CN110078816A - A kind of preparation method of Suo Malu peptide - Google Patents
A kind of preparation method of Suo Malu peptide Download PDFInfo
- Publication number
- CN110078816A CN110078816A CN201910482593.2A CN201910482593A CN110078816A CN 110078816 A CN110078816 A CN 110078816A CN 201910482593 A CN201910482593 A CN 201910482593A CN 110078816 A CN110078816 A CN 110078816A
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- Prior art keywords
- otbu
- fmoc
- tbu
- aeea
- gly
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- HOZZVEPRYYCBTO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)-2-methylpropanoic acid Chemical compound C1=CC=C2C(COC(=O)NC(C)(C)C(O)=O)C3=CC=CC=C3C2=C1 HOZZVEPRYYCBTO-UHFFFAOYSA-N 0.000 description 1
- HXMVNCMPQGPRLN-UHFFFAOYSA-N 2-hydroxyputrescine Chemical compound NCCC(O)CN HXMVNCMPQGPRLN-UHFFFAOYSA-N 0.000 description 1
- ZHGNHOOVYPHPNJ-UHFFFAOYSA-N Amigdalin Chemical compound FC(F)(F)C(=O)OCC1OC(OCC2OC(OC(C#N)C3=CC=CC=C3)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C2OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C1OC(=O)C(F)(F)F ZHGNHOOVYPHPNJ-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 108010011459 Exenatide Proteins 0.000 description 1
- HTQBXNHDCUEHJF-XWLPCZSASA-N Exenatide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)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)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 HTQBXNHDCUEHJF-XWLPCZSASA-N 0.000 description 1
- 241001062009 Indigofera Species 0.000 description 1
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)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](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)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=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 description 1
- 108010019598 Liraglutide Proteins 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 1
- AHVYPIQETPWLSZ-UHFFFAOYSA-N N-methyl-pyrrolidine Natural products CN1CC=CC1 AHVYPIQETPWLSZ-UHFFFAOYSA-N 0.000 description 1
- 108700020479 Pancreatic hormone Proteins 0.000 description 1
- 102000052651 Pancreatic hormone Human genes 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 229960001519 exenatide Drugs 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000030136 gastric emptying Effects 0.000 description 1
- 230000002641 glycemic effect Effects 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000000859 incretin Substances 0.000 description 1
- MGXWVYUBJRZYPE-YUGYIWNOSA-N incretin Chemical class C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O)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=1C=CC(O)=CC=1)[C@@H](C)O)[C@@H](C)CC)C1=CC=C(O)C=C1 MGXWVYUBJRZYPE-YUGYIWNOSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229960002701 liraglutide Drugs 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
- 229960003105 metformin Drugs 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 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
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
- 230000003442 weekly effect Effects 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
-
- 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 present invention provides a kind of preparation methods of Suo Malu peptide, by using short peptide chain Fmoc-Thr13‑Ser14‑Asp15- OH lacks impurity caused by directly avoiding because of 14 site Ser difficulty reaction sites；Side chain Fmoc-AEEA-AEEA-OH and Octa (OtBu)-Glu (α-OtBu)-γ-COOH is coupled by using two-step method, solve the problems, such as that side chain segments indissoluble and the low caused yield of reactivity and purity are relatively low, substantially reduce Suo Malu peptide purification difficulty, improve product yield and purity, gained fine work purity is greater than 99.0%, the cost for preparing Suo Malu peptide is also reduced, there is extensive practical value and application prospect.
Description
Technical field
The invention belongs to polypeptide drugs preparation method technical fields, and in particular to a kind of preparation method of Suo Malu peptide.
Background technique
Suo Malu peptide is a kind of GLP-1 (glucagon-like peptide) analog, has a structure that His-Aib-Glu-
Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-
AEEA- γ-Glu-N α-Octa-OH)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with people
GLP-1 has 97% sequence homology.
People GLP-1 can be combined and be activated GLP-1 receptor.GLP-1 receptor is the target spot of natural GLP-1, and GLP-1 is a kind of
Endogenous gut incretin hormones, pancreatic beta cell concentration of glucose dependence can be promoted excreting insulin.With natural GLP-
Unlike 1, Suo Malu peptide pharmacokinetics in human body and pharmacodynamic characteristics be suitable for once a week to prescription
Case.After subcutaneous administrations, the mechanism of extended durations of action includes: to make to absorb the self association slowed down；In conjunction with albumin；
There is higher enzyme stability to DPP IV (DPP-IV) and neutral endopeptidase (NEP), to have longer blood plasma
Half-life period.
By it, specifically interaction mediates the activity of Suo Malu peptide between GLP-1 receptor, leads to cyclic adenosine monophosphate
(cAMP) increase.The secretion for the pattern stimuli insulin that Suo Malu peptide can be relied on concentration of glucose, while with glucose
The mode of concentration dependant reduces the secretion of excessively high glicentin.Therefore, when blood glucose rise, insulin secretion is stimulated,
Glicentin secretion simultaneously is suppressed.In contrast, in hypoglycemia, Suo Malu peptide can reduce insulin secretion, and not shadow
Ring the secretion of glicentin.The hypoglycemic mechanism of Suo Malu peptide further includes the mild prolonged gastric emptying time.Suo Malu peptide can lead to
It crosses mitigation hunger and Energy intaking loses weight and body fat.
Subcutaneous injection is primary weekly, and type 2 diabetic patient's blood glucose level can be made to greatly improve, and risk of hypoglycemia compared with
It is low.Meanwhile Suo Malu peptide can also be by reducing appetite and reducing food intake dose, induction weight-reducing.Compared with Liraglutide, rope
The fat of Ma Shandong peptide connects longer, and hydrophobicity is strong, but Suo Malu peptide is modified through the PEG of too short chain, and hydrophily greatly enhances.PEG
It can not only combine closely with albumin after modification, cover DPP-4 enzyme hydrolysis site, moreover it is possible to reduce renal excretion, biology can be extended
Half-life period reaches macrocyclic effect.Suo Malu peptide provides better glycemic control and weight subtracts compared with Exenatide
Gently.Suo Malu peptide has reduction blood glucose, loses weight, promotes a variety of effects such as pancreatic cell regeneration and cardiovascular system protection
It answers, potential applicability in clinical practice is wide.
This product controls blood glucose for adults with type 2 diabetes: being suitable for metformin alone or sulfonylurea drugs are maximum
Blood glucose still controls bad patient after tolerable dose treatment, with melbine or sulfonylurea drugs use in conjunction.
There are many report both at home and abroad about Suo Malu peptide preparation report, as Chinese patent CN201811590004.4 is provided
A kind of solid-phase synthesis, using fmoc-protected amino acid as monomer, successively connects amino using Wang resin as starting material one by one
Acid obtains linear Suo Malu peptide resin, is cracked using TFA and obtains crude product, is finally purified, is obtained using reversed-phased high performace liquid chromatographic
Get Suo Malu peptide, does not evade Thr13-Ser14-Asp15The peptide disappearance impurity of difficult fragment sequence；Chinese patent
CN201811466181.1 is synthesized using segment method, and segment is excessive, and amino acid starting material uses excessively, at high cost.
Above-mentioned preparation method has the following disadvantages, during preparing Suo Malu peptide, sequence Thr13-Ser14-Asp15Place by
In β-pleated sheet, amino acid condensation is difficult, a large amount of missing impurity occurs, in addition, main chain take off be coupled after Lys protecting group side chain AEEA and
It is excessive that there are steric hindrances when the unnatural amino acids such as Glu-OtBu, is condensed phenomena such as incomplete, these are not exclusively produced because being condensed
Raw impurity polarity and Suo Malu peptide is very close, brings difficulty to purifying crude, reduces product yield.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides a kind of preparation methods of Suo Malu peptide, it includes following step
It is rapid:
(1) Fmoc-Gly- resin is taken, by polypeptide solid-state reaction method, by Suo Malu Peptide C end to N-terminal sequence, in the resin
On successively coupling have the fmoc-protected amino acid of N-terminal to the 22nd Val, obtain following sequence peptide resin:
Fmoc-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-
Ala-Lys(R1)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-
Resin；
Wherein, one of R1 Alloc, Dde, Mtt or Mmt；
(2) after the Fmoc protecting group for removing the 22nd Val, it is coupled short peptide chain Fmoc-Thr (tBu)-Ser (tBu)-Asp
(OtBu)-OH, and by Suo Malu Peptide C end to N-terminal sequence, continuing coupling has the fmoc-protected amino acid of N-terminal to the 29th His
(Trt), following sequence peptide resin is obtained:
Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp
(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys
(R1)-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly- resin；
(3) after the protecting group R1 for removing the 12nd Lys, using two-step method in Lys side chain coupling short peptide chain Fmoc-AEEA-
AEEA-OH and Octa (OtBu)-Glu (α-OtBu)-γ-COOH；Wherein, it is first coupled short peptide chain Fmoc-AEEA-AEEA-OH, then
It is coupled short peptide chain Octa (OtBu)-Glu (α-OtBu)-γ-COOH；
Alternatively, using one-step method in Lys side chain coupling short peptide chain Octa (OtBu)-Glu (α-OtBu)-AEEA-AEEA-
OH；
Obtain Suo Malu peptide full guard peptide resin, structure are as follows:
Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp
(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys
(AEEA-AEEA-γ-Glu(α-OtBu)-Octa(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-
Arg (Pbf)-Gly-Arg (Pbf)-Gly- resin；
(4) Suo Malu peptide full guard peptide resin is after lysate cracks up to Suo Malu peptide crude product；
(5) the inverted high performance liquid chromatography purifying of Suo Malu peptide crude product, then salt is changed to get Suo Malu peptide fine work.
Further, in step (1), the Fmoc-Gly- resin is Fmoc-Gly-Wang or Fmoc-Gly-CTC；When
When for Fmoc-Gly-Wang, substitution degree is 0.15~0.45mmol/g, preferably 0.28mmol/g；When for Fmoc-Gly-CTC
When, substitution degree is 0.25~0.45mmol/g.
Further, in step (2), coupling short peptide chain Fmoc-Thr (tBu)-Ser (tBu)-Asp (the OtBu)-OH
Condensing agent be selected from Cl-HOBt/DIC, HOBt/DIC, PyBOP/Cl-HOBt/DIEA, PyBOP/HOAt/DIEA or COMU/
DIEA；It is described coupling short peptide chain Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH solvent be selected from DMF, DCM, NMP or
One of DMSO or a variety of.
Further, in step (2), the condensing agent is Cl-HOBt/DIC；The solvent is by DMF, DCM and DMSO group
At；
Wherein, the molar ratio of Cl-HOBt and DIC is 2:3~5, preferably 2:3 in the condensing agent；In the solvent
The volume ratio of DMF, DCM and DMSO are 2:1~2:1~2, preferably 2:1:1.
Further, in step (3), the method for the deprotection base R1 is as follows:
Work as R1When for Alloc, the morpholine of 5~10 equivalents or the phenylsilane of 5~10 equivalents are selected in protecting group removing, then plus
Enter the tetrakis triphenylphosphine palladium of 0.1~0.3 equivalent, selection DCM is solvent, elimination reaction 3 minutes, heavy after reaction solution is filtered
It operates 1 time again；
Work as R1When for Dde, the DMF solution for containing 2% hydrazine hydrate is selected, elimination reaction 3 minutes, after reaction solution is filtered, then
Repetitive operation 1 time；
Work as R1When for Mtt, the DCM solution containing 5%TFA is selected, elimination reaction 30 minutes, after reaction solution is filtered, then is weighed
It operates 1 time again；
Work as R1When for Mmt, the DCM solution containing 5%TFA is selected, elimination reaction 30 minutes, after reaction solution is filtered, then is weighed
It operates 1 time again.
Further, described to use two-step method in Lys side chain coupling short peptide chain Fmoc-AEEA-AEEA-OH in step (3)
When with Octa (OtBu)-Glu (α-OtBu)-γ-COOH, the condensing agent of coupling is selected from PyBOP/Cl-HOBt/DIEA, PyBOP/
HOAt/DIEA,COMU/DIEA；It is described to use one-step method in Lys side chain coupling short peptide chain Octa (OtBu)-Glu (α-OtBu)-
When AEEA-AEEA-OH, the condensing agent of coupling is selected from Cl-HOBt/DIC, HOBt/DIC, COMU/DIEA.
Further, described to use two-step method in Lys side chain coupling short peptide chain Fmoc-AEEA-AEEA-OH in step (3)
When with Octa (OtBu)-Glu (α-OtBu)-γ-COOH, the condensing agent of coupling is selected from PyBOP/HOAt/DIEA；Preferably, institute
The molar ratio for stating PyBOP, HOAt and DIEA is 2:2:3.
Further, in step (3), solvent selected by the two-step method and one-step method is selected from DMF, DCM, NMP or DMSO
One of or it is a variety of, be added following surfactant in solvent: the Qula of 1~3% (v/v) is logical or the sulphur of 10-15% (v/v)
Hydrofining.
Further, in step (4), the lysate is by TFA and thioanisole, methyl sulfide, phenol, 1,2- ethylene dithiol
One of alcohol, tri isopropyl silane and water or a variety of compositions；
Preferably, the lysate is made of TEA, phenol, 1,2- dithioglycol and tri isopropyl silane；Wherein TEA, benzene
The volume ratio of phenol, 1,2- dithioglycol and tri isopropyl silane is 90:5:2.5:2.5.
Further, described to change salt method are as follows: mobile phase A in step (5): 0.5-2% acetic acid/water solution (v/v)；Stream
Dynamic phase B: acetonitrile；
Chromatographic column: the chromatographic column of 10 μm of reverse phase C18,77mm*250mm；
Detection wavelength: 220nm；
Gradient elution: using the mixed liquor of A phase and B phase, first balancing 30-80min with the acetonitrile that volume ratio is 5-15%, then
The volume ratio of B phase is risen to 80% by 10% within 30-60min, and keeps 20-60min.
V/v represents the ratio of volume and volume in the present invention.
Polypeptide solid-state reaction method is method commonly used in the art, the protection ammonia for as reacting back in the present invention
After base acid-resin sloughs Fmoc protecting group, then with next protected amino acid coupling reaction.
Rx in the present invention in "-amino acid (Rx)-" bracket is the side chain protecting group of amino acid；" Fomc- in the present invention
Amino acid (Rx)-OH " is protected amino acid, and Fomc and OH are the blocking group of amino acid.
Compared with prior art, the present invention has an advantage that
(1) present invention synthesizes short peptide chain using segment method, uses short peptide chain Fmoc-Thr13-Ser14-Asp15- OH, directly
Caused by avoiding because of 14 site Ser difficulty reaction sites impurity is lacked, is conducive to remaining amino acid and continues to be coupled, mention significantly
The high purity and yield of Suo Malu peptide crude product, reduces synthesis cost.
(2) segment, but these methods are generally coupled using the coupling side chain of method one by one or directly in previous disclosed patent
All not can avoid segment indissoluble or reactivity it is low caused by yield and the relatively low problem of purity.The present invention is even using two-step method
Joining side chain, that is, the protected amino acid used is Fmoc-AEEA-AEEA-OH and Octa (OtBu)-Glu (α-OtBu)-γ-COOH,
Segment solubility problem is improved, the introducing of surfactant even more greatly improves reactivity, improves the receipts of finished product
Rate and purity improve the rate of recovery and lower difficulty of rear end purifying preparation.
To sum up, Suo Malu peptide preparation method of the present invention directly avoids Thr13-Ser14-Asp15And it is stranded at AEEA-AEEA two
Difficult condensing site single amino acid missing matter class impurity generates, and substantially reduces Suo Malu peptide purification difficulty, improves product receipts
Rate and purity, products obtained therefrom purity are greater than 99.0%, also reduce the cost for preparing Suo Malu peptide, have extensive practical value
And application prospect.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is the MS map of Suo Malu peptide prepared by the present invention.
Fig. 2 is the HPLC map of Suo Malu peptide prepared by the present invention.
Specific embodiment
The raw material that partially uses in the specific embodiment of the invention, equipment are known product, pass through purchase commercial product
It obtains.
Chinese corresponding to english abbreviation involved in the present invention is shown in Table 1:
Chinese corresponding to english abbreviation involved in 1 present invention of table
The preparation of embodiment 1, Suo Malu peptide of the present invention
Using the Fmoc-Gly-Wang that uniform substitution degree is 0.28mmol/g, (purchase, which knows scientific and technological new material from Xi'an indigo plant, to be had
Limit company, lot number UN104F08-190221) be initial resin carrier, by go Fmoc protect and coupling reaction, successively with table 2
Shown in protected amino acid coupling, be made Suo Malu peptide peptide resin.
The protected amino acid that 2 present invention of table uses
| Meet peptide sequence n= | Protected amino acid | Molecular weight |
| 2 | Fmoc-Arg(Pbf)-OH | 648 |
| 3 | Fmoc-Gly-OH | 297 |
| 4 | Fmoc-Arg(Pbf)-OH | 648 |
| 5 | Fmoc-Val-OH | 339 |
| 6 | Fmoc-Leu-OH | 353 |
| 7 | Fmoc-Trp(Boc)-OH | 526 |
| 8 | Fmoc-Ala-OH | 311 |
| 9 | Fmoc-Ile-OH | 353 |
| 10 | Fmoc-Phe-OH | 387 |
| 11 | Fmoc-Glu(OtBu)-OH | 425 |
| 12 | Fmoc-Lys(Alloc)-OH | 452 |
| 13 | Fmoc-Ala-OH | 311 |
| 14 | Fmoc-Ala-OH | 311 |
| 15 | Fmoc-Gln(Trt)-OH | 610 |
| 16 | Fmoc-Gly-OH | 297 |
| 17 | Fmoc-Glu(OtBu)-OH | 425 |
| 18 | Fmoc-Leu-OH | 353 |
| 19 | Fmoc-Tyr(tBu)-OH | 459 |
| 20 | Fmoc-Ser(tBu)-OH | 384 |
| 21 | Fmoc-Ser(tBu)-OH | 384 |
| 22 | Fmoc-Val-OH | 339 |
| 23 | Make segment Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH by oneself | 712 |
| 24 | Fmoc-Phe-OH | 387 |
| 25 | Fmoc-Thr(tBu)-OH | 397 |
| 26 | Fmoc-Gly-OH | 297 |
| 27 | Fmoc-Glu(OtBu)-OH | 425 |
| 28 | Fmoc-Aib-OH | 325 |
| 29 | Boc-His(Trt)-OH | 498 |
| 30 | Make segment Fmoc-AEEA-AEEA-OH by oneself | 531 |
| 31 | Make segment Octa (OtBu)-Glu- (α-OtBu)-OH by oneself | 556 |
It is specific the preparation method is as follows:
1, the preparation of segment is made by oneself
(1) preparation of Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH
A. it is swollen: the CTC Resin resin that 100g (90mmol) substitution degree is 0.9mmol/g (is bought from gill biochemistry
(Shanghai) Co., Ltd., lot number GLS190202-48101), 1500ml methylene chloride is added, after mixing 40min, filters out dichloromethane
Alkane, the resin are spare.
B. it substitutes resin: Fmoc-Asp (OtBu)-OH of 100mmol being dissolved in 1500ml methylene chloride, 300mmol is added
DIEA, solution is stirred at 0-10 DEG C activation 5min after, pour into the CTC resin that step A is obtained, in 20-25 DEG C of condition
After lower mixing 10min, the DIEA of 20ml is added, 50min is continuesd to mix.To after reaction, methanol 100ml be added, continues to mix
Close 30min.After reaction, it filters, resin is washed 5 times with methylene chloride, each 1500ml.Obtain Fmoc-Asp (OtBu)-
CTC。
C. it deprotects: 20% piperidines/DMF solution (v/v) 1000ml being added in Fmoc-Asp (OtBu)-CTC, in 20-
After mixing 5min under the conditions of 30 DEG C, drain；DMF1000ml is added, after mixing 5min, drains；20% piperidines/DMF solution is added
(v/v) 1000ml is drained after mixing 10min under the conditions of 20-30 DEG C；DMF1500ml is added, after mixing 5min, drains.Weight
It is multiplexed DMF to wash 8 times, each 1500ml mixes 5min every time.
D. it is coupled: sequentially adding Fmoc-Ser (tBu)-OH, 300mmolTBTU the and 300mmol HOBT of 300mmol, add
The DIEA of 400mmol is added in the DMF/DCM solution 1200ml for entering 3:1 under the conditions of 0-10 DEG C, activates 5min.It will activate above
Liquid is slowly added into reaction kettle, and 2h is mixed under the conditions of 20-25 DEG C.To after reaction, drain, DMF1500ml is added, mixes
After closing 5min, drain.Repetition is washed 6 times with DMF, and each 1500ml mixes 5min every time.Finally yin is detected as with ninhydrin
Property to get arrive Fmoc-Ser (tBu)-Asp (OtBu)-CTC.
E. it is coupled Fmoc-Thr (tBu)-OH: repeating above-mentioned C and D operation, by the Fmoc-Ser (tBu)-in above-mentioned D operation
OH is changed to Fmoc-Thr (tBu)-OH, additional amount 300mmol, remaining reagent and its ratio are constant to get arriving 181g's
Fmoc-Thr(tBu)-Ser(tBu)-Asp(OtBu)-CTC。
F. it cracks: 1000ml trifluoroethanol/DCM solution (v/v=1:3) is added, after being stirred to react 2h under the conditions of 25 DEG C,
Filtering, the DCM solution of filter cake trifluoroethanol wash 3 times, and merging filtrate is concentrated into residual volume about under the conditions of 30 DEG C
The concentrate of 200ml volume.In the tertiary ether of the first that the concentrate is poured slowly into freezing, after stirring 30min sufficiently analyses solid, mistake
Filter after filter residue washes 3 times with the tertiary ether of first, which is dried in vacuo to get 75g short peptide chain Fmoc-Thr (tBu)-Ser is arrived
(tBu)-Asp (OtBu)-OH, yield 94.50%, HPLC purity are 98.51%.
(2) preparation of Fmoc-AEEA-AEEA-OH
A. it is swollen: the CTC Resin resin that 100g (90mmol) substitution degree is 0.9mmol/g (is bought from gill biochemistry
(Shanghai) Co., Ltd., lot number GLS190202-48101) 1500ml methylene chloride is added, after mixing 40min, filter out dichloromethane
Alkane, the resin are spare.
B. it substitutes resin: the Fmoc-AEEA-OH of 100mmol is dissolved in 1500ml methylene chloride, remaining step and " (1)
In the preparation of Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH " step B operation it is identical to get arrive Fmoc-
AEEA-CTC。
C. it deprotects: being operated with step C in " preparation of (1) Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH "
It is identical.
D. it is coupled: sequentially adding Fmoc-AEEA-OH, 300mmolTBTU and 300mmol HOBT of 300mmol, remaining step
Suddenly identical with step D operation in " preparation of (1) Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH ".Finally use
Ninhydrin is detected as negative to get the Fmoc-AEEA-AEEA-CTC for arriving 155g.
E. it cracks: having been operated with step F in " preparation of (1) Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH "
It is exactly the same to get arrive 51g short peptide chain Fmoc-AEEA-AEEA-OH, yield 89.61%, HPLC purity be 99.13%.
(3) preparation of Octa (OtBu)-Glu- (α-OtBu)-OH
A. it is swollen: the CTC Resin resin that 100g (90mmol) substitution degree is 0.9mmol/g (is bought from gill biochemistry
(Shanghai) Co., Ltd., lot number GLS190202-48101) 1500ml methylene chloride is added, after mixing 40min, filter out dichloromethane
Alkane, the resin are spare.
B. it substitutes resin: Fmoc-Glu- (α-OtBu)-OH of 100mmol being dissolved in 1500ml methylene chloride, remaining step
It is identical with step B operation in " preparation of (1) Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH " to get arriving
Fmoc-Glu-(α-OtBu)-CTC。
C. it deprotects: being operated with step C in " preparation of (1) Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH "
It is identical.
D. be coupled: sequentially add Fmoc-Octa (OtBu)-OH of 300mmol, the Cl-HOBT of 300mmol and
400mmolDIC, remaining step are grasped with step D in " preparation of (1) Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH "
Make identical.It is finally detected as with ninhydrin negative to get Fmoc-Octa (the OtBu)-Glu- (α-OtBu)-for arriving 166g
CTC。
E. it cracks: having been operated with step F in " preparation of (1) Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH "
It is exactly the same to get Fmoc-Octa (OtBu)-Glu- (α-the OtBu)-OH for arriving 55g, yield 91.1%, HPLC purity is
98.97%.
2, the synthesis of Suo Malu peptide peptide resin
1)Fmoc-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-
Ala-Lys(R1)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-
The preparation of Wang (intermediate 1)
The Fmoc-Gly-Wang for taking the 0.28mmol/g of 40mmol, is all poured into reaction kettle, molten with 1500ml DCM
It drains after swollen mixing 15min, is carried out by following deprotection and COUPLING PROCEDURE:
A. it deprotects: 20% piperidines/DMF solution (v/v) 1000ml is added, after mixing 5min under the conditions of 20-30 DEG C, take out
It is dry, DMF1000ml is added, after mixing 5min, drains.20% piperidines/DMF solution (v/v) 1000ml is added, in 20-30 DEG C of item
After mixing 10min under part, drain.DMF1500ml is added, after mixing 5min, drains.It repeats to wash 8 times with DMF, every time
1500ml mixes 5min every time.
B. it is coupled: sequentially adding Fmoc-Arg (Pbf)-OH, 80mmolTBTU the and 80mmol HOBT of 80mmol, be added
The DIEA of 120mmol is added in the DMF/DCM solution 1200ml of 3:1 under the conditions of 0-10 DEG C, activates 5min.By the above activating fluid
It is slowly added into reaction kettle, 2h is mixed under the conditions of 20-25 DEG C.To after reaction, drain, DMF1500ml, mixing is added
After 5min, drain.Repetition is washed 6 times with DMF, and each 1500ml mixes 5min every time.It finally is detected as feminine gender with ninhydrin,
Obtain Fmoc-Arg (Pbf)-Gly-Wang.
C. by the coupling method of the deprotection method of above-mentioned a and b, according to main chain from C-terminal to N-terminal sequence, sequentially according to table 2,
It is successively coupled remaining protected amino acid respectively, i.e., since table 2 connects peptide sequence 3, is coupled to No. 22 Val one by one to get rope Ma is arrived
The Wang peptide resin of Shandong peptide intermediate A, it is spare.
2) short peptide chain Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH, the as preparation of intermediate 2 are accessed
By the Wang peptide resin of above-mentioned Suo Malu peptide intermediate A according to step a the method in " 1) preparation of intermediate 1 "
After deprotection operation, short peptide chain Fmoc-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH, the 80mmol of 80mmol are sequentially added
Cl-HOBt, be added DMF/DCM/DMSO (v:v:v=2:1:1) solution 1200ml, 120mmol is added under the conditions of 0-10 DEG C
DIC, activate 5min.The above activating fluid is slowly added into reaction kettle, 2h is mixed under the conditions of 20-25 DEG C.Wait react knot
Shu Hou is drained, and DMF 1500ml is added, and after mixing 5min, is drained.Repetition is washed 6 times, each 1500ml with DMF, is mixed every time
5min.Feminine gender finally, which is detected as, with ninhydrin obtains intermediate 2, it is spare.
3) preparation of main chain (intermediate 3)
By the coupling method of the deprotection method and steps b of step a in above-mentioned " 1) preparation of intermediate 1 ", according to main chain from C
End is successively coupled remaining protected amino acid according to 2 sequence of table to N-terminal sequence respectively, i.e., since table 2 connects peptide sequence 24, one by one
Be coupled to No. 29 His to get arrive Suo Malu peptide backbone (intermediate 3) Wang peptide resin, it is spare.
(4) side chain short peptide chain Fmoc-AEEA-AEEA-OH is accessed, intermediate 4 is prepared
A. Alloc is taken off
The DCM of 1500ml is added in intermediate 3, sequentially adds 150ml phenylsilane and 15g tetrakis triphenylphosphine palladium, in
After leading to nitrogen bubbling 30min under the conditions of 20-30 DEG C, drains, washed 1 time with the DCM of 1500ml.Add 150ml phenylsilane and
8g tetrakis triphenylphosphine palladium is drained, is washed 5 times with the DCM of 1500ml after leading to nitrogen bubbling 30min under the conditions of 20-30 DEG C,
Resin is spare.
B. side chain short peptide chain Fmoc-AEEA-AEEA-OH is accessed
After deprotection operation by step a in " 1) preparation of intermediate 1 ", the short peptide chain Fmoc- of 80mmol is sequentially added
The HOAt of the PyBOP of AEEA-AEEA-OH, 80mmol, 80mmol are dissolved in DMF/DCM/NMP (v:v:v=5:3:2) solution
1200ml, it is logical to be added the Qula that surfactant is 2% (v/v) in solution, is added 120mmol's under the conditions of 0-10 DEG C
DIEA activates 5min.The above activating fluid is slowly added into reaction kettle, 2h is mixed under the conditions of 20-25 DEG C.To the end of reacting
Afterwards, it drains, DMF1500ml is added, after mixing 5min, drain.Repetition is washed 6 times, each 1500ml with DMF, is mixed every time
5min.Feminine gender finally, which is detected as, with ninhydrin obtains intermediate 4, it is spare.
(5) side chain short peptide chain Fmoc-Octa (OtBu)-Glu- (α-OtBu)-OH is accessed
After deprotection operation of the intermediate 4 by step a in " 1) preparation of intermediate 1 ", the short of 80mmol is sequentially added
Peptide chain Fmoc-Octa (OtBu)-Glu- (α-OtBu)-OH, the HOAt of the PyBOP of 80mmol, 200mmol is dissolved in DMF/DCM/
NMP (v:v:v=5:3:2) solution 1200ml, it is logical to be added the Qula that surfactant is 2% (v/v) in solution, in 0-10 DEG C of item
The DIEA of 120mmol is added under part, activates 5min.The above activating fluid is slowly added into reaction kettle, under the conditions of 20-25 DEG C
Mix 2h.To after reaction, drain, DMF1500ml is added, after mixing 5min, drains.It repeats to wash 6 times with DMF, every time
1500ml mixes 5min every time.Finally feminine gender is detected as with ninhydrin.It is finally washed 5 times with methylene chloride, each 800ml；It washes
Finish, is washed twice with methanol, each 800ml；It is washed 2 times with methylene chloride again, each 800ml；3 times finally are washed with alcohol, every time
800ml is opened until resin is fully dispersed.
The resin is dried in vacuum oven to 4h under the conditions of 20-30 DEG C, until constant weight (it weighs twice in succession, error
Lower than the full guard peptide resin 324g for 1%), obtaining Wang resin.The peptide resin of Suo Malu peptide is obtained, it is spare.
3, the preparation of Suo Malu peptide crude product
The peptide resin of the above-mentioned Suo Malu peptide being prepared is taken, addition 3240ml volume ratio is TFA: phenol: 1,2- second two
Mercaptan: tri isopropyl silane=90:5:2.5:2.5 lytic reagent (10mL/ grams of resin of lytic reagent) stirs evenly, room temperature
It is stirred to react 3 hours, reaction mixture is filtered using sand core funnel, collects filtrate, and resin is washed 3 times with a small amount of TFA again, is merged
It is concentrated under reduced pressure after filtrate, anhydrous ether precipitating is added, then wash precipitating 3 times with anhydrous ether, drains to obtain off-white powder, as rope
Ma Shandong peptide crude product 174g, yield 98.71%, the HPLC purity of crude product are 73.35%.
4, the preparation of Suo Malu peptide fine work
A. the above-mentioned Suo Malu peptide crude product being prepared is taken, is added water and stirred, with ammonium hydroxide tune pH8.5 to being completely dissolved, solution
With 0.45 μm of mixing filtering with microporous membrane, purify spare；
It is purified using high performance liquid chromatography, the reverse phase C18 that purifying is 10 μm with chromatograph packing material, flow phase system is
The column flow rate of 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solution, 77mm*250mm is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, and starting mobile phase elution collects main peak and boils off acetonitrile
Afterwards, get Suo Malu peptide purification intermediate concentrate, content 45.9mg/mL, HPLC purity are 99.25%.
B. Suo Malu peptide purification intermediate concentrate is taken, it is spare with 0.45 μm of filter membrane filtration；
It carries out changing salt using high performance liquid chromatography:
Mobile phase A: 1% acetic acid/water solution (v/v)；Mobile phase B: acetonitrile；
Chromatographic column: the chromatographic column of 10 μm of reverse phase C18,77mm*250mm；
Detection wavelength: 220nm；
Gradient elution: eluant, eluent is the mixed liquor of A phase and B phase, first with 10% acetonitrile balance 60min, then by the body of B phase
Product ratio rises to 80% by 10% within 45min, and keeps 30min.
Column flow rate is 90mL/min (can adjust corresponding flow velocity according to the chromatographic column of different size)；Using gradient
It elutes, quadrat method in circulation is splined in chromatographic column, and starting mobile phase elution acquires map, observes the variation of trap, receives
Collection changes salt main peak and detects purity with analysis liquid phase, and salt main peak solution is changed in merging, is concentrated under reduced pressure, it is water-soluble to obtain Suo Malu peptide acetic acid
Liquid, freeze-drying, get Suo Malu peptide fine work 94.83g, total recovery 54.5%.
MS and HPLC detection is carried out to Suo Malu peptide fine work is prepared, gained MS schemes as indicated with 1, HPLC map such as Fig. 2
It is shown.
The Suo Malu peptide sterling molecular weight: 1029.29 ([M+4H]/4)；Purity: 99.41%；Maximum single contaminant
0.3%.
To sum up, Suo Malu peptide preparation method of the present invention directly avoids Thr13-Ser14-Asp15And it is stranded at AEEA-AEEA two
Difficult condensing site single amino acid missing matter class impurity generates, and substantially reduces Suo Malu peptide purification difficulty, improves product receipts
Rate and purity, products obtained therefrom purity are greater than 99.0%, also reduce the cost for preparing Suo Malu peptide, have extensive practical value
And application prospect.
Claims (10)
1. a kind of preparation method of Suo Malu peptide, it is characterised in that: it includes the following steps:
(1) take Fmoc-Gly- resin, by polypeptide solid-state reaction method, by Suo Malu Peptide C end to N-terminal sequence, over the resin according to
Secondary coupling has the fmoc-protected amino acid of N-terminal to the 22nd Val, obtains following sequence peptide resin:
Fmoc-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-
Lys (R1)-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly- tree
Rouge；
Wherein, one of R1 Alloc, Dde, Mtt or Mmt；
(2) after the Fmoc protecting group for removing the 22nd Val, it is coupled short peptide chain Fmoc-Thr (tBu)-Ser (tBu)-Asp
(OtBu)-OH, and by Suo Malu Peptide C end to N-terminal sequence, continuing coupling has the fmoc-protected amino acid of N-terminal to the 29th His
(Trt), following sequence peptide resin is obtained:
Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-
Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(R1)-Glu
(OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly- resin；
(3) after the protecting group R1 for removing the 12nd Lys, using two-step method in Lys side chain coupling short peptide chain Fmoc-AEEA-AEEA-
OH and Octa (OtBu)-Glu (α-OtBu)-γ-COOH；Wherein, it is first coupled short peptide chain Fmoc-AEEA-AEEA-OH, then is coupled
Short peptide chain Octa (OtBu)-Glu (α-OtBu)-γ-COOH；
Alternatively, using one-step method in Lys side chain coupling short peptide chain Octa (OtBu)-Glu (α-OtBu)-AEEA-AEEA-OH；
Obtain Suo Malu peptide full guard peptide resin, structure are as follows:
Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-
Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-
AEEA-γ-Glu(α-OtBu)-Octa(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg
(Pbf)-Gly-Arg (Pbf)-Gly- resin；
(4) Suo Malu peptide full guard peptide resin is after lysate cracks up to Suo Malu peptide crude product；
(5) the inverted high performance liquid chromatography purifying of Suo Malu peptide crude product, then salt is changed to get Suo Malu peptide fine work.
2. preparation method according to claim 1, it is characterised in that: in step (1), the Fmoc-Gly- resin is
Fmoc-Gly-Wang or Fmoc-Gly-CTC；When for Fmoc-Gly-Wang, substitution degree is 0.15~0.45mmol/g, preferably
For 0.28mmol/g；When for Fmoc-Gly-CTC, substitution degree is 0.25~0.45mmol/g.
3. preparation method according to claim 1, it is characterised in that: in step (2), the coupling short peptide chain Fmoc-Thr
(tBu) condensing agent of-Ser (tBu)-Asp (OtBu)-OH is selected from Cl-HOBt/DIC, HOBt/DIC, PyBOP/Cl-HOBt/
DIEA, PyBOP/HOAt/DIEA or COMU/DIEA；Coupling short peptide chain Fmoc-Thr (tBu)-Ser (the tBu)-Asp
(OtBu) solvent of-OH is selected from one of DMF, DCM, NMP or DMSO or a variety of.
4. preparation method according to claim 3, it is characterised in that: in step (2), the condensing agent is Cl-HOBt/
DIC；The solvent is made of DMF, DCM and DMSO；
Wherein, the molar ratio of Cl-HOBt and DIC is 2:3~5, preferably 2:3 in the condensing agent；DMF, DCM in the solvent
Volume ratio with DMSO is 2:1~2:1~2, preferably 2:1:1.
5. preparation method according to claim 1, it is characterised in that: in step (3), the method for the deprotection base R1
It is as follows:
Work as R1When for Alloc, the morpholine of 5~10 equivalents or the phenylsilane of 5~10 equivalents are selected in protecting group removing, add 0.1
The tetrakis triphenylphosphine palladium of~0.3 equivalent, selection DCM be solvent, elimination reaction 3 minutes, after reaction solution is filtered, repetitive operation
1 time；
Work as R1When for Dde, the DMF solution for containing 2% hydrazine hydrate is selected, elimination reaction 3 minutes, after reaction solution is filtered, repeats behaviour
Make 1 time；
Work as R1When for Mtt, the DCM solution containing 5%TFA is selected, elimination reaction 30 minutes, after reaction solution is filtered, repeats operation
1 time；
Work as R1When for Mmt, the DCM solution containing 5%TFA is selected, elimination reaction 30 minutes, after reaction solution is filtered, repeats operation
1 time.
6. preparation method according to claim 1, it is characterised in that: described to use two-step method in the side Lys in step (3)
When chain is coupled short peptide chain Fmoc-AEEA-AEEA-OH and Octa (OtBu)-Glu (α-OtBu)-γ-COOH, the condensing agent of coupling
Selected from PyBOP/Cl-HOBt/DIEA, PyBOP/HOAt/DIEA, COMU/DIEA；It is described to use one-step method in Lys side chain coupling
When short peptide chain Octa (OtBu)-Glu (α-OtBu)-AEEA-AEEA-OH, the condensing agent of coupling is selected from Cl-HOBt/DIC, HOBt/
DIC、COMU/DIEA。
7. preparation method according to claim 6, it is characterised in that: described to use two-step method in the side Lys in step (3)
When chain is coupled short peptide chain Fmoc-AEEA-AEEA-OH and Octa (OtBu)-Glu (α-OtBu)-γ-COOH, the condensing agent of coupling
Selected from PyBOP/HOAt/DIEA；Preferably, the molar ratio of described PyBOP, HOAt and DIEA are 2:2:3.
8. preparation method according to claim 1, it is characterised in that: in step (3), selected by the two-step method and one-step method
Solvent is selected from one of DMF, DCM, NMP or DMSO or a variety of, and following surfactant: 1~3% (v/ is added in solvent
V) Qula is led to or the potassium bisulfide of 10-15% (v/v).
9. preparation method according to claim 1, it is characterised in that: in step (4), the lysate is by TFA and benzene first
One of thioether, methyl sulfide, phenol, 1,2- dithioglycol, tri isopropyl silane and water or a variety of compositions；
Preferably, the lysate is made of TEA, phenol, 1,2- dithioglycol and tri isopropyl silane；Wherein TEA, phenol,
The volume ratio of 1,2- dithioglycol and tri isopropyl silane is 90:5:2.5:2.5.
10. preparation method according to claim 1, it is characterised in that: described to change salt method are as follows: mobile phase in step (5)
A:0.5-2% acetic acid/water solution (v/v)；Mobile phase B: acetonitrile；
Chromatographic column: the chromatographic column of 10 μm of reverse phase C18,77mm*250mm；
Detection wavelength: 220nm；
Gradient elution: using the mixed liquor of A phase and B phase, 30-80min first is balanced with the acetonitrile that volume ratio is 5-15%, then by B
The volume ratio of phase rises to 80% by 10% within 30-60min, and keeps 20-60min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910482593.2A CN110078816B (en) | 2019-06-04 | 2019-06-04 | Preparation method of somalupeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN110372785A (en) * | 2019-07-25 | 2019-10-25 | 成都诺和晟泰生物科技有限公司 | A kind of synthetic method of Suo Malu peptide |
| CN110922470A (en) * | 2019-12-26 | 2020-03-27 | 杭州肽佳生物科技有限公司 | Preparation method of somaglutide |
| CN111217901A (en) * | 2019-10-31 | 2020-06-02 | 成都圣诺生物制药有限公司 | Preparation method of Somalutide |
| CN113423723A (en) * | 2020-01-19 | 2021-09-21 | 深圳市健元医药科技有限公司 | Preparation method of somaglutide |
| CN113754753A (en) * | 2021-09-30 | 2021-12-07 | 深圳翰宇药业股份有限公司 | Synthetic method of somaglutide |
| CN114014925A (en) * | 2021-12-30 | 2022-02-08 | 浙江湃肽生物有限公司南京分公司 | Somaltulipide main peptide chain and preparation method thereof |
| CN114031680A (en) * | 2021-09-01 | 2022-02-11 | 浙江湃肽生物有限公司 | Somalulet sodium salt and preparation method and application thereof |
| CN114685645A (en) * | 2020-12-30 | 2022-07-01 | 深圳翰宇药业股份有限公司 | Synthetic method of somaglutide |
| WO2023012709A1 (en) * | 2021-08-05 | 2023-02-09 | Usv Private Limited | An improved process for fmoc synthesis of semaglutide |
| CN118344462A (en) * | 2024-06-18 | 2024-07-16 | 苏州易合医药有限公司 | Soxhlet Ma Lutai mutant, fusion protein and related products for pulmonary administration |
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| CN110922470A (en) * | 2019-12-26 | 2020-03-27 | 杭州肽佳生物科技有限公司 | Preparation method of somaglutide |
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| CN114685645A (en) * | 2020-12-30 | 2022-07-01 | 深圳翰宇药业股份有限公司 | Synthetic method of somaglutide |
| WO2023012709A1 (en) * | 2021-08-05 | 2023-02-09 | Usv Private Limited | An improved process for fmoc synthesis of semaglutide |
| CN114031680A (en) * | 2021-09-01 | 2022-02-11 | 浙江湃肽生物有限公司 | Somalulet sodium salt and preparation method and application thereof |
| CN114031680B (en) * | 2021-09-01 | 2024-03-26 | 浙江湃肽生物有限公司 | Sodamide sodium salt and preparation method and application thereof |
| CN113754753A (en) * | 2021-09-30 | 2021-12-07 | 深圳翰宇药业股份有限公司 | Synthetic method of somaglutide |
| CN114014925A (en) * | 2021-12-30 | 2022-02-08 | 浙江湃肽生物有限公司南京分公司 | Somaltulipide main peptide chain and preparation method thereof |
| CN118344462A (en) * | 2024-06-18 | 2024-07-16 | 苏州易合医药有限公司 | Soxhlet Ma Lutai mutant, fusion protein and related products for pulmonary administration |
| CN118344462B (en) * | 2024-06-18 | 2024-11-08 | 苏州易合医药有限公司 | Soxhlet Ma Lutai mutant, fusion protein and related products for pulmonary administration |
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