A CN114031680B CN 114031680 B CN114031680 B CN 114031680B CN 202111020743 A CN202111020743 A CN 202111020743A CN 114031680 B CN114031680 B CN 114031680B
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- resin
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- sodium salt
- amino
- somalundum
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- -1 Sodamide sodium salt Chemical class 0.000 title description 4
- 159000000000 sodium salts Chemical class 0.000 claims abstract description 59
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- 238000000034 method Methods 0.000 claims description 44
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- 125000006239 protecting group Chemical group 0.000 claims description 15
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 13
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 13
- 238000000746 purification Methods 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 11
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 10
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- DLSWIYLPEUIQAV-UHFFFAOYSA-N Semaglutide Chemical group CCC(C)C(NC(=O)C(Cc1ccccc1)NC(=O)C(CCC(O)=O)NC(=O)C(CCCCNC(=O)COCCOCCNC(=O)COCCOCCNC(=O)CCC(NC(=O)CCCCCCCCCCCCCCCCC(O)=O)C(O)=O)NC(=O)C(C)NC(=O)C(C)NC(=O)C(CCC(N)=O)NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(CC(C)C)NC(=O)C(Cc1ccc(O)cc1)NC(=O)C(CO)NC(=O)C(CO)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(Cc1ccccc1)NC(=O)C(NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(C)(C)NC(=O)C(N)Cc1cnc[nH]1)C(C)O)C(C)O)C(C)C)C(=O)NC(C)C(=O)NC(Cc1c[nH]c2ccccc12)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CCCNC(N)=N)C(=O)NCC(O)=O DLSWIYLPEUIQAV-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000008484 agonism Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- ZIUSEGSNTOUIPT-UHFFFAOYSA-N ethyl 2-cyanoacetate Chemical compound CCOC(=O)CC#N ZIUSEGSNTOUIPT-UHFFFAOYSA-N 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000030136 gastric emptying Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 238000002868 homogeneous time resolved fluorescence Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229960002701 liraglutide Drugs 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 108010060325 semaglutide Proteins 0.000 description 1
- 229950011186 semaglutide Drugs 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000007921 solubility assay Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
Abstract
The application discloses a sodium salt of somalupeptide, a preparation method and application thereof. The molar ratio of sodium ions to the somalupeptide in the sodium salt of the somalupeptide is 1-7:1. The stability of the sodium salt of the somalundum is greatly improved compared with other salt forms.
Description
Technical Field
The application relates to a sodium salt of somalundum, a preparation method and application thereof, belonging to the technical field of diabetes medicines.
Background
Diabetes is a global disease, and a global population of 4.22 million diabetics is reported to be about 8.5% of the global population in 2014, with type II diabetes accounting for about 90%. The somalunin is a modifier of human glucagon-like peptide-1 (GLP-1) for treating type II diabetes, has GLP-1 receptor agonism, and is similar to GLP-1 in molecular structure, bioactivity, action target point, immunogenicity and the like.
The English name of the somalupeptide is semaglutide, and the sequence of the somalupeptide is:
His 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys 20 (PEG2-PEG2-γ-Glu-Octadecanedioic)-Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 。
the somalundum is a long-acting dosage form developed based on the basic structure of the rilalundum. Compared with liraglutide, the modified sorlutide has stronger hydrophilicity, can inhibit the hydrolysis of DPP-4 enzyme, prolong gastric emptying, reduce kidney clearance, prolong biological half-life, promote islet cell regeneration, reduce blood sugar for a long time and the like, and has wide application prospect.
The current synthesis of somalupeptide has the following problems: the individual amino acid in the Somamuno sequence is difficult to couple, the carbon end amino acid is easy to be deleted, the purity of the crude peptide is low, the number of impurities is large, and the separation and the purification are difficult. In addition, the stability of the trifluoroacetate and acetate forms of the cable marlutide reported at present is poor, and the polymer impurities are difficult to control, so that the factors are unfavorable for industrial production.
Disclosure of Invention
According to one aspect of the application, a sodium salt of the somalundum is provided, wherein the sodium salt of the somalundum contains sodium ions in a proper proportion, so that the stability of the final product is greatly improved compared with other salt forms.
The sodium salt of the somalundum, wherein the molar ratio of sodium ions to the somalundum in the sodium salt of the somalundum is 1-7:1.
According to one aspect of the present application, there is provided a method for preparing a sodium salt of somalundum, the method comprising the steps of:
and drying the mixture containing the somalupeptide and sodium ions to obtain the sodium salt of the somalupeptide.
Optionally, the somalupeptide sequence is:
His 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys 20 (PEG2-PEG2-γ-Glu-Octadecanedioic)-Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 。
optionally, the molar ratio of the sodium ions to the somalundin is 1-7:1.
Optionally, the mixture is obtained by:
(S1) obtaining crude peptide of the somalundum through a solid phase synthesis method, and purifying the crude peptide of the somalundum to obtain wet somalundum;
(S2) mixing the wet product of somalundum with a sodium salt solution to obtain the mixture.
Optionally, the sodium salt in the sodium salt solution comprises at least one of sodium carbonate, sodium bicarbonate and sodium hydroxide;
alternatively, the sodium salt solution has a concentration of 0.01 to 1mol/L in terms of sodium ions.
Optionally, the purification comprises the steps of:
and purifying the crude somatide by using a reversed-phase high performance liquid chromatography to obtain a somatide fraction, regulating the pH value of the somatide fraction to 4-6.5, and collecting precipitate to obtain the somatide wet product.
Optionally, the purity of the somalupeptide fraction reaches more than 99%, and the single impurity is less than 0.10%.
Optionally, the regulator for regulating the pH value of the Sodamlutide fraction comprises at least one of potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium carbonate and potassium bicarbonate;
the collected precipitate is: centrifuging to remove supernatant, washing precipitate with water to obtain the cable-marlutide wet product.
Optionally, the method of solid phase synthesis comprises the steps of:
r is R a -N(Gly 31 -O R b )R c Coupling with amino resin to obtain amino acid resin I, and protecting and synthesizing according to the C-terminal to N-terminal of the main chain of the Somar rupeptide on the amino acid resin IPeptide sequence, finish the coupling of amino acid of 30-20 th site; at amino acid Lys in position 20 20 The coupling of PEG2, gamma-Glu and octadecanedioic acid mono-tert-butyl ester is completed; at amino acid Lys in position 20 20 The coupling of the 19 th to 1 st amino acid is completed on the main chain amino group to obtain the full-protection cable-Mar Lu Taitai resin; the full-protection cable-mar Lu Taitai resin is cracked to obtain the cable-mar-lutide crude peptide;
wherein the R is a Is Gly 31 A protecting group for a main chain amino group selected from any one of amino protecting groups;
the R is b Is Gly 31 A protecting group for a main chain carboxyl group of (a) a carboxyl group selected from any one of the protecting groups for carboxyl groups;
the R is c To be connected to Gly 31 A linker arm on the N atom of (2), said linker arm bearing a free carboxyl group;
the R is a -N(Gly 31 -O R b )R c By R c Is coupled to the amino group of the amino resin.
Alternatively, the R is a -N(Gly 31 -O R b )R c Coupling to an amino resin comprises the steps of:
will contain amino resin, R a -N(Gly 31 -O R b )R c The raw materials of condensing agent and solvent react to obtain the amino acid resin I;
optionally, the condensing agent comprises HOBt, DIC;
the solvent includes DMF, DCM, NMP, DCE;
optionally, the amino resin, R a -N(Gly 31 -O R b )R c The ratio of the condensing agent to the solvent is 2mmol to 4-12 mmol to 20-60 ml;
optionally, the ratio of HOBt to DIC is 1-2 mmol/1 mmol.
Optionally, the amino Resin is selected from any one of AM Resin, MBHA Resin, rink Amide MBHA Resin, rink Amide AM Resin;
the amino protecting group is selected from any one of H, fmoc, boc, dde, ivDde, mtt, MMt, alloc;
the carboxyl protecting group is selected from t Bu, OAll, OBzl;
the protection synthesis strategy is selected from at least one of Fmoc strategy and Boc strategy.
Optionally, the R a -N(Gly 31 -O R b )R c Fmoc-N (Gly-O) t Bu)Hmb Butyric Acid。
Optionally, the sodium salt of somalundum is any one of the sodium salts of somalundum described above.
According to one aspect of the application, the application of the sodium salt of the somalundum, which is prepared by the preparation method of any one of the above, in preparing the diabetes medicine is provided.
As an embodiment, the present application provides a novel method for preparing and converting somalundum, which has the following advantages compared with the prior art:
according to the invention, the modified Gly is adopted to load the first amino acid, and the stable amino Resin is used as the initial Resin, so that the problems that the Wang Resin is easy to cause the impurity problem of deleting one or more amino acids at the carboxyl end and the yield is low due to unstable CTC Resin are avoided, the Resin polycondensation phenomenon is improved, the coupling efficiency of the amino acid is improved, and the purity and the yield of the crude peptide are greatly improved.
The invention directly separates out the polypeptide by adjusting the pH value to isoelectric point of the purified and collected Somar lupeptide qualified fraction, thereby removing the original acetate, completely eliminating the need of desalting by HPLC reverse phase chromatographic column as in the prior art, simplifying the operation steps and reducing the cost of labor and materials.
According to the invention, the precipitated polypeptide is redissolved by sodium salt to directly complete salt type conversion, so that the concentration of a sample can be controlled at will, the freeze-drying volume of a product is greatly reduced, the concentration of the product is improved by avoiding using HPLC column concentration or rotary evaporation concentration in the traditional process, meanwhile, the concentration process is reduced, so that the polymer impurities in the product are well controlled, the convenience of the process flow is greatly improved, and a larger space is provided for subsequent large-scale production.
According to the invention, the Somar lupeptide is converted into a sodium salt form, and the number of sodium ions combined with the product and the pH value of the product are controlled within a certain range, so that the stability of the final product is greatly improved compared with other salt forms, the content of polymer impurities is controlled below 0.15%, and the stability is obviously reduced compared with other processes.
Specifically, the scheme provided by the application comprises the following steps:
(1) The resin carrier was reacted with Fmoc-N (Gly) 31 -O t Bu) Hmb butyl Acid coupling to Fmoc-N (Gly) 31 -O t Bu) Hmb butyl-resin;
(2) Taking Fmoc-N (Gly) obtained in step (1) 31 -O t Bu) Hmb butyl-resin, sequentially coupling amino acids with N-terminal Fmoc protection to Lys according to the C-terminal to N-terminal peptide sequence of the cable marlutide backbone by solid phase Fmoc synthesis 20 Obtaining Dde-Lys 20 (Fmoc)-Glu 21 (O t Bu)-Phe 22 -Ile 23 -Ala 24 -Trp 25 (Boc)-Leu 26 -Val 27 -Arg 28 (Pbf)-Gly 29 -Arg 30 (Pbf)-N(Gly-O t Bu) Hmb butyl-resin.
(3) Removing Fmoc, and sequentially coupling side chain amino acid and octadecanedioic acid mono-tert-butyl ester to obtain the following structure:
Dde-Lys 20 (PEG2-PEG2-γ-Glu(O t Bu)-Octadecanedioic(O t Bu))-Glu 21 (O t Bu)-Phe 22 -Ile 23 -Ala 24 -Trp 25 (Boc)-Leu 26 -Val 27 -Arg 28 (Pbf)-Gly 29 -Arg 30 (Pbf)-N(Gly 31 -O t bu) Hmb butyl-resin;
(4) Removing Dde protecting group, sequentially coupling residual amino acids with protecting groups, and completing main chain synthesis to obtain full-protection cable-Mar Lu Taitai resin, wherein His 1 -Aib 2 Using Boc-His (Trt) -Aib-OH, glu 3 -Gly 4 And Glu (Glu) 15 -Gly 16 Fmoc-Glu (O) t Bu) -Gly-OH dipeptide for connection;
(5) The fully protected peptide resin is subjected to pyrolysis by a pyrolysis liquid, and then sedimentation, centrifugation and drying are carried out to obtain the crude peptide of the cable marlutide;
(6) Purifying the crude peptide of the somalunin by a reversed phase high performance liquid chromatography to obtain a qualified sample fraction (the qualified sample fraction refers to a first purification collection fraction);
(7) Purifying the qualified fraction in the step (6) by a reverse phase high performance liquid chromatography to obtain a second purified and collected fraction, wherein the purity of the sample reaches more than 99%, and the single impurity is less than 0.10%;
(8) Regulating the pH value of the second purified and collected fraction to an isoelectric point to separate out a sample, centrifuging to remove supernatant, washing lower-layer solid by using purified water, and centrifuging for 2 times to obtain a salt-free cable-malux wet product;
(9) Mixing the wet product of the somalundum and the sodium salt solution, and then directly freeze-drying to obtain a finished product of the sodium salt of the somalundum.
Further, in the step (1), the Resin is AM Resin, MBHA Resin, rink Amide MBHA Resin, rink Amide AM Resin, etc.;
further, in the step (4), the removal conditions are as follows: solvent A (2-4% hydroxylamine hydrochloride/DMF solution) to solvent B (3-8% DIPEA/DMF solution) =1:1 (v/v) for 60min, the resin was drained, the protection solution was filtered off, and the above procedure was repeated 1 time;
further, in the step (4), the resin washing conditions after Dde removal are as follows: washing with 2-5% DIPEA/DMF solution, and repeating the steps for 2 times, and then washing with DMF, and repeating the steps for 3 times;
further, in the step (4), a DEPBT/NMM coupling reagent is adopted for Boc-His (Trt) -Aib-OH coupling;
further, in the step (5), the lysate is TFA, TIS, EDT, phOH, H 2 O=90∶2.5∶2.5∶2.5∶2.5;
Further, in the step (6), the conditions for purification by reverse-phase high performance liquid chromatography are as follows:
mobile phase: phase A is TEAP (pH 6.5), and phase B is acetonitrile;
chromatographic column: c8 reverse phase chromatography column;
detection wavelength: 220nm;
gradient elution conditions: the eluent is a mixed solution of phase A and phase B, wherein the volume percentage of phase B is increased from 33% to 38% in 60min, and then the phase B is kept unchanged at 38%.
Further, in the step (7), the conditions for purification by reverse-phase high performance liquid chromatography are as follows:
mobile phase: phase A is 0.5% HAc/water and phase B is acetonitrile;
chromatographic column: c8 reverse phase chromatography column;
detection wavelength: 220nm;
gradient elution conditions: the eluent is a mixed solution of phase A and phase B, wherein the volume percentage of phase B is increased from 30% to 35% in 60min, and then the phase B is kept unchanged at 35%.
Further, in the step (8), the reagent for adjusting the pH to the isoelectric point is 0.05mol/L aqueous potassium hydroxide solution;
further, in the step (9), the wet product of the somatide is redissolved by using a sodium salt solution (such as 0.05mol/L sodium carbonate);
further, in the step (9), m (sodium ions) to m (somalupeptide) =1 to 7, i.e. the mole number of added sodium ions is between 1 and 7 times of the mole number of the soma Lu Taima.
From this, it can be seen that the synthetic scheme of the somalupeptide of the present application has the following characteristics:
the modified Gly is adopted to load the first amino acid, the stable amino Resin is used as the initial Resin, the problems that the Wang Resin is used to easily cause the impurity problem of deleting one or more amino acids at the carboxyl end and the yield is low due to the instability of CTC Resin are avoided, meanwhile, the Resin polycondensation phenomenon is improved, the coupling efficiency of the amino acid is improved, and the purity and the yield of the crude peptide are greatly improved.
The isoelectric point sedimentation somalupeptide polypeptide, the somalupeptide wet product and sodium salt solution mixing salt transferring process has the following characteristics:
1. without passing through the column, save equipment
In the traditional process, HPLC salt replacement concentration or decompression concentration is used, so that production equipment resources are occupied, and the use and control flow of equipment are increased;
the application utilizes the principle of isoelectric point sedimentation to directly precipitate polypeptide by regulating pH to isoelectric point, so that original acetate is removed, HPLC (high performance liquid chromatography) reverse-phase chromatographic column is not needed to be carried out for desalting like the prior art, operation can be completed by using simple equipment (a stirrer and a container), convenience of a process flow is greatly improved, operation steps are simplified, and labor and material cost are reduced.
2. The process is easy to control and simple to operate
The traditional process uses HPLC to change salt for concentration, and has a certain limit on the loading amount. Excessive loading can cause insufficient elution, a large amount of mobile phase is needed to be used for elution, the production cost and the process control difficulty are increased, and the output of waste liquid is increased; too little loading can reduce batch production, increase equipment frequency and control flow, and is inconvenient for production operation. The use of reduced pressure concentration increases the risk of product stability and is not convenient for large-scale industrial production.
The method utilizes the principle of isoelectric point sedimentation to precipitate the polypeptide, and then uses sodium salt to redissolve to directly complete the conversion of salt types, thereby controlling the concentration of the sample at will, greatly reducing the freeze-drying volume of the product, avoiding the concentration of the product by using HPLC column concentration or rotary evaporation concentration in the traditional process, simultaneously greatly controlling the polymer impurities in the product due to the reduction of concentration procedures, greatly improving the convenience of the process flow, and being easy to control, simple to operate and convenient for large-scale industrial production.
3. High product stability and low content of polymer impurities
According to the method, the somalupeptide is converted into a sodium salt form, and the number of sodium ions combined with the product is controlled within a certain range, so that the stability of the final product is greatly improved compared with other salt forms, the content of polymer impurities is controlled below 0.15%, and the stability is obviously reduced compared with other processes.
In this application, the English/abbreviations are illustrated below:
Fmoc-N(Gly-O t bu) Hmb butyl Acid:4- (2- (((9-fluorenylmethoxycarbonyl) (2- (tert-butoxy) -2-oxoethyl) amino) methyl) -5-methoxyphenoxy) butanoic acid;
AM Resin: aminomethyl polystyrene cross-linked resin;
MBHA Resin: polymer-bonded 4-methylbenzylamine hydrochloride;
rink Amide MBHA Resin:4- (2 ',4' -dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl) -phenoxyacetamido-methyl benzhydryl amine resin;
rink Amide AM Resin:4- (2 ',4' -dimethoxyphenyl-FMOC-aminomethyl) phenoxyacetamido aminomethyl resin;
wang Resin: polymer-bonded poly benzyloxy benzyl alcohol;
CTC Resin: 2-chlorotrityl polystyrene cross-linked resin;
his: histidine;
aib: 2-methylalanine;
glu: glutamic acid;
gly: glycine;
thr: threonine;
phe: phenylalanine;
ser: serine;
asp: aspartic acid;
val: valine;
tyr: tyrosine;
leu: leucine;
gln: glutamine;
ala: alanine;
lys: lysine;
PEG2:2- (2- (2-aminoethoxy) ethoxy) acetic acid;
octadecanadioic: octadecanedioic acid;
lie: isoleucine;
trp: tryptophan;
arg: arginine;
fmoc: 9-fluorenylmethoxycarbonyl;
t bu: a tertiary butyl group;
DIPEA: n, N-diisopropylethylamine;
DMF: n, N-dimethylformamide;
HOBt: 1-hydroxybenzotriazole;
DIC: n, N' -diisopropylcarbodiimide;
DCM: dichloromethane;
oxyma: 2-oxime cyanoacetic acid ethyl ester;
DEPBT:3- (diethoxyphthalyloxy) -1,2, 3-benzotriazin-4-one;
pbf:2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl;
boc: t-butoxycarbonyl;
dde:1, 1-dichloro-2, 2-bis (4-chlorophenyl) ethylene;
trt: a trityl group;
TFA: trifluoroacetic acid;
TIS: triisopropylsilane;
EDT:1, 2-ethanedithiol;
HPLC: high performance liquid chromatography;
IBMX: 3-isobutyl-1-methyl-xanthine.
The beneficial effects that this application can produce include:
(1) The sodium salt of the somalundum provided by the application has reasonable sodium ion content, so that the stability of the final product is greatly improved compared with other salt forms.
(2) According to the preparation method of the sodium salt of the somalundum, provided by the application, the number of sodium ions combined by the product and/or the pH value of the product are controlled within a certain range, so that the stability of the final product is greatly improved compared with other salt forms.
(3) According to the preparation method of the sodium salt of the somalundum, provided by the application, the pH value is regulated to a specific range, the polypeptide is precipitated and separated out, and then the sodium salt solution is used for redissolving to directly complete salt type conversion, so that the concentration of a sample can be controlled at will, the freeze-drying volume of a product can be greatly reduced, the concentration of the product is improved by using an HPLC column for concentration or rotary evaporation concentration in the traditional process is avoided, meanwhile, the concentration procedure is reduced, so that the polymer impurities in the product are well controlled, the convenience of the process flow is greatly improved, the process is easy to control, the operation is simple, and the large-scale industrial production is convenient.
In addition, salt impurities introduced in the preparation and purification processes of the polypeptide can be effectively removed by a method of adjusting the pH value to precipitate the polypeptide, and the concentration of a final product is also facilitated.
(4) According to the preparation method of the sodium salt of the soruloside, the modified Gly is adopted to load the first amino acid, and the stable amino Resin is used as the initial Resin, so that the problems that the Wang Resin is easy to cause the impurity problem of missing one or more amino acids at the carboxyl end and the yield is low due to unstable CTC Resin are avoided, the Resin polycondensation phenomenon is improved, the coupling efficiency of the amino acid is improved, the purity and the yield of crude peptide are greatly improved, and the purity of the final sodium salt of the soruloside is improved.
Drawings
FIG. 1 shows the HPLC detection result of the sodium salt of the cable-to-roup peptide prepared in example 1.
Detailed Description
The present application is described in detail below with reference to examples, but the present application is not limited to these examples.
Unless otherwise indicated, all starting materials in the examples of the present application were purchased commercially.
Example 1
1.1 Somar Lupeptide peptide resin Synthesis
AM Resin (3.8 g,2mmol, degree of substitution 0.53 mmol/g) was weighed into the reactor, swollen with 200ml of 5% (v/v) DIPEA in DMF and removed by suction filtration. Washing with DMF 50ml for 4 times, draining the solvent, and qualitatively detecting the free amino group by using ninhydrin as the amino group.
Fmoc-N (Gly-O) was additionally weighed t Bu) Hmb butyl Acid (3.45 g,6 mmol) and HOBt (0.81 g,6 mmol) were added to a beaker, dissolved in DMF 20ml, controlled at 10℃and pre-activated by adding DIC (0.94 ml,6 mmol)The reaction was carried out for 10min. Adding the preactivated solution into a reactor, stirring and reacting for 2 hours, and using ninhydrin as amino qualitative detection to track the reaction end point, wherein the reaction solution is colorless and transparent, namely the detection is negative, so that the reaction is complete. The liquid was removed by suction filtration, and the resin was washed 4 times with DMF to complete Fmoc-N (Gly) 31 -O t Bu) coupling of Hmb butyl Acid.
80ml of a proper amount of deprotection solution (20% (v/v) piperidine in DMF) is added, the reaction is carried out for 20min at 20 ℃, fmoc of the deprotection group is removed, the solution is pumped out, the deprotected amino acid resin is washed 6 times with a proper amount of DMF, and the sample is detected by tetrachloroquinone to be positive for standby.
Fmoc-Arg was additionally weighed 30 (Pbf) -OH (3.89 g,6 mmol) and Oxyma (0.85 g,6 mmol) were added to a beaker, dissolved in 20ml DMF, the temperature was controlled at 9℃and DIC (0.94 ml,6 mmol) was added to pre-activate for 8min. Adding the preactivated solution into a reactor, stirring and reacting for 2 hours, and using tetrachlorobenzoquinone as amino qualitative detection to track the reaction end point, wherein the reaction solution is colorless and transparent, namely the detection is negative, so that the reaction is complete. Removing liquid by suction filtration, washing resin with DMF for 4 times to complete Arg 30 Is a coupling of (a) and (b).
Repeating the operations of Fmoc removal, preactivation, coupling, DMF washing and the like, and sequentially coupling Fmoc-Gly 29 -OH、Fmoc-Arg 28 (Pbf)-OH、Fmoc-Val 27 -OH、Fmoc-Leu 26 -OH、Fmoc-Trp 25 (Boc)-OH、Fmoc-Ala 24 -OH、Fmoc-Ile 23 -OH、Fmoc-Phe 22 -OH、Fmoc-Glu 21 ( t Bu)-OH、Dde-Lys 20 (Fmoc)-OH、Fmoc-PEG2-OH、Fmoc-PEG2-OH、Fmoc-Glu-O t Bu, octadecanedioic acid mono-tert-butyl ester to obtain peptide resin Dde-Lys 20 (PEG2-PEG2-γ-Glu(O t Bu)-Octadecanedioic(O t Bu))-Glu 21 (O t Bu)-Phe 22 -Ile 23 -Ala 24 -Trp 25 (Boc)-Leu 26 -Val 27 -Arg 28 (Pbf)-Gly 29 -Arg 30 (Pbf)-N(Gly-O t Bu)Hmb Butyriyl-AM Resin。
The peptide resin is added with 40ml of DMF solution containing 1mol/L hydroxylamine hydrochloride and 40ml of DMF solution containing 1mol/L DIPEA, the protective group Dde is removed after 60min reaction at 20 ℃, the solution is drained, the peptide resin is washed 2 times with proper amount of DMF, 2% (v/v) DIPEA/DMF is washed 2 times, DMF is washed 4 times, and ninhydrin is used for detection to show positive standby.
Fmoc-Ala was additionally weighed 19 OH (1.87 g,6 mmol) and HOBt (0.81 g,6 mmol) were added to a beaker, dissolved in 20ml DMF, and the temperature was controlled at 10℃and DIC (0.94 ml,6 mmol) was added to pre-activate for 10min. The preactivated solution is added into a reactor to be stirred for reaction for 1.5 hours, and the reaction end point is detected and tracked by ninhydrin. Removing liquid by suction filtration, washing resin with DMF for 4 times to finish Ala 19 Is a coupling of (a) and (b).
Repeating Fmoc removal, preactivation, coupling and DMF washing operation, and sequentially coupling Fmoc-Ala 18 -OH、Fmoc-Gln 17 (Trt)-OH、Fmoc-Glu 15 ( t Bu)-Gly 16 -OH、Fmoc-Leu 14 -OH、Fmoc-Tyr 13 ( t Bu)-OH、Fmoc-Ser 12 ( t Bu)-OH、Fmoc-Ser 11 ( t Bu)-OH、Fmoc-Val 10 -OH、Fmoc-Asp 9 ( t Bu)-OH、Fmoc-Ser 8 ( t Bu)-OH、Fmoc-Thr 7 ( t Bu)-OH、Fmoc-Thr 5 ( t Bu)-Phe 6 -OH、Fmoc-Glu 3 ( t Bu)-Gly 4 -OH、Boc-His 1 (Trt)-Aib 2 -OH. The resulting product was washed 2 times with DCM, 2 times with methyl tert-butyl ether, dried under suction to give a fully protected cable Lu Taitai resin (2 mmol,16.8 g).
1.2 cleavage of fully protected Sodamlutide peptide resin
The full protective cable ma Lu Taitai resin (16.8 g,2 mmol) was weighed and added with the lysate (TFA: TIS: EDT: H 2 O volume ratio=90:2.5:2.5:2.5, 100 ml), reaction temperature 25℃was controlled, and the reaction was stirred for 3 hours. The mixture was filtered and the cake resin was washed with 20ml TFA. Mixing the filtrates, adding methyl tert-butyl ether (800 ml) at 10deg.C, stirring, settling, centrifuging, adding methyl tert-butyl ether (500 ml) into the filter cake, pulping, and centrifuging; the cake was repeatedly beaten and centrifuged with the same volume of methyl t-butyl ether. The filter cake was dried in vacuo to give crude somalundin (8.42 g, hplc purity 74.1%).
1.3 purification and lyophilization of crude peptide
1.3.1 dissolution of crude product
8.42g of crude somalundin was dissolved with 4g/L ammonium acetate and filtered through a 0.45um membrane.
1.3.2 purification
The reverse-phase high-efficiency preparation column is adopted for crude purification, the packing is Kromasil C8, the mobile phase A of the first purification is 1% triethylamine phosphate aqueous solution (pH 6.5), the mobile phase B is pure acetonitrile, and the purity of the sample reaches about 95%. The mobile phase A of the second purification is 0.5% acetic acid/water, the mobile phase B is pure acetonitrile, and the purity of the sample reaches more than 99.5%.
1.3.3 precipitation
Adding the secondarily purified Somalus peptide fraction into a stainless steel precipitation tank, cooling to 7 ℃, mechanically stirring for 150 revolutions per minute, adding 0.05mol/L potassium hydroxide into the precipitation tank at a flow rate of 400ml/min, regulating the pH of the solution to an isoelectric point, precipitating a large amount of white precipitate, centrifuging, and washing the precipitate with purified water for 3 times to obtain a Somalus peptide wet product.
1.3.4 salt-changing lyophilization
Mixing the wet product of the somalundum and 0.05mol/L sodium carbonate aqueous solution, adding sodium ions with the number of 6.5 times of that of the somama Lu Taima, and freeze-drying to obtain 2.56g of finished product of the sodium somalundum salt, wherein the total yield reaches about 32%.
Purity detection of sodium salt of somalu peptide
Through HPLC detection, the purity of the sodium salt of the somalundum reaches 99.69 percent, and the single impurity is less than 0.10 percent. The results are shown in Table 1 and FIG. 1.
Table 1 HPLC detection of sodium salt of somalundin
Solubility detection of sodium salt of somalupeptide
The solubility of 3 batches of sodium salt of somalupeptide prepared according to the method of example 1 was tested, the test method was referred to the pharmacopoeia of 2020 edition, and the results are shown in table 2.
Table 2 solubility assay of sodium salt of somalundin
The results show that the obtained sodium salt of the somalundum has very good solubility in water, ethanol and methanol and good batch-to-batch stability.
Polymer impurity content detection of sodium salt of somalundum
For 3 batches of finished product of the sodium salt of the somalundin prepared according to the method of example 1, the content of the polymer impurities is detected by adopting a universal SEC chromatographic column, and meanwhile, the commercial injection of the somalundin Ma Lutai is purchased for quality comparison, and the result is shown in Table 3. Wherein, the reference substance-1 is commercial Soxhlet Ma Lutai injection (batch number: JP 50872), and the reference substance-2 is commercial Soxhlet Ma Lutai injection (batch number: JP 54343), both manufactured by Noand Nord company.
TABLE 3 detection of Polymer impurity content
Name of the name | Polymer content/% |
Homemade first batch | 0.11 |
Homemade second batch | 0.11 |
Self-made third batch | 0.10 |
Reference substance-1 | 0.29 |
Reference substance-2 | 0.58 |
The results show that the process of the invention is stable, the content of the polymer impurity in the sodium salt of the somalundum is lower than that of the commercial injection of the Ma Lutai, the difference between batches is smaller, and the process is stable.
Stability detection of sodium salt of somalu peptide
The finished product of the sodium salt of the somalundum was left at 2-8deg.C for 60 days, and the purity and the maximum single impurity content of the sample were measured on day 0, day 30 and day 60, respectively, and the results are shown in Table 4. Wherein, the acetate reference substance and the hydrochloride reference substance are self-made samples of the same process, namely, in the step 1.3.4 of the example 1, 0.05mol/L sodium carbonate aqueous solution is respectively replaced by 0.3mol/L acetic acid and 0.05mol/L hydrochloric acid, and the rest steps are the same as the example 1.
Table 4 stability assay of sodium salt of somalundum
The results show that the purity of the finished product of the sodium salt of the somalundum is higher, the purity of the product and the content of the maximum single impurity are basically stable and unchanged after the product is placed for 60 days, the purity of an acetate/hydrochloride reference substance is gradually reduced along with the extension of time, and the content of the maximum single impurity is started to be higher, so that the stability of the finished product of the sodium salt of the somalundum is better than that of other salt types.
Biological Activity test
3 batches of finished sodium salt of Soxhlet Ma Lutai prepared in accordance with the method of example 1 and a control (Soxhlet Ma Lutai injection, norand Norde, batch number: JP 50872) were subjected to in vitro biological activity detection by cAMP-HTRF Assay (detection by GLP1R/CRE-Luc/HEK293 cells using CAMP-GS DYNAMIC KIT kit from Cisbio Co., according to the instructions) and the results are shown in FIG. 5.
Table 5 in vitro bioactivity assay of sodium salt of Ma Lutai
The results show that the in vitro activity data of the finished sodium salt of the cable Ma Lutai has better performance compared with the control, and the sodium salt of the cable Ma Lutai has better anti-diabetic activity.
The foregoing description is only a few examples of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred examples, it is not intended to limit the present application, and any person skilled in the art may make some changes or modifications to the disclosed technology without departing from the scope of the technical solution of the present application, and the technical solution is equivalent to the equivalent embodiments.
Claims (11)
1. A method for preparing a sodium salt of somalundum, comprising the steps of:
drying a mixture containing the somalundum and sodium ions to obtain sodium salt of the somalundum;
the molar ratio of sodium ions to the somalupeptide in the sodium salt of the somalupeptide is 1-7:1;
the mixture is obtained by the following steps:
(S1) obtaining crude peptide of the somalundum through a solid phase synthesis method, and purifying the crude peptide of the somalundum to obtain wet somalundum;
(S2) mixing the wet product of somalundum with a sodium salt solution to obtain the mixture;
the concentration of the sodium salt solution is 0.01-1 mol/L based on sodium ions;
the purification comprises the following steps:
purifying the crude somatide with reversed-phase high performance liquid chromatography to obtain a somatide fraction, regulating the pH value of the somatide fraction to 4-6.5, and collecting precipitate to obtain the wet somatide;
the method for solid phase synthesis comprises the following steps:
r is R a -N(Gly 31 -O R b )R c Coupling to amino resin to obtain amino acid resin I, and adopting a protection synthesis strategy to finish coupling of amino acids at the 30 th to 20 th positions on the amino acid resin I according to the peptide sequence from the C end to the N end of the main chain of the Somar Lupeptide; at amino acid Lys in position 20 20 The coupling of PEG2, gamma-Glu and octadecanedioic acid mono-tert-butyl ester is completed; at amino acid Lys in position 20 20 The coupling of the 19 th to 1 st amino acid is completed on the main chain amino group to obtain the full-protection cable-Mar Lu Taitai resin; the full-protection cable-mar Lu Taitai resin is cracked to obtain the cable-mar-lutide crude peptide;
wherein the R is a Is Gly 31 A protecting group for a main chain amino group selected from any one of amino protecting groups;
the R is b Is Gly 31 A protecting group for a main chain carboxyl group of (a) a carboxyl group selected from any one of the protecting groups for carboxyl groups;
the R is c To be connected to Gly 31 A linker arm on the N atom of (2), said linker arm bearing a free carboxyl group;
the R is a -N(Gly 31 -O R b )R c By R c Is coupled to the amino group of the amino resin.
2. The method according to claim 1, wherein the sodium salt in the sodium salt solution comprises at least one of sodium carbonate, sodium bicarbonate, and sodium hydroxide.
3. The process according to claim 1, wherein the fraction of somalupeptide has a purity of 99% or more and a single impurity of less than 0.10%.
4. The process according to claim 1, wherein the regulator for regulating the pH of the somalundum fraction comprises at least one of potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate;
the collected precipitate is: centrifuging to remove supernatant, washing precipitate with water to obtain the cable-marlutide wet product.
5. The method of claim 1, wherein R is a -N(Gly 31 -OR b )R c Coupling to an amino resin comprises the steps of:
will contain amino resin, R a -N(Gly 31 -O R b )R c And (3) reacting the raw materials of the condensing agent and the solvent to obtain the amino acid resin I.
6. The method of claim 5, wherein the condensing agent comprises HOBt and DIC;
the solvent is DMF, DCM, NMP or DCE.
7. The method according to claim 5, wherein the amino resin, R a -N(Gly 31 -O R b )R c The ratio of the condensing agent to the solvent is 2 mmol/4-12 mmol/20-60 ml.
8. The process according to claim 6, wherein the ratio of HOBt to DIC is 1-2 mmol/1 mmol.
9. The method of claim 1, wherein the amino Resin is selected from any one of AM Resin, MBHA Resin, rink Amide MBHA Resin, rink Amide AM Resin;
the amino protecting group is selected from any one of H, fmoc, boc, dde, ivDde, mtt, MMt, alloc;
the carboxyl protecting group is selected from t Bu, OAll, OBzl;
the protection synthesis strategy is selected from at least one of Fmoc strategy and Boc strategy.
10. The method of claim 1, wherein R is a -N(Gly 31 -OR b )R c Fmoc-N (Gly-O) t Bu)Hmb Butyric Acid。
11. The use of the sodium salt of somalupeptide prepared by the preparation method according to any one of claims 1 to 10 in the preparation of a medicament for diabetes.
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