CN111944037B - Synthetic method of somalupeptide - Google Patents

Synthetic method of somalupeptide Download PDF

Info

Publication number
CN111944037B
CN111944037B CN201910358894.4A CN201910358894A CN111944037B CN 111944037 B CN111944037 B CN 111944037B CN 201910358894 A CN201910358894 A CN 201910358894A CN 111944037 B CN111944037 B CN 111944037B
Authority
CN
China
Prior art keywords
fmoc
otbu
glu
fragment
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910358894.4A
Other languages
Chinese (zh)
Other versions
CN111944037A (en
Inventor
李新宇
姚林
朱亮平
付玉清
邱心敏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SHENZHEN JYMED TECHNOLOGY CO LTD
Original Assignee
SHENZHEN JYMED TECHNOLOGY CO LTD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SHENZHEN JYMED TECHNOLOGY CO LTD filed Critical SHENZHEN JYMED TECHNOLOGY CO LTD
Priority to CN201910358894.4A priority Critical patent/CN111944037B/en
Publication of CN111944037A publication Critical patent/CN111944037A/en
Application granted granted Critical
Publication of CN111944037B publication Critical patent/CN111944037B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

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
    • 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)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a synthesis method of a somalupeptide, which comprises the following steps: the S1-S2 fragment and the S3-S6 fragment which are fully protected by the somalupeptide are respectively synthesized and used for synthesizing the somalupeptide resin, and the somalupeptide is obtained through cracking and purifying. When Fmoc-Lys (Dde) -OH is selected, the cost for synthesizing the crude product of the somalunin is greatly reduced. The S19-S20 polypeptide fragment or the S18-S20 polypeptide fragment is selected to be connected into the main chain, so that the coupling of the main chain of the cable-mar-lutide is facilitated, the purity and the yield of the crude cable-mar-lutide are greatly improved, and the synthesis cost is reduced. The strategy of coupling dipeptide and tetrapeptide fragments greatly reduces the generation of D-His racemization impurity, +Gly impurity, D-Thr impurity and D-Phe impurity, greatly reduces the difficulty of product purification, improves the yield and has wide application prospect.

Description

Synthetic method of somalupeptide
Technical Field
The invention relates to the field of solid-phase synthesis of polypeptides, in particular to a solid-phase synthesis method of somalupeptide.
Technical Field
Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by human intestinal L cells, and has effects of promoting insulin secretion, inhibiting glucagon secretion, and lowering blood sugar concentration, and can be used for treating type II diabetes. However, natural GLP-1 is unstable in vivo and is susceptible to rapid degradation by dipeptidyl peptidase-IV (DPP-IV).
Somamunotide, named Semaglutide, is a novel long-acting glucagon-like peptide-1 (GLP-1) analog developed and produced by Daneno and Norde corporation for the treatment of type II diabetes. The somalupeptide has the effects of reducing blood sugar, losing weight and protecting cardiovascular system, and is approved by FDA in 12 months of 2017. After the Lys side chain of the somalundum is modified by PEG, glu and octadecadicarboxylic acid, the hydrophilicity is greatly improved, and the binding force with albumin is enhanced; meanwhile, after Ala at the 2 nd position of the N end is mutated into Aib, the inactivation caused by DPP-IV enzymolysis is effectively avoided, the half life reaches 40h, patients only need to inject once a week, and the oral dosage form of the medicine is also under development at present. The CAS number of the somalundum is 910463-68-2, the molecular formula is C187H291N45O59, the molecular weight is 4113.64g/mol, and the peptide sequence is:
H- 1 His- 2 Aib- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser- 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln - 18 Ala- 19 Ala- 20 Lys(Octadecanedioic-γ-Glu-PEG-PEG)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 Gly-OH。
the methods for preparing somalundin reported so far are roughly divided into two categories: one is to directly access Lys containing a side chain as a fragment into a main chain of the somalundum to complete synthesis, and patent CN104356224A discloses a method for preparing the somalundum by grafting a side chain on epsilon-N H2 of Lys by a liquid phase method and then gradually condensing amino acid on resin. The other is to finish the coupling of the main chain and the side chain of the cable-marlutide one by one, and patent CN 201511027176 discloses that the cable-marlutide main chain peptide is synthesized gradually in a solid phase, a side chain modification group is synthesized, a protecting group of Lys is removed, the side chain modification group is coupled, and finally the polypeptide product is obtained by cleavage. Because the sequence of the somalunin is longer and has more hydrophobic amino acids, when the somalunin is synthesized by adopting an amino acid gradual condensation method, the somalunin is easy to form folding, the shrinkage of resin is serious, the reaction time is prolonged, and more impurities which are very similar to the properties of products, such as racemized impurities of D-His, are generated in the crude peptide:
H-D-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Oct adecanedioic-γ-Glu-PEG-PEG)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH；
+gly impurity:
H-His-Aib-Glu-Gly-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(O ctadecanedioic-γ-Glu-PEG-PEG)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH；
racemic impurity of D-Thr:
H-His-Aib-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Oct adecanedioic-γ-Glu-PEG-PEG)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH；
racemic impurity of D-Phe:
H-His-Aib-Glu-Gly-Thr-D-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (Oct adecaneioic-gamma-Glu-PEG-PEG) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; D-His racemization impurities are similar to physical and chemical properties of the somalundum, so that the separation and purification difficulty of the somalundum product is greatly increased, and the product yield is greatly reduced. Therefore, there is an urgent need for a synthetic method of somalundin with high purity and yield and low cost.
Disclosure of Invention
The invention provides a synthesis method of the simaroubm peptide with high yield and high purity, which aims to solve the problems of more impurities, low purity and yield, high cost, complex operation steps and adverse industrial production in the existing synthesis process. The strategy of dipeptide and dipeptide fragment is adopted to reduce the generation of D-His racemization impurity and +Gly impurity, thus having considerable economic application value and wide application prospect.
In order to achieve the aim of the invention, the invention provides a synthesis method of the somalundum, which is used for respectively synthesizing a somalundum full-protection S1-S2 fragment and a somalundum full-protection S3-S4 fragment, and is applied to the synthesis of a somalundum full-protection peptide resin, and the somalundum is obtained through cracking and purification.
It should be noted that in the present invention, S1 is the first amino acid at the N-terminal in the somalupeptide peptide sequence, and similarly S2, S3, S4, S5, S6, S18, S19, S20 are the 2 nd, 3 rd, 4 th, 5 th, 6 th, 18 th, 19 th, 20 th amino acids at the N-terminal, respectively, and so on.
Preferably, the fully protected S1-S2 fragment is R1-His (R2) -Aib-OH and the fully protected S3-S4 fragment is R3-Glu (R4) -Gly-OH. Wherein R1 is selected from: boc or Fmoc; r2 is selected from: trt or Boc; r3 is selected from Fmoc; r4 is selected from OtBu.
As a more preferred embodiment, the full protection S1-S2 fragment is Boc-His (Trt) -Aib-OH and the full protection S3-S4 fragment is Fmoc-Glu (OtBu) -Gly-OH.
The dipeptide fragment Boc-His (Trt) -Aib-OH of the full protection S1-S2 can effectively reduce the generation of D-His racemization impurities; the generation of +Gly impurities can be effectively reduced by adopting the dipeptide fragment Fmoc-Glu (OtBu) -Gly-OH of full protection S3-S4;
preferably, the synthesis of the somalupeptide adopts the full-protection S5-S6 fragment as a raw material. More preferably, the fully protected S5-S6 fragment is Fmoc-Thr (tBu) -Phe-OH.
The adoption of The dipeptide fragment Fmoc-Thr (tBu) -Phe-OH of full protection S5-S6 can effectively reduce The generation of D-The impurity and D-Phe impurity.
Preferably, fmoc-Lys (Dde) -OH is used as a raw material for the synthesis of the somalupeptide. Wherein, the method for removing Fmoc-Lys (Dde) -OH side chain Dde protecting group adopts DMF solution and NH mixed by hydroxylamine hydrochloride and imidazole 2 NH 2 DMF solution of (b).
Preferably, a DMF solution of hydroxylamine hydrochloride and imidazole is used, the mass ratio of hydroxylamine hydrochloride to imidazole is 1-3:1-3, more preferably the ratio is 2:1.
Hydrazine hydrate has potential genotoxicity, and the use of hydroxylamine hydrochloride and imidazole has no potential genotoxicity risk should be avoided in industry as much as possible.
As a preferred scheme, S19-S20 fragment Fmoc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH is used as a raw material for the synthesis of the somalupeptide.
The scheme can effectively reduce the generation of impurities in the +Ala process and can improve the total synthesis yield.
Preferably, the S18-S20 peptide fragment Fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH is used as a raw material for the synthesis of the somalupeptide.
The scheme can effectively reduce the generation of impurities in the +Ala process and can improve the total synthesis yield.
Preferably, the synthesis method of the somalundin comprises the following steps: synthesizing Fmoc-Gly-resin, synthesizing the Fmoc-Gly-resin according to the peptide sequence from the C end to the N end of the main chain of the sorulon by a solid phase synthesis method, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys (Dde) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Phe-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (OtBu) -OH, fmoc-Glu (tBu) -OH, fmoc-Glu (OtBu) -OH, fmoc-Glu (tBu) -Glu (OtBu) -OH, obtaining a cable-marlutide main chain peptide resin; removing a side chain Dde protecting group of Lys20, and sequentially coupling side chain amino acid sequences Fmoc-AEEA-OH, fmoc-Glu (OH) -OtBu and octadecanedioic acid mono-tert-butyl ester to obtain the cable marlutide full-protection peptide resin; and obtaining the somalupeptide through cracking and purifying.
As another preferable scheme, the synthesis method of the somalundin comprises the following steps: synthesizing Fmoc-Gly-resin, according to the peptide sequence from C end to N end of the cable MALUO peptide chain through a solid phase synthesis method, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Lys (AEEa-eea-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl-er) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Phe-OH, fmoc-Ser (tBu) -OH, fmoc-Glu-OH, fmoc-Asp (tBu) -OH, fmoc-Glu (Boc) -OH, obtaining the total protective peptide resin of the somalupeptide, and obtaining the somalupeptide through cracking and purifying.
As a third preferred scheme, the synthesis method of the somalundin comprises the following steps: fmoc-Gly-resin is synthesized, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl-estrer) -OH, fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu-OH), fmoc-OH, fmoc-Phe-OH, fmoc-Tyr (tBu) -OH, fmoc-Bou-OH, fmoc-Boc-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Ala-Ala-Lys (AEE-A-gamma-Glu (OtButBu) -OH) is sequentially coupled by solid phase synthesis from the C-end to N-end peptide chain to the end peptide, and the peptide is purified.
Preferably, the carrier resin used for the synthesis of the somalundin is selected from the group consisting of 2-CTC resin, wang-resin.
The 2-CTC resin and the Wang-resin are relatively stable in the synthesis process, and side reactions are not easy to occur. The price is relatively low, the method has advantages in control of synthesis cost, and is suitable for industrialized mass production.
Preferably, the lysate of the total protective peptide resin for splitting the somalundum is a mixed solution of TFA, EDT, DMS, phenylsulfide and TIS.
More preferably, the volume ratio of TFA, EDT, DMS, phenyl sulfide and TIS is 80-90:1-5:1-5:2-6:1-5.
More preferably the volume ratio of TFA, EDT, DMS, phenylsulfide, TIS is 90:2:2:2:2:2.
The invention adopts a segment-by-segment condensation method, greatly simplifies the process steps, has short synthesis period and low cost, and is very beneficial to industrialized mass production. When Fmoc-Lys (Dde) -OH is selected, the cost for synthesizing the crude product of the somalunin is greatly reduced. Fmoc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH polypeptide fragments or Fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH polypeptide fragments are selected to access the main chain, so that the coupling of the main chain of the cable marlutide is facilitated, the purity and yield of a crude product of the cable marlutide are greatly improved, and the synthesis cost is reduced. The purity of the crude product reaches 70 percent. The strategy of coupling dipeptide and dipeptide fragments is adopted, so that the generation of D-His racemization impurities and +Gly impurities is greatly reduced, the difficulty of product purification is greatly reduced, the yield is improved, and the method has wide application prospect.
Drawings
FIG. 1 is a HPLC chromatogram of crude somalupeptide prepared in example 19
FIG. 2 is a HPLC chromatogram of purified somalupeptide of example 21
Detailed Description
The foregoing of the invention will be described in further detail with reference to specific embodiments. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques realized based on the above description of the present invention are within the scope of the present invention.
The meanings of the abbreviations used in the present invention are listed in the following table:
Fmoc fluorene methoxycarbonyl group
Fmoc-AA Fluorene methoxycarbonyl protected amino acids
TBTU 2- (1H-Benzotrisazo L-1-yl) -1, 3-tetramethylurea tetrafluoroborate
HOBT 1-hydroxybenzotriazoles
DIEA N, N-diisopropylethylamine
DIC N, N-diisopropylcarbodiimide
DCC N, N-dicyclohexylcarbodiimide
tBu Tert-butyl group
Boc Boc-group
HOSU N-hydroxysuccinimide
DMF N, N-dimethylformamide
TFE Trifluoroethanol
DCM Dichloromethane (dichloromethane)
EXAMPLE 1 preparation of the fully protected S1-S2 fragment dipeptide Boc-His (Trt) -Aib-OH
Preparation of Boc-His (Trt) -OH active ester
A100 ml single vial was placed in a cryogenically stirred reactor, 2.50g Boc-His (Trt) -OH and 25ml DCM solvent were added, and 0.69g HOSU was added. After clarification by stirring at 0 ℃, 1.34g dcc solution in 10ml dcm was added dropwise. After the completion of the dropwise addition for 10min, the temperature is raised to 25 ℃ for reaction for 3h. The reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1, 2 additional drops of acetic acid were added). After completion of the reaction, suction filtration, washing with 5ml of DCM was added, the filtrates were combined and the solvent was removed by rotary evaporation to give 2.63g of a viscous mass.
Preparation of Boc-His (Trt) -Aib-OH
25mL of a single-necked flask was placed in a stirred reactor at a low temperature and constant temperature, and 0.14. 0.14g H-Aib-OH, 1.5mL of an aqueous solution of sodium carbonate of 0.087g/mL and 3mL (v/v=1:1) of THF/H were added 2 And (3) mixing the solution and cooling to 0 ℃.0.59g of Boc-His (Trt) -OH active ester obtained in step A was weighed out in 1.5ml of THF and added dropwise to a single vial. After the completion of the 5min dropwise addition, the temperature was raised to 25℃for 4h of reaction, and the reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1, 2 drops of acetic acid were additionally added). After completion of the reaction, ph=5 was adjusted by adding aqueous citric acid, and extracted with EA 2 times, 10ml each. The organic phase is collected and washed 2 times with 20ml of aqueous citric acid each time. After washing with 20ml of saturated brine once, the solvent was removed by rotary evaporation after drying over anhydrous sodium sulfate to obtain a viscous solid. 4ml (v/v=1:1) of petroleum ether/isopropyl ether mixed solvent was added to pulp for 30min, and suction filtration was performed to obtain 0.25g of white solid.
EXAMPLE 2 preparation of the fully protected S1-S2 fragment dipeptide Fmoc-His (Trt) -Aib-OH
Preparation of Fmoc-His (Trt) -OH active esters
A100 ml single vial was placed in a low temperature stirred reactor, 3.10g Fmoc-His (Trt) -OH and 25ml DCM solvent were added, and 1.1g pentafluorophenol was added. After clarification by stirring at 0 ℃, 1.34g dcc solution in 10ml dcm was added dropwise. After the completion of the dropwise addition for 10min, the temperature is raised to 25 ℃ for reaction for 3h. The reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1, 2 additional drops of acetic acid were added). After completion of the reaction, it was suction filtered, washed with 5ml of DCM, the filtrates were combined and the solvent was removed by rotary evaporation to give 3.24g of a viscous mass.
Preparation of Fmoc-His (Trt) -Aib-OH
25mL of a single-necked flask was placed in a stirred reactor at a low temperature and constant temperature, and 0.14. 0.14g H-Aib-OH, 1.5mL of an aqueous solution of sodium carbonate of 0.087g/mL and 3mL (v/v=1:1) of THF/H were added 2 And (3) mixing the solution and cooling to 0 ℃. 0.87g of Fmoc-His (Trt) -OH active ester obtained in step A was weighed out and dissolved in 1.5ml of THF and added dropwise to a single vial. After completion of the 5min dropwise addition, the temperature was raised to 25℃for 4h of reaction, and the reaction was monitored by TLC (Petroleum ether: ethyl acetate=1:1, 2 additional drops of acetic acid were added). After completion of the reaction, ph=5 was adjusted by adding aqueous citric acid, and extracted with EA 2 times, 10ml each. The organic phase was collected and washed 2 times with 20ml of aqueous citric acid. After washing with 20ml of saturated brine once, the solvent was removed by rotary evaporation after drying over anhydrous sodium sulfate to obtain a viscous solid. 4ml (v/v=1:1) of petroleum ether/isopropyl ether mixed solvent was addedPulping for 30min, and suction filtering to obtain yellow sticky material 0.32g.
EXAMPLE 3 preparation of the fully protected S1-S2 fragment dipeptide Fmoc-His (Boc) -Aib-OH
Preparation of Fmoc-His (Boc) -OH active esters
A100 ml single vial was placed in a low temperature stirred reactor, 3.50g Fmoc-His (Boc) -OH and 25ml DCM solvent were added, and 1.3g pentafluorophenol was added. After clarification by stirring at 0 ℃, 1.34g dcc solution in 10ml dcm was added dropwise. After the completion of the dropwise addition for 10min, the temperature is raised to 25 ℃ for reaction for 3h. The reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1, 2 additional drops of acetic acid were added). After completion of the reaction, it was suction filtered, washed with 5ml of DCM, the filtrates were combined and the solvent was removed by rotary evaporation to give 3.72g of a viscous mass.
Preparation of Fmoc-His (Boc) -Aib-OH
25mL of a single-necked flask was placed in a stirred reactor at a low temperature and constant temperature, and 0.14. 0.14g H-Aib-OH, 1.5mL of an aqueous solution of sodium carbonate of 0.087g/mL and 3mL (v/v=1:1) of THF/H were added 2 And (3) mixing the solution and cooling to 0 ℃. 0.73g of Fmoc-His (Boc) -OH active ester obtained in step A was weighed out in 1.5ml of THF and added dropwise to a single vial. After completion of the 5min dropwise addition, the temperature was raised to 25℃for 4h of reaction, and the reaction was monitored by TLC (Petroleum ether: ethyl acetate=1:1, 2 additional drops of acetic acid were added). After completion of the reaction, ph=5 was adjusted by adding aqueous citric acid, and extracted with EA 2 times, 10ml each. The organic phase was collected and washed 2 times with 20ml of aqueous citric acid. After washing with 20ml of saturated brine once, the solvent was removed by rotary evaporation after drying over anhydrous sodium sulfate to obtain a viscous solid. 4ml (v/v=1:1) of petroleum ether/isopropyl ether mixed solvent was added to pulp for 30min, and suction filtration was performed to obtain 0.28g of yellow dope.
EXAMPLE 4 preparation of the fully protected S3-S4 fragment dipeptide Fmoc-Glu (OtBu) -Gly-OH
Preparation of Fmoc-Glu (OtBu) -OH active ester
A100 ml single vial was placed in a low temperature stirred reactor, 2.13g Fmoc-Glu (OtBu) -OH and 25ml DCM solvent were added, and 1.1g pentafluorophenol was added. After clarification by stirring at 0 ℃, 1.34g dcc solution in 10ml dcm was added dropwise. After the completion of the dropwise addition for 10min, the temperature is raised to 25 ℃ for reaction for 3h. The reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1, 2 additional drops of acetic acid were added). After completion of the reaction, it was suction filtered, washed with 5ml of DCM, the filtrates were combined and the solvent was removed by rotary evaporation to give 2.78g of a viscous mass.
Preparation of Fmoc-Glu (OtBu) -Gly-OH
25mL of a single-necked flask was placed in a stirred reactor at a low temperature and constant temperature, and 0.10. 0.10g H-Gly-OH, 1.5mL of an aqueous solution of sodium carbonate of 0.087g/mL and 3mL (v/v=1:1) of THF/H were added 2 And (3) mixing the solution and cooling to 0 ℃. 0.36g of Fmoc-Glu (OtBu) -OH active ester obtained in step A was weighed out in 1.5ml of THF and added dropwise to a single vial. After the completion of the 5min dropwise addition, the temperature was raised to 25℃for 4h of reaction, and the reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1, 2 drops of acetic acid were additionally added). After completion of the reaction, ph=5 was adjusted by adding aqueous citric acid, and extracted with EA 2 times, 10ml each. The organic phase was collected and washed 2 times with 20ml of aqueous citric acid. After washing with 20ml of saturated brine once, the solvent was removed by rotary evaporation after drying over anhydrous sodium sulfate to obtain a viscous solid. 4ml (v/v=1:1) of petroleum ether/isopropyl ether mixed solvent was added to pulp for 30min, and suction filtration was performed to obtain 0.21g of yellow dope.
EXAMPLE 5 preparation of Fmoc-Gly-Wang resin with substitution of 0.3mmol/g
A. 10g (8 mmol) of Wang resin with a substitution degree of 0.8mmol/g are added to the reaction vessel, 100ml of dichloromethane are added, after mixing for 2min, the dichloromethane is filtered off, 100ml of dichloromethane are added again, after mixing for 40min, the dichloromethane is filtered off, finally 100ml of dichloromethane is added again, after mixing for 2min, the dichloromethane is filtered off and the resin is ready for use.
B. 7.14g Fmoc-Gly-OH and 3.89g HOBT were weighed into a beaker, 100ml DMF and 3.97ml DIEA were added, the solution was stirred at 0-10℃for 5min, then poured into the Wang resin obtained in step A, 0.15g DMAP was added, and the mixture was mixed at 20-25℃for 4h. After the reaction was completed, 7.55ml of acetic anhydride was added thereto, and mixing was continued for 1 hour. After the reaction is finished, carrying out suction filtration, and washing the resin with DMF for 5 times, wherein 100ml of the resin is used each time; after washing, methanol is used for washing twice, and 100ml of methanol is used for each time; washing with dichloromethane for 2 times, each time with 100ml; finally, the resin was washed 3 times with 100ml portions of methanol until the resin was sufficiently dispersed.
C. And (3) drying the resin obtained in the step (B) in a vacuum drying oven at 20-30 ℃ for 4 hours until the weight is constant (twice continuous weighing, and the error is lower than 1%). After drying, 13.8g of Fmoc-Gly-Wang resin was obtained, and the substitution degree by ultraviolet detection was 0.3mmol/g.
EXAMPLE 6 preparation of Fmoc-Gly-Wang resin with substitution of 0.6mmol/g
A. 10g (14 mmol) of Wang resin with a substitution degree of 1.4mmol/g are added to the reaction vessel, 100ml of dichloromethane are added, after mixing for 2min, the dichloromethane is filtered off, 100ml of dichloromethane are added again, after mixing for 40min, the dichloromethane is filtered off, finally 100ml of dichloromethane is added again, after mixing for 2min, the dichloromethane is filtered off and the resin is ready for use.
B. 12.49g Fmoc-Gly-OH and 6.81g HOBT were weighed into a beaker, 100ml DMF and 6.95ml DIEA were added, the solution was stirred at 0-10℃for 5min, then poured into the Wang resin obtained in step A, 0.26g DMAP was added, and the mixture was mixed at 20-25℃for 4h. After the reaction was completed, 13.21ml of acetic anhydride was added thereto, and mixing was continued for 1 hour. After the reaction is finished, carrying out suction filtration, and washing the resin with DMF for 5 times, wherein 100ml of the resin is used each time; after washing, methanol is used for washing twice, and 100ml of methanol is used for each time; washing with dichloromethane for 2 times, each time with 100ml; finally, the resin was washed 3 times with 100ml portions of methanol until the resin was sufficiently dispersed.
C. And (3) drying the resin obtained in the step (B) in a vacuum drying oven at 20-30 ℃ for 4 hours until the weight is constant (twice continuous weighing, and the error is lower than 1%). After drying, 12.7g of Fmoc-Gly-Wang resin was obtained, and the degree of substitution was detected to be 0.6mmol/g.
EXAMPLE 7 preparation of Fmoc-Gly-CTC resin with degree of substitution of 0.45mmol/g
A. 10g (10 mmol) of CTC resin with a substitution degree of 1.0mmol/g was added to the reaction vessel, 100ml of dichloromethane was added, after mixing for 2min, dichloromethane was filtered off, 100ml of dichloromethane was added again, after mixing for 40min, dichloromethane was filtered off, finally 100ml of dichloromethane was added again, after mixing for 2min, dichloromethane was filtered off and the resin was ready for use.
B. 8.92g Fmoc-Gly-OH and 4.86g HOBT were weighed into a beaker, 100ml DMF and 4.96ml DIEA were added, the solution was stirred at 0-10℃for 5min, then poured into the CTC resin obtained in step A, and mixed for 4h at 20-25 ℃. After the reaction is finished, DMF is filtered off. A mixed solution of 4ml methanol and 50ml DMF, a mixed solution of 5ml DIEA and 50ml DMF was added to the resin and mixing was continued for 1h. After the reaction is finished, carrying out suction filtration, washing the resin with DMF for 5 times, and 100ml each time; after washing, methanol is used for washing twice, and 100ml of methanol is used for each time; washing with dichloromethane for 2 times, each time with 100ml; finally, the resin was washed 3 times with 100ml portions of methanol until the resin was sufficiently dispersed.
C. And (3) drying the resin obtained in the step (B) in a vacuum drying oven at 20-30 ℃ for 4 hours until the weight is constant (twice continuous weighing, and the error is lower than 1%). After drying, 13.2g of Fmoc-Gly-CTC resin was obtained, and the degree of substitution was detected to be 0.45mmol/g.
EXAMPLE 8 preparation of the fully protected S5-S6 fragment dipeptide Fmoc-Thr (tBu) -Phe-OH
Preparation of Fmoc-Thr (tBu) -OH active ester
A100 ml single vial was placed in a low temperature stirred reactor and 1.99g Fmoc-Thr (tBu) -OH and 25ml DCM solvent were added followed by 1.1g pentafluorophenol. After clarification by stirring at 0 ℃, 1.34g dcc solution in 10ml dcm was added dropwise. After the completion of the dropwise addition for 10min, the temperature is raised to 25 ℃ for reaction for 3h. The reaction was monitored by TLC (petroleum ether: ethyl acetate=1:1, 2 additional drops of acetic acid were added). After completion of the reaction, it was suction filtered, washed with 5ml of DCM, the filtrates were combined and the solvent was removed by rotary evaporation to give 2.55g of a viscous mass.
Preparation of Fmoc-Thr (tBu) -Phe-OH
25mL of a single-necked flask was placed in a stirred reactor at a low temperature and constant temperature, and 0.19. 0.19g H-Phe-OH, 1.5mL of an aqueous solution of sodium carbonate of 0.087g/mL and 3mL (v/v=1:1) of THF/H were added 2 And (3) mixing the solution and cooling to 0 ℃. 0.34g of Fmoc-Thr (tBu) -OH active ester obtained in step A was weighed out and dissolved in 1.5ml of THF and added dropwise to a single vial. After completion of the 5min dropwise addition, the temperature was raised to 25℃for 4h of reaction, and the reaction was monitored by TLC (Petroleum ether: ethyl acetate=1:1, 2 additional drops of acetic acid were added). After completion of the reaction, ph=5 was adjusted by adding aqueous citric acid, and extracted with EA 2 times, 10ml each. The organic phase was collected and washed 2 times with 20ml of aqueous citric acid. Washing with 20ml saturated saline solution once, drying with anhydrous sodium sulfate, and steamingThe solvent was removed to give a viscous solid. 4ml (v/v=1:1) of petroleum ether/isopropyl ether mixed solvent was added to pulp for 30min, and suction filtration was performed to obtain 0.25g of yellow dope.
EXAMPLE 9 Synthesis of Somarlutide backbone peptide resin Using Fmoc-Lys (Dde) -OH as a starting Material
A. 10g of Fmoc-Gly-Wang resin obtained in example 5 was poured into the reaction kettle, swollen with 100ml of DCM and mixed for 15min, and then drained. 100ml of a 20% piperidine/DMF solution was added and mixed at 20-30℃for 5min, followed by draining. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. 100ml of a 20% piperidine/DMF solution was added and mixed at 20-30℃for 10min, followed by draining. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. And washing with DMF repeatedly for 8 times and 100ml each time, mixing for 5min each time, and detecting filtrate with PH test paper after the seventh washing, wherein the result shows that the PH is 6.5-7.0.
B. 3.89g of Fmoc-Arg (Pbf) -OH, 1.16g of TBTU and 0.49g of HOBT are weighed in sequence into a clean 1L beaker, 100ml of DMF/DCM solution in a volume ratio of 1:1 is added, the mixture is placed into ice water and stirred and dissolved by a mechanical stirrer at 0-10 ℃, after the temperature is constant, 0.50ml of DIEA is added, the temperature is continuously maintained and stirring and activation are continued for 5min. Slowly adding the activating solution into a reaction kettle, and mixing for 2 hours at 20-25 ℃. After the reaction is finished, the mixture is pumped to dryness, 100ml of DMF is added, and after 5 minutes of mixing, the mixture is pumped to dryness. The DMF washes were repeated 6 times, 100ml each time, and 5min each time mixed. And finally, detecting the obtained product as negative by ninhydrin to obtain Fmoc-Arg (Pbf) -Gly-Wang resin.
C. According to the deprotection method of the step A and the coupling method of the step B, the residual amino acids are coupled in sequence according to the sequence of the main chain, namely: fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys (Dde) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-OH Fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -Phe-OH obtained in example 8, fmoc-Glu (OtBu) -Gly-OH obtained in example 4, boc-His (Trt) -Aib-OH obtained in example 1. Wherein, a DIC/Cl-HOBt coupling system and a DMF solvent are adopted in the coupling of Fmoc-Gly-OH, fmoc-Val-OH and Fmoc-Lys (Dde) -OH; coupling Fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH and Fmoc-Ile-OH by using a TBTU/HOBt/DIEA coupling system and a DCM solvent; the Fmoc-Glu (OtBu) -OH coupling system is adopted in the coupling process of TBTU/Cl-HOBt/DIEA; the Fmoc-Phe-OH coupling is performed by adopting a TBTU/HOAt/DIEA coupling system; the Fmoc-Ala-OH coupling is performed by adopting a TBTU/DIEA coupling system; the Fmoc-Ser (tBu) -OH and Fmoc-Glu (OtBu) -Gly-OH are coupled by using a PyBop/DIEA coupling system; the Fmoc-Thr (tBu) -OH coupling is performed by adopting a PyAop/DIEA coupling system; coupling Boc-His (Trt) -Aib-OH was performed using a COMU/DIEA coupling system with a NMP/DMSO=1:1 mixed solvent. Finally, washing with dichloromethane for 5 times, each time 100ml; after washing, methanol is used for washing twice, and 100ml of methanol is used for washing each time; washing with dichloromethane for 2 times, each time with 100ml; finally, the resin was washed 3 times with 100ml of alcohol until the resin was sufficiently dispersed. The resin was dried in a vacuum oven at 20-30 ℃ for 4h to constant weight (twice weighing in succession, error lower than 1%). 21.9g of a cable MALU peptide backbone peptide resin was obtained.
EXAMPLE 10 Synthesis of Somarlutide fully protected peptide resin Using Fmoc-Lys (Dde) -OH as a raw Material
A. 15g of the cable-marlutide main chain peptide resin obtained in example 9 was added to a reaction vessel, 150ml of methylene chloride was added, and after mixing for 15 minutes, methylene chloride was filtered off, and the resin was used.
B. A clear solution of hydroxylamine hydrochloride (12 g) and imidazole (6 g) in DMF (150 ml) was added, and the mixture was mixed at 20-30℃for 1 hour, followed by draining. 150ml of DMF was added and after mixing for 5min, the mixture was drained. 150ml of DMF was added and after mixing for 5min, the mixture was drained. And washing with DMF repeatedly for 8 times and 150ml each time, mixing for 5min each time, and detecting filtrate with PH test paper after the seventh washing, wherein the result shows that the PH is 6.5-7.0.
C. 1.54g Fmoc-AEEA-OH, 1.54g TBTU and 0.81g HOBT were weighed in a clean 500mL beaker, 150mL of DMF/DCM solution was added in a volume ratio of 1:1, the mixture was placed in ice water and stirred with a mechanical stirrer at 0-10℃to dissolve, after the temperature was constant, 0.66mL DIEA was added, and the temperature was maintained and stirring was continued for 5min. Slowly adding the activating solution into a reaction kettle, and mixing for 2 hours at 20-25 ℃. After the reaction is finished, the mixture is pumped to dryness, 150ml of DMF is added, and after 5 minutes of mixing, the mixture is pumped to dryness. The DMF washes were repeated 6 times at 150ml each time and mixed for 5min each time. And finally, detecting negative by ninhydrin.
D. After Fmoc-AEEA-OH was coupled as above, fmoc-Glu (OH) -OtBu, octadecanedioic acid mono-tert-butyl ester was coupled sequentially. In the coupling of mono-tert-butyl octadecanedioate, a PyBop/DIEA system is used, namely 2.50g of PyBop and 0.66mL of DIEA are added, and DMF is used as a solvent: nmp=1:1V/V, the remaining reaction conditions were unchanged. After the coupling is completed, methanol is used for shrinking, and the synthesis of S20 side chain of the Somalux is completed, thus 14.31g of Somalux full-protection peptide resin is obtained.
EXAMPLE 11 Synthesis of Somarlutide fully protected peptide resin Using Fmoc-Lys (Dde) -OH as a raw Material
A. 10g of the cable MALUO-peptide main chain peptide resin obtained in example 9 was added to a reaction vessel, 100ml of methylene chloride was added, and after mixing for 15 minutes, methylene chloride was filtered off, and the resin was used.
B. 100ml of a clear DMF solution of 12g hydroxylamine hydrochloride/4 g imidazole was added, and the mixture was stirred at 20-30℃for 1 hour and then dried. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. And washing with DMF repeatedly for 8 times and 100ml each time, mixing for 5min each time, and detecting filtrate with PH test paper after the seventh washing, wherein the result shows that the PH is 6.5-7.0.
C. 1.03g Fmoc-AEEA-OH, 1.03g TBTU and 0.54g HOBT were weighed in a clean 500mL beaker, 100mL of DMF/DCM solution was added in a volume ratio of 1:1, the mixture was placed in ice water and stirred with a mechanical stirrer at 0-10℃to dissolve, after the temperature was constant, 0.44mL DIEA was added, and the temperature was maintained and stirring was continued for 5min. Slowly adding the activating solution into a reaction kettle, and mixing for 2 hours at 20-25 ℃. After the reaction, the mixture was drained, 100ml of DMF was added thereto, and after 5 minutes of mixing, the mixture was drained. The DMF washes were repeated 6 times, 100ml each time, and 5min each time mixed. And finally, detecting negative by ninhydrin.
D. After Fmoc-AEEA-OH was coupled as above, fmoc-Glu (OH) -OtBu, octadecanedioic acid mono-tert-butyl ester was coupled sequentially. In the coupling of mono-tert-butyl octadecanedioate, a PyBop/DIEA system is used, i.e. 1.67g of PyBop and 0.44mL of DIEA are added, and DMF is used as solvent: nmp=1:1V/V, the remaining reaction conditions were unchanged. After the coupling is finished, methanol is used for shrinking, and the synthesis of the S20 side chain of the Somalux is finished, so that 13.85g of Somalux full-protection peptide resin is obtained.
EXAMPLE 12 Synthesis of Somarlutide fully protected peptide resin Using Fmoc-Lys (Dde) -OH as a raw Material
A. 10g of the cable MALUO-peptide main chain peptide resin obtained in example 9 was added to a reaction vessel, 100ml of methylene chloride was added, and after mixing for 15 minutes, methylene chloride was filtered off, and the resin was used.
B. A clear solution of hydroxylamine hydrochloride (4 g/12 g) in imidazole in DMF (100 ml) was added, and the mixture was stirred at 20-30℃for 1 hour and then dried. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. And washing with DMF repeatedly for 8 times and 100ml each time, mixing for 5min each time, and detecting filtrate with PH test paper after the seventh washing, wherein the result shows that the PH is 6.5-7.0.
C. 1.03g Fmoc-AEEA-OH, 1.03g TBTU and 0.54g HOBT were weighed in a clean 500mL beaker, 100mL of DMF/DCM solution was added in a volume ratio of 1:1, the mixture was placed in ice water and stirred with a mechanical stirrer at 0-10℃to dissolve, after the temperature was constant, 0.44mL DIEA was added, and the temperature was maintained and stirring was continued for 5min. Slowly adding the activating solution into a reaction kettle, and mixing for 2 hours at 20-25 ℃. After the reaction is finished, the mixture is pumped to dryness, 100ml of DMF is added, and after 5 minutes of mixing, the mixture is pumped to dryness. The DMF washes were repeated 6 times, 100ml each time, and 5min each time mixed. And finally, detecting negative by ninhydrin.
D. After Fmoc-AEEA-OH was coupled as above, fmoc-Glu (OH) -OtBu, octadecanedioic acid mono-tert-butyl ester was coupled sequentially. In the coupling of mono-tert-butyl octadecanedioate, a PyBop/DIEA system is used, i.e. 1.67g of PyBop and 0.44mL of DIEA are added, and DMF is used as solvent: nmp=1:1V/V, the remaining reaction conditions were unchanged. After the coupling is finished, methanol is used for shrinking, and the synthesis of the S20 side chain of the somalu peptide is finished, so that 13.55g of the somalu peptide full-protection peptide resin is obtained.
EXAMPLE 13 Synthesis of Somarlutide fully protected peptide resin Using Fmoc-Lys (Dde) -OH as a raw Material
A. 10g of the cable MALUO-peptide main chain peptide resin obtained in example 9 was added to a reaction vessel, 100ml of methylene chloride was added, and after mixing for 15 minutes, methylene chloride was filtered off, and the resin was used.
B. Adding 2% NH 2 NH 2 After 10 minutes of mixing at 20-30℃the solution was drained. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. Repeated DMF washing 8 times, 100ml each time, mixing 5min each time, and after the seventh washing, the filtrate was checked with PH test paper, which showed that PH was acceptable at 6.5-7.0.
C. 1.03g Fmoc-AEEA-OH, 1.03g TBTU and 0.54g HOBT were weighed in a clean 500mL beaker, 100mL of DMF/DCM solution was added in a volume ratio of 1:1, the mixture was placed in ice water and stirred with a mechanical stirrer at 0-10℃to dissolve, after the temperature was constant, 0.44mL DIEA was added, and the temperature was maintained and stirring was continued for 5min. Slowly adding the activating solution into a reaction kettle, and mixing for 2 hours at 20-25 ℃. After the reaction is finished, the mixture is pumped to dryness, 100ml of DMF is added, and after 5 minutes of mixing, the mixture is pumped to dryness. The DMF washes were repeated 6 times, 100ml each time, and 5min each time mixed. And finally, detecting negative by ninhydrin.
D. After Fmoc-AEEA-OH was coupled as above, fmoc-Glu (OH) -OtBu, octadecanedioic acid mono-tert-butyl ester was coupled sequentially. In the coupling of mono-tert-butyl octadecanedioate, a PyBop/DIEA system was used, i.e.1.67 g of PyBop and 0.44ml of DIEA were added, and DMF was used as solvent: nmp=1:1V/V, the remaining reaction conditions were unchanged. After the coupling is completed, methanol is used for shrinking, and the synthesis of S20 side chain of the Somalux is completed, thus 13.43g of Somalux full-protection peptide resin is obtained.
Example 14: preparation of Boc-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH
A. 150g (165 mmol) of 2-CTC resin with a substitution degree of 1.1mmol/g are added to the reaction kettle, 160ml of dichlorosilane are added, after mixing for 2min, dichloromethane is filtered off, 160ml of dichloromethane is added again, after mixing for 40min, dichloromethane is filtered off, finally 160ml of dichloromethane is added again, after mixing for 2min, dichloromethane is filtered off, and the resin is ready for use.
B. 127.17g of Fmoc-AEEA-OH was weighed into a beaker, 160ml of DMF and 80ml of DIEA were added, the solution was stirred at 0-10℃for 5min, and after stirring, poured into the CTC resin obtained in step A and mixed for 4h at 20-25 ℃. After the reaction is finished, DMF is filtered off. A mixed solution of 27ml of methanol and 100ml of DMF was added to the resin, and the mixture of 38ml of DIEA and 100ml of DMF was mixed for a further 1h. After the reaction is finished, carrying out suction filtration, and washing the resin with DMF for 5 times, wherein 160ml of the resin is used each time; after washing, methanol is used for washing twice, 160ml each time; washing with dichloromethane for 2 times, each time 160ml; finally, the resin was washed with methanol 3 times, 160ml each time, until the resin was sufficiently dispersed.
C. And (3) drying the resin obtained in the step (B) in a vacuum drying oven at 20-30 ℃ for 4 hours until the weight is constant (twice continuous weighing, and the error is lower than 1%). After drying, 201.5g of Fmoc-AEEA-CTC resin was obtained. The substitution degree was 0.6mmol/g by detection.
D. The Fmoc-AEEA-CTC resin obtained in step C was poured into a reaction vessel, swelled and mixed with 200ml DCM for 15min, and then drained. 200ml of 20% piperidine/DMF solution was added and mixed at 20-30℃for 5min, followed by draining. 200ml of DMF was added and the mixture was mixed for 5 minutes and then drained. 200ml of 20% piperidine/DMF solution was added and mixed at 20-30℃for 10min, followed by draining. 200ml of DMF was added and the mixture was mixed for 5 minutes and then drained. The washing with DMF was repeated 8 times at 200ml each time and mixed for 5min each time, and after the seventh washing the filtrate was checked with pH paper and the result showed that the pH was acceptable at 6.5-7.0.
E. 93.19g of Fmoc-AEEA-OH, 45.43mL of DIC and 39.20g of HOBT are weighed in sequence in a clean 1L beaker, 200mL of DMF/DCM solution in a volume ratio of 1:1 is added, the mixture is placed in ice water and stirred with a mechanical stirrer at 0-10 ℃ for dissolution, and after the temperature is constant, the temperature is kept continuously and stirring is continued for 5min for activation. Slowly adding the activating solution into a reaction kettle, and mixing for 2 hours at 20-25 ℃. After the reaction, the mixture was drained, 200ml of DMF was added, and after 5 minutes of mixing, the mixture was drained. The DMF washes were repeated 6 times, 200ml each time, and mixed 5min each time. Finally, detecting the obtained product as negative by ninhydrin to obtain Fmoc-AEEA-AEEA-CTC resin.
F. According to the deprotection method of the step D and the coupling method of the step E, the amino acid Fmoc-Glu (OH) -OtBu and the octadecanedioic acid mono-tert-butyl ester are respectively coupled in sequence. Finally, washing with dichloromethane for 5 times, 200ml each time; after washing, methanol is used for washing twice, 200ml each time; washing with dichloromethane for 2 times, 200ml each time; finally, alcohol was used 3 times, 200ml each time, until the resin was sufficiently dispersed. The resin was dried in a vacuum oven at 20-30 ℃ for 4h to constant weight (twice weighing in succession, error lower than 1%). 291.48g of Octadecanedioic acid mono-tert-butyl ester-. Gamma. -Glu (OtBu) -AEEA-AeEA-CTC resin was obtained.
G. The lysate ratio is TFE: DCM=1:4 (volume ratio), 291.48g of the full-protection peptide resin of the CTC resin obtained in the step F is added into 3L lysate at 15 ℃, the temperature is raised to 30 ℃, the reaction is continued for 3 hours by stirring, then the filtered resin is filtered by a sand core funnel, the filtered resin is washed by 500mL of DCM, the filtrate is combined after repeated operation for two times, the filtrate is decompressed and concentrated to 30% of the volume of the original volume, then the concentrated solution is slowly added into precooled 3L isobutyl ether, the mixture is centrifuged for 5 times after settling overnight, 500mL of isobutyl ether is used each time, white solid powder is obtained, the mixture is dried by a vacuum drying box for 10 hours after being dried by nitrogen, and Octadecanedioic acid mono-tert-butyl ester-gamma-Glu (OtBu) -AEEA-AEEA-OH crude product 96.80g is obtained after weighing.
H. The crude Octadecanedioic acid mono-tert-butyl ester-. Gamma. -Glu (OtBu) -AEEa-AEEa-OH product obtained in step G (10G) was dissolved in 20mL of DCM, and 4.4G of pentafluorophenol was added. 20mL of DCC solution in DCM containing 4.8g of DCC was slowly added dropwise. The reaction was stirred for 1.0h, and after completion of the TLC detection, it was filtered. The filtrate was washed once with saturated brine, once with water, and the DCM solution was dried over anhydrous sodium sulfate, concentrated to dryness, and dissolved in an appropriate amount of acetonitrile. In addition, 12.15g of Boc-Lys-OH.HCl was weighed and dissolved in acetonitrile/water (acetonitrile/water=1/2), 15mL of DIEA was added, and the mixture was stirred for 15 minutes to obtain a Boc-Lys-OH solution. The above reaction solution was slowly added dropwise to the Boc-Lys-OH solution and reacted for 1.5 hours with stirring. Dilute hydrochloric acid was added to adjust the pH to about 6, and a small amount of DCM was added for extraction. Purification gave Boc-Lys (AEEA-AEEA-. Gamma. -Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH 3.48g.
Example 15: preparation of Fmoc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH
A. 10mL of 4M HCl in 1, 4-dioxane solution was cooled in an ice-water bath, and 1.30g of Boc-Lys (AEEA-AEEA-. Gamma. -Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH obtained in example 14 was added. The reaction mixture was warmed to room temperature and then reacted for 2 hours. The solvent was removed by rotary evaporation, dissolved with 15mL of DCM and filtered, and washed twice with 15mL each. The filtrates were combined and the solvent was removed by rotary evaporation to give crude oil which was used directly in the next reaction without purification.
B. The crude H-Lys (AEEA-AEEA-. Gamma. -Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH obtained in step A was dissolved in 20mL DCM and 0.66g pentafluorophenol was added. 0.74g of DCC was weighed and dissolved in 20mL of DCM, and the DCC solution was slowly added dropwise to the reaction solution, followed by stirring for 1.0h and filtration after completion of the TLC detection. The filtrate was washed once with saturated brine, once with water, and the DCM solution was dried over anhydrous sodium sulfate, concentrated to dryness, and dissolved in an appropriate amount of acetonitrile. In addition, 0.75g of Fmoc-Ala-OH.HCl was weighed and dissolved in acetonitrile/water (acetonitrile/water=1/2), 0.50mL of DIEA was added, and stirred for 15 minutes to obtain Fmoc-Ala-OH solution. The above reaction solution was slowly added dropwise to Fmoc-Ala-OH solution and the reaction was stirred for 1.5h. Dilute hydrochloric acid was added to adjust the pH to about 6, and a small amount of DCM was added for extraction. Fmoc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH 1.54g was obtained by purification.
EXAMPLE 16 Sodama Lu Quanbao Propeptide resin was synthesized using Fmoc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH as the starting material
A. 10g of Fmoc-Gly-Wang resin obtained in example 6 was poured into the reaction kettle, swollen with 100ml of DCM and mixed for 15min, and then drained. 100ml of a 20% piperidine/DMF solution was added and mixed at 20-30℃for 5min, followed by draining. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. 100ml of a 20% piperidine/DMF solution was added and mixed at 20-30℃for 10min, followed by draining. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. Washing with DMF was repeated 8 times, 100ml each time, mixing 5min each time, and after the seventh washing, the filtrate was checked with pH paper, which showed that the pH was acceptable at 6.5-7.0.
B. 3.89g of Fmoc-Arg (Pbf) -OH, 1.16g of TBTU and 0.49g of HOBT are weighed in sequence into a clean 1L beaker, 100ml of DMF/DCM solution in a volume ratio of 1:1 is added, the mixture is placed into ice water and stirred and dissolved by a mechanical stirrer at 0-10 ℃, after the temperature is constant, 0.50ml of DIEA is added, the temperature is continuously maintained and stirring and activation are continued for 5min. Slowly adding the activating solution into a reaction kettle, and mixing for 2 hours at 20-25 ℃. After the reaction is finished, the mixture is pumped to dryness, 100ml of DMF is added, and after 5 minutes of mixing, the mixture is pumped to dryness. The DMF washes were repeated 6 times, 100ml each time, and 5min each time mixed. And finally, detecting the obtained product as negative by ninhydrin to obtain the Fmoc-Arg-Gly-Wang resin.
C. According to the deprotection method of the step A and the coupling method of the step B, the residual amino acids are coupled in sequence according to the sequence of the main chain, namely: fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH prepared in example 15 Fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -Phe-OH obtained in example 8, fmoc-Glu (OtBu) -Gly-OH obtained in example 4, boc-His (Trt) -Aib-OH obtained in example 1. Wherein, when Fmoc-Gly-OH and Fmoc-Val-OH are coupled, a DIC/Cl-HOBt coupling system and a DMF solvent are adopted; coupling Fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH and Fmoc-Ile-OH by using a TBTU/HOBt/DIEA coupling system and a DCM solvent; the Fmoc-Glu (OtBu) -OH coupling system is adopted in the coupling process of TBTU/Cl-HOBt/DIEA; the coupling system TBTU/HOAt/DIEA is adopted for coupling Fmoc-Phe-OH, fmoc-Glu (OtBu) -Gly-OH and Boc-His (Trt) -Aib-OH; the Fmoc-Ala-OH coupling is performed by adopting a TBTU/DIEA coupling system; the Fmoc-Ser (tBu) -OH coupling is performed by using a PyBop/DIEA coupling system; the Fmoc-Thr (tBu) -OH coupling is performed by adopting a PyAop/DIEA coupling system; fmoc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH was coupled using a COMU/DIEA coupling system with a mixed solvent NMP/DMSO=1:1. Finally, washing with dichloromethane for 5 times, each time 100ml; after washing, methanol is used for washing twice, and 100ml of methanol is used for each time; washing with dichloromethane for 2 times, each time with 100ml; finally, the resin was washed 3 times with 100ml of alcohol until the resin was sufficiently dispersed. The resin was dried in a vacuum oven at 20-30 ℃ for 4h to constant weight (twice weighing in succession, error lower than 1%). 25.4g of Somam Lu Taitai resin are obtained.
Example 17 preparation of Fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH.
A. 10mL of 4M HCl in 1, 4-dioxane solution was cooled in an ice-water bath, and 1.30g of Boc-Lys (AEEA-AEEA-. Gamma. -Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH obtained in example 13 was added. The reaction mixture was warmed to room temperature and then reacted for 2 hours. The solvent was removed by rotary evaporation, dissolved with 15mL of DCM and filtered, and washed twice with 15mL each. The filtrates were combined and the solvent was removed by rotary evaporation to give crude oil which was used directly in the next reaction without purification.
B. The crude H-Lys (AEEA-AEEA-. Gamma. -Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH obtained in step A was dissolved in 20mL DCM and 0.66g pentafluorophenol was added. 0.74g of DCC was weighed and dissolved in 20mL of DCM, and the DCC solution was slowly added dropwise to the reaction solution, followed by stirring for 1.0h and filtration after completion of the TLC detection. The filtrate was washed once with saturated brine, once with water, and the DCM solution was dried over anhydrous sodium sulfate, concentrated to dryness, and dissolved in an appropriate amount of acetonitrile. In addition, 0.68g of Boc-Ala-OH.HCl was weighed and dissolved in acetonitrile/water (acetonitrile/water=1/2), 0.50mL of DIEA was added, and the mixture was stirred for 15 minutes to obtain a Boc-Ala-OH solution. The above reaction solution was slowly added dropwise to the Boc-Ala-OH solution and reacted for 1.5 hours with stirring. Dilute hydrochloric acid was added to adjust the pH to about 6, and a small amount of DCM was added for extraction. Boc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH 1.44g was obtained by purification.
C. 10mL of 4M HCl in 1, 4-dioxane solution was cooled in an ice-water bath, and 1.44g of Boc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH obtained in step B was added. The reaction mixture was warmed to room temperature and then reacted for 2 hours. The solvent was removed by rotary evaporation, dissolved with 15mL of DCM and filtered, and washed twice with 15mL each. The filtrates were combined and the solvent was removed by rotary evaporation to give crude oil which was used directly in the next reaction without purification.
D. The crude H-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH obtained in step C was dissolved in 20mL DCM and 0.66g pentafluorophenol was added. 0.74g of DCC was weighed and dissolved in 20mL of DCM, and the DCC solution was slowly added dropwise to the reaction solution, followed by stirring for 1.0h and filtration after completion of the TLC detection. The filtrate was washed once with saturated brine, once with water, and the DCM solution was dried over anhydrous sodium sulfate, concentrated to dryness, and dissolved in an appropriate amount of acetonitrile. In addition, 0.75g of Fmoc-Ala-OH.HCl was weighed and dissolved in acetonitrile/water (acetonitrile/water=1/2), 0.50mL of DIEA was added, and stirred for 15 minutes to obtain Fmoc-Ala-OH solution. The above reaction solution was slowly added dropwise to Fmoc-Ala-OH solution and the reaction was stirred for 1.5h. Dilute hydrochloric acid was added to adjust the pH to about 6, and a small amount of DCM was added for extraction. Fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH 1.63g was obtained by purification.
EXAMPLE 18 Synthesis of Sodama Lu Quanbao Propeptide resin Using Fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH as a starting material
A. 10g of Fmoc-Gly-Wang resin with substitution of 0.3 mmol/g was poured into the reaction kettle, swollen with 100ml of DCM and mixed for 15min, and then pumped down. 100ml of a 20% piperidine/DMF solution was added and mixed at 20-30℃for 5min, followed by draining. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. 100ml of a 20% piperidine/DMF solution was added and mixed at 20-30℃for 10min, followed by draining. DMF (100 ml) was added thereto, and after mixing for 5 minutes, the mixture was drained. And washing with DMF repeatedly for 8 times and 100ml each time, mixing for 5min each time, and detecting filtrate with PH test paper after the seventh washing, wherein the result shows that the PH is 6.5-7.0.
B. 3.89g of Fmoc-Arg (Pbf) -OH, 1.16g of TBTU and 0.49g of HOBT are weighed in sequence into a clean 1L beaker, 100ml of DMF/DCM solution in a volume ratio of 1:1 is added, the mixture is placed into ice water and stirred and dissolved by a mechanical stirrer at 0-10 ℃, after the temperature is constant, 0.50ml of DIEA is added, the temperature is continuously maintained and stirring and activation are continued for 5min. Slowly adding the activating solution into a reaction kettle, and mixing for 2 hours at 20-25 ℃. After the reaction is finished, the mixture is pumped to dryness, 100ml of DMF is added, and after 5 minutes of mixing, the mixture is pumped to dryness. The DMF washes were repeated 6 times, 100ml each time, and 5min each time mixed. And finally, detecting the obtained product as negative by ninhydrin to obtain the Fmoc-Arg-Gly-Wang resin.
C. According to the deprotection method of the step A and the coupling method of the step B, the residual amino acids are coupled in sequence according to the sequence of the main chain, namely: fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Ala-Lys (AEEA-Aea-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl-estrer) -OH, fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH prepared in example 17 Fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -Phe-OH obtained in example 8, fmoc-Glu (OtBu) -Gly-OH obtained in example 4, boc-His (Trt) -Aib-OH obtained in example 1. Wherein, when Fmoc-Gly-OH and Fmoc-Val-OH are coupled, a DIC/Cl-HOBt coupling system and a DMF solvent are adopted; coupling Fmoc-Arg (Pbf) -OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH and Fmoc-Ile-OH by using a TBTU/HOBt/DIEA coupling system and a DCM solvent; the Fmoc-Glu (OtBu) -OH coupling system is adopted in the coupling process of TBTU/Cl-HOBt/DIEA; the coupling system TBTU/HOAt/DIEA is adopted for coupling Fmoc-Phe-OH, fmoc-Glu (OtBu) -Gly-OH and Boc-His (Trt) -Aib-OH; the Fmoc-Ala-OH coupling is performed by adopting a TBTU/DIEA coupling system; the Fmoc-Ser (tBu) -OH coupling is performed by using a PyBop/DIEA coupling system; the Fmoc-Thr (tBu) -OH coupling is performed by adopting a PyAop/DIEA coupling system; fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH was coupled using a COMU/DIEA coupling system with a mixed solvent NMP/DMSO=1:1. Finally, washing with dichloromethane for 5 times, each time 100ml; after washing, methanol is used for washing twice, and 100ml of methanol is used for each time; washing with dichloromethane for 2 times, each time with 100ml; finally, the resin was washed 3 times with 100ml of alcohol until the resin was sufficiently dispersed. The resin was dried in a vacuum oven at 20-30 ℃ for 4h to constant weight (twice weighing in succession, error lower than 1%). 25.7g of Somam Lu Taitai resin are obtained.
EXAMPLE 19 preparation of crude Sodamantane peptide
The mixture ratio of the lysate is TFA, EDT, DMS, phenylsulfide, tis and H 2 O=80:4:4:4:4:4 (volume ratio), 10g of the full-protection peptide resin obtained in example 10 is added into 100mL of lysate at 15 ℃, the temperature is raised to 30 ℃, the reaction is continued for 3 hours while stirring, then a sand core funnel is used for filtering, the filtered resin is washed by 30mL of TFA, the filtrate is combined after repeated operation for two times, the filtrate volume is reduced to 30% of the original volume, then the concentrated solution is slowly added into pre-cooled 300mL of isobutyl ether, the mixture is centrifuged for 5 times after sedimentation overnight, 200mL of isobutyl ether is used each time to obtain white solid powder, the white solid powder is dried by a vacuum drying box for 10 hours after being dried by nitrogen for 4 hours, 5.9g of crude product of the cable-marlutide is obtained after being taken out and weighed, the HPLC purity is 65.4%, and the HPLC chromatogram is shown in figure 1.
EXAMPLE 20 preparation of crude Sodamantane peptide
The mixture ratio of the lysate is TFA, EDT, DMS, phenylsulfide, tis and H 2 O=85:5:3:4:2:1 (volume ratio), 10g of the full-protection peptide resin obtained in example 16 is added into 100mL of pyrolysis liquid at 15 ℃, the temperature is raised to 30 ℃, the stirring reaction is continued for 3 hours, then a sand core funnel is used for filtering, the filtered resin is washed by 30mL of TFA, the repeated operation is carried out twice, the filtrate is combined, the pressure is reduced, the volume of the filtrate is 30% of the original volume, then the concentrated solution is slowly added into pre-cooled 300mL of isobutyl ether, the mixture is centrifuged for 5 times after sedimentation overnight, 200mL of isobutyl ether is used for each time to obtain white solid powder, the white solid powder is dried by a vacuum drying box for 10 hours after 4 hours, and the crude product of the cable-marlutide is obtained after taking out and weighing 5.2g, HPLC purity 58.3% and HPLC chromatogram similar to FIG. 1.
EXAMPLE 21 preparation of crude Sodamantane peptide
The mixture ratio of the lysate is TFA to EDT to DMS to Tis to H2O=90:2:2:2:2:2 (volume ratio), 10g of the full-protection peptide resin obtained in the example 18 is added into 100mL of lysate at 15 ℃, the temperature is raised to 30 ℃, the reaction is continued for 3 hours, then a sand core funnel is used for filtering, the filtered resin is washed by 30mL of TFA, the filtrate is combined after repeated operation for two times, the filtrate volume is reduced to 30% of the original volume, then the concentrated solution is slowly added into pre-cooled 300mL of isobutyl ether, the mixture is settled overnight and centrifuged for 5 times, 200mL of isobutyl ether is used each time, white solid powder is obtained, after the mixture is dried by nitrogen for 4 hours, the mixture is dried by a vacuum drying box for 10 hours, 7.1g of a crude product of the cable-marlutide is obtained after being taken out and weighed, the HPLC purity is 71.1%, and the HPLC chromatogram is similar to that of FIG. 1.
EXAMPLE 22 preparation of Sodamantane spermidine
Dissolving 5.42g of crude Somar Lupeptide obtained in example 19 in acetonitrile water solution, using octadecyl bonded silica gel as stationary phase, and NaClO 4 And (3) carrying out HPLC gradient elution on the salt solution and acetonitrile which are flowing relative to the crude peptide solution of the somalupeptide, collecting the fraction of the somalupeptide, and removing part of the acetonitrile by rotary evaporation by using a rotary evaporator to obtain a primary purified solution of the somalupeptide. The primary purification liquid of the somalundum takes octadecyl bonded silica gel as a stationary phase, takes TFA aqueous solution and acetonitrile as a mobile phase for HPLC linear elution, the somalundum fraction is collected, acetonitrile and most of water are removed by rotary evaporation by a rotary evaporator, 2.16g of the somalundum essential peptide is obtained by freeze drying, the HPLC purity is 99.5%, the HPLC chromatogram is shown in figure 2, and the purification yield is 61.2%.

Claims (7)

1. A synthesis method of the somalundin is characterized in that:
full protection of S1-S2 fragment, S3-S4 fragment, S19-20 fragment was used: synthesizing Fmoc-Gly-resin, synthesizing the Fmoc-Gly-resin according to the peptide sequence from the C end to the N end of the main chain of the sorulon by a solid phase synthesis method, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Lys (AEEa-eeA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl-er) -OH, fmoc-Ala-OH Fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -Phe-OH, S3-S4 fragment, S1-S2 fragment, obtaining a cable-marlutide main chain peptide resin; obtaining the somalunin through pyrolysis and purification;
or full protection S1-S2 fragment, S3-S4 fragment and S18-20 fragment is adopted: synthesizing Fmoc-Gly-resin, synthesizing the Fmoc-Gly-resin according to the peptide sequence from the C end to the N end of the main chain of the sorulon by a solid phase synthesis method, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH, fmoc-Ala-OH Fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -Phe-OH, S3-S4 fragment, S1-S2 fragment, obtaining a cable-marlutide main chain peptide resin; obtaining the somalunin through pyrolysis and purification;
Or full protection S1-S2 fragment, S3-S4 fragment, fmoc-Lys (Dde) -OH: synthesizing Fmoc-Gly-resin, synthesizing the Fmoc-Gly-resin according to the peptide sequence from the C end to the N end of the main chain of the sorulon by a solid phase synthesis method, fmoc-Arg (Pbf) -OH, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys (Dde) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys (Dde) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH Fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -Phe-OH, S3-S4 fragment, S1-S2 fragment, obtaining a cable-marlutide main chain peptide resin; removing a side chain Dde protecting group of Lys20, and sequentially coupling side chain amino acid sequences Fmoc-AEEA-OH, fmoc-Glu (OH) -OtBu and octadecanedioic acid mono-tert-butyl ester to obtain the cable marlutide full-protection peptide resin; obtaining the somalunin through pyrolysis and purification;
wherein the full protection S1-S2 fragment is R1-His (R2) -Aib-OH, and the full protection S3-S4 fragment is R3-Glu (R4) -Gly-OH; wherein R1 is selected from: boc or Fmoc; r2 is selected from: trt or Boc; r3 is selected from Fmoc; r4 is selected from OtBu;
The fully protected S19-20 fragment is Fmoc-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH;
the fully protected S18-20 fragment is Fmoc-Ala-Ala-Lys (AEEA-AEEA-gamma-Glu (OtBu) -Octadecanedioic acid mono-tert-butyl ester) -OH.
2. The method for synthesizing the somalupeptide according to claim 1, wherein: the full-protection S1-S2 fragment is Boc-His (Trt) -Aib-OH, and the full-protection S3-S4 fragment is Fmoc-Glu (OtBu) -Gly-OH.
3. The method for synthesizing the somalupeptide according to claim 1, wherein: the removing solution of Fmoc-Lys (Dde) -OH side chain Dde protecting group is selected from DMF solution mixed by hydroxylamine hydrochloride and imidazole, NH 2 NH 2 DMF solution of (b).
4. A method of synthesizing somalundum according to claim 3, wherein: the mass ratio of hydroxylamine hydrochloride to imidazole is 1-3:1-3.
5. The method for synthesizing the somalundum according to claim 1, wherein: the resin is selected from 2-CTC resin and Wang-resin.
6. The method for synthesizing the somalundum according to claim 1, wherein: the cracking solution of the total protective peptide resin for cracking the somalundum is a mixed solution of TFA, EDT, DMS, phenylsulfide and TIS.
7. The method for synthesizing the somalundum according to claim 6, wherein: the volume ratio of TFA, EDT, DMS to phenyl sulfide to TIS in the pyrolysis liquid is 80-90:1-5:1-5:2-6:1-5.
CN201910358894.4A 2019-04-30 2019-04-30 Synthetic method of somalupeptide Active CN111944037B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910358894.4A CN111944037B (en) 2019-04-30 2019-04-30 Synthetic method of somalupeptide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910358894.4A CN111944037B (en) 2019-04-30 2019-04-30 Synthetic method of somalupeptide

Publications (2)

Publication Number Publication Date
CN111944037A CN111944037A (en) 2020-11-17
CN111944037B true CN111944037B (en) 2023-06-13

Family

ID=73335447

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910358894.4A Active CN111944037B (en) 2019-04-30 2019-04-30 Synthetic method of somalupeptide

Country Status (1)

Country Link
CN (1) CN111944037B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115197312B (en) * 2022-08-09 2023-06-09 奥锐特药业(天津)有限公司 Preparation method and intermediate of cable Ma Lutai

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104418949A (en) * 2013-08-22 2015-03-18 深圳翰宇药业股份有限公司 Preparation method of teduglutide
CN107960079A (en) * 2016-08-19 2018-04-24 深圳市健元医药科技有限公司 A kind of synthetic method of low racemization impurity Liraglutide
CN108359006A (en) * 2018-05-25 2018-08-03 扬子江药业集团四川海蓉药业有限公司 A kind of preparation method of Suo Malu peptides
CN109369798A (en) * 2018-12-25 2019-02-22 苏州天马医药集团天吉生物制药有限公司 A method of synthesis Suo Malu peptide

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104650219B (en) * 2015-02-15 2017-11-14 兰州大学 The method that fragment condensation prepares Liraglutide
CN105524143B (en) * 2016-03-10 2019-01-22 成都圣诺生物制药有限公司 A method of synthesis Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
CN106699871B (en) * 2016-12-27 2020-06-12 哈药集团技术中心 Preparation method of liraglutide
CN109180801B (en) * 2018-11-01 2021-08-13 汉肽生物医药集团有限公司 Method for synthesizing Somalutide
CN109456401B (en) * 2018-12-03 2019-06-25 成都诺和晟泰生物科技有限公司 A kind of synthetic method of Suo Malu peptide
CN109627317B (en) * 2019-02-01 2020-11-27 兰州大学 Method for preparing Somalutide by fragment condensation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104418949A (en) * 2013-08-22 2015-03-18 深圳翰宇药业股份有限公司 Preparation method of teduglutide
CN107960079A (en) * 2016-08-19 2018-04-24 深圳市健元医药科技有限公司 A kind of synthetic method of low racemization impurity Liraglutide
CN108359006A (en) * 2018-05-25 2018-08-03 扬子江药业集团四川海蓉药业有限公司 A kind of preparation method of Suo Malu peptides
CN109369798A (en) * 2018-12-25 2019-02-22 苏州天马医药集团天吉生物制药有限公司 A method of synthesis Suo Malu peptide

Also Published As

Publication number Publication date
CN111944037A (en) 2020-11-17

Similar Documents

Publication Publication Date Title
CN106699871B (en) Preparation method of liraglutide
KR101087859B1 (en) Insulinotropic peptide synthesis
CN113135991B (en) Method for preparing somaglutide
CN111944039A (en) Synthetic method of somaglutide
CN101463081B (en) GLP-1 derivative
CN108059666A (en) A kind of method that solid-liquid combination prepares Suo Malu peptides
CN110372785B (en) Synthesis method of Somalutide
CN112010961B (en) Solid-liquid synthesis method of somalupeptide
CN111732651B (en) Method for preparing Somalutide through continuous flow solid phase reaction
CN110317258A (en) A kind of novel polypeptide segment of Suo Malu peptide and preparation method thereof
CN110922470A (en) Preparation method of somaglutide
US20130030148A1 (en) Process for the synthesis of (aib8,35)hglp-1(7-36)-nh2
CN112679602B (en) Solid phase synthesis method of cable Ma Lutai
WO2021070202A1 (en) A method for preparing glp-1 analogue by solid-phase peptide synthesis
CN111944037B (en) Synthetic method of somalupeptide
CN111944038B (en) Synthetic method of somalupeptide
CN108070030B (en) Preparation method of loxenapeptide and analogue thereof
CN103214568A (en) Solid phase method of secretin
CN113637064B (en) Synthetic method of somalupeptide
CN110615836B (en) Solid-phase synthesis method of liraglutide
CN115677827A (en) Peptide compounds
CN110845600B (en) Method for preparing liraglutide
CN113135988B (en) Preparation method of thymosin beta 4
CN102558340B (en) Glucagon-like peptide (GLP)-1 derivative
CN112679600B (en) Method for preparing thymalfasin by solid-liquid phase combination

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant