CN116836258A - Solid phase synthesis method of liraglutide - Google Patents
Solid phase synthesis method of liraglutide Download PDFInfo
- Publication number
- CN116836258A CN116836258A CN202210297659.2A CN202210297659A CN116836258A CN 116836258 A CN116836258 A CN 116836258A CN 202210297659 A CN202210297659 A CN 202210297659A CN 116836258 A CN116836258 A CN 116836258A
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- CN
- China
- Prior art keywords
- fmoc
- glu
- otbu
- liraglutide
- wang resin
- 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.)
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- 108010019598 Liraglutide Proteins 0.000 title claims abstract description 56
- 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 title claims abstract description 54
- 229960002701 liraglutide Drugs 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000010532 solid phase synthesis reaction Methods 0.000 title claims abstract description 28
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- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000000746 purification Methods 0.000 claims abstract description 13
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 29
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- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 14
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- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 claims description 12
- MWRZFXOQMUTNRK-OAHLLOKOSA-N 9h-fluoren-9-ylmethyl n-[(2s)-1-[(2-methylpropan-2-yl)oxy]-3-oxopropan-2-yl]carbamate Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C=O)C3=CC=CC=C3C2=C1 MWRZFXOQMUTNRK-OAHLLOKOSA-N 0.000 claims description 10
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- DBTMQODRSDEGRZ-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-(2-oxoethyl)carbamate Chemical compound C1=CC=C2C(COC(=O)NCC=O)C3=CC=CC=C3C2=C1 DBTMQODRSDEGRZ-UHFFFAOYSA-N 0.000 claims description 9
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 claims description 8
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- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims 2
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- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- 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
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- 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
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- Life Sciences & Earth Sciences (AREA)
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- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a solid-phase synthesis method of liraglutide, belonging to the technical field of preparation methods and purification of polypeptide medicaments. The method comprises the following steps: firstly, 31 peptides of a liraglutide main chain are split into 5 fragments, the 5 fragments are synthesized one by adopting a solid-phase synthesis method by taking Wang-resin as an initial part in the presence of an activator system, and the sequences of the 5 fragments are respectively as follows: his-Ala-Glu-Gly, thr-Phe-Thr-Ser-Asp-Val-Ser, ser-Tyr-Leu-Glu-Gly, gln-Ala-Ala-Lys-Glu, phe-Ile-Ala-Trp-Leu-Val-Arg-Gly; then, the 5 fragments are sequentially connected to form a liraglutide main peptide chain structure. Wherein the 4 th fragment Lys side chain needs to be modified, fmoc-Glu-OtBu, palmitoyl chloride and carboxyl are sequentially coupled for activation, and the synthesis is carried out according to a liquid phase method. The invention has the advantages of less impurity, easy purification, high product yield, short synthesis time and low production cost.
Description
Technical Field
The invention belongs to the technical field of polypeptide medicine preparation methods and purification, and particularly relates to a solid-phase synthesis method of liraglutide.
Background
Liraglutide, called Liraglutide in English, its peptideThe sequence is as follows: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (N) ε -(N α -Palmitoyl)-Lγ-Glutamyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH。
The liraglutide is a glucagon-like peptide receptor agonist, and can play a good role in reducing blood sugar as a subcutaneous injection preparation.
The liraglutide of original research manufacturer is mainly prepared by biological methods such as genetic engineering, and has high production cost, great technical difficulty, long research and development period and no contribution to the industrialized production of the liraglutide. The traditional solid phase synthesis method of liraglutide has the defects of complicated steps, more waste liquid, high cost and the like.
In the solid phase synthesis process, as the number of peptide bonds increases, the terminal amino acids present a number of problems. His residues are prone to racemization during peptide formation, resulting in epimerized products. At the same time, ala 2 -and Thr 5 Easy loss in the synthesis of the whole liraglutide, very similar properties of the lost hetero peptide and the target peptide, and difficult separation. Based on the problems, the liraglutide is synthesized by a fragment method, the length of a single peptide chain is artificially reduced, the number of peptide bonds of each fragment is obviously reduced compared with that of single chains, and the occurrence of abnormal problems is effectively reduced. In the process of selecting fragments, we avoid the synthesis of a difficult site like Ser-Ser, split the fragments into two fragments, realize the connection between the fragments instead of direct connection, and improve the connection efficiency.
Disclosure of Invention
The invention aims to solve the problem of providing a solid-phase synthesis method of liraglutide, which has the advantages of less impurities, easy purification, high product yield, and reduced synthesis time and production cost.
In order to solve the technical problems, the invention adopts the following technical scheme: a solid phase synthesis method of liraglutide, comprising the following steps:
step one, preparing a lysine tripeptide fragment Fmoc-Lys- (Glu (N) by a liquid phase synthesis method α -Palmitoyl)-OtBu)-OH；
Step two, under the action of an activator environment, fmoc-Gly-Wang resin, fmoc-Ser (tBu) -Wang resin and Fmoc-Glu (OtBu) -Wang resin are prepared by respectively coupling Fmoc-Gly-OH, fmoc-Ser (tBu) -OH and Fmoc-Glu (OtBu) -OH from Wang resin serving as starting ends;
coupling the amino acids with N-end Fmoc protection and side chain protection in sequence according to the sequence of 5 fragments of liraglutide by a solid phase synthesis method, wherein the lysine tripeptide fragment adopts Fmoc-Lys- (Glu (N) α -Palmitoyl)-OtBu)-OH；
Step four, sequentially connecting 5 fragments according to the main chain sequence of the liraglutide;
and fifthly, cracking, purifying and freeze-drying to obtain the liraglutide.
In step one, the fragment Fmoc-Lys- (Glu (N) α The liquid phase synthesis method of-Palmitoyl) -OtBu) -OH is as follows: coupling n-hexadecanoic acid, HOSu and DCC to obtain Palmitoyl-OSu activated lipid, and then reacting with H-Glu-OtBu to obtain a dipeptide fragment Palmitoyl-Glu-OtBu; palmitonyl-Glu-OtBu, HOSu, DCC is coupled to yield Palmitonyl-Glu (OSu) -OtBu activated lipid, which is then reacted with Fmoc-Lys-OH to yield the lysine tripeptide fragment Fmoc-Lys- (Glu (N) α -Palmitoyl)-OtBu)-OH。
In the second step, the resin solid carrier adopts Wang resin, the activator system is selected from DIC, HOBt and DMF, and the Wang resin has a substitution degree of 0.2-0.4 mmol/g.
In step three, the solid phase synthesis method comprises the steps of:
1) Under the action of an activator environment, fmoc-Gly-Wang resin, fmoc-Ser (tBu) -Wang resin and Fmoc-Glu (OtBu) -Wang resin are prepared by respectively coupling Fmoc-Gly-OH, fmoc-Ser (tBu) -OH and Fmoc-Glu (OtBu) -OH from Wang resin as starting ends;
2) The volume ratio of the adopted components is 1:3, removing Fmoc protecting groups on the Fmoc-Gly-Wang resin, the Fmoc-Ser (tBu) -Wang resin and the Fmoc-Glu (OtBu) -Wang resin respectively by the deprotected solution composed of PIP and DMF to obtain H-Gly-Wang resin, H-Ser (tBu) -Wang resin and H-Glu (OtBu) -Wang resin;
3) Coupling the H-Gly-Wang resin, the H-Ser (tBu) -Wang resin and the H-Glu (OtBu) -Wang resin with Fmoc-protected and side chain-protected amino acids in the presence of a coupling agent system to give Fmoc-amino acid residue-Gly-Wang resin, fmoc-amino acid residue-Ser (tBu) -Wang resin and Fmoc-amino acid residue-Glu (OtBu) -Wang resin, respectively;
4) Repeating the above steps, and respectively coupling amino acids according to the peptide sequences of 5 fragments of liraglutide, wherein the lysine tripeptide fragment adopts Fmoc-Lys- (Glu (N) α Palmitoyl) -OtBu) -OH. The sequence of the 1 st fragment coupling amino acid is: 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; the sequence of the coupling amino acids of the 2 nd fragment is as follows: fmoc-Glu (OtBu) -OH, fmoc-Lys- (Glu (N) α -Palmitoyl) -OtBu) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH; the sequence of the 3 rd fragment coupling amino acids is: fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH; the sequence of the 4 th fragment coupling amino acid is: fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH, fmoc-Thr (tBu) -OH; the sequence of the 5 th fragment coupling amino acid is: fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH, boc-His (Boc) -OH.
In the fourth step, the synthesized 5 fragments are subjected to shearing-connection operation, and the sequence is 5- & gt 4- & gt 3- & gt 2- & gt 1, and the fragments are sequentially connected to form the liraglutide peptide chain structure. The shearing agent was a 1% TFA in DCM.
In step five, screening of the lysing agent:
TABLE 1
Table 1 shows: among the above cleavage agents, the most preferred cleavage agent is TFA: phSMe: TES: EDT: h 2 O (90:1:2:2:5), trifluoroacetic acid: anisole: triethylsilane: ethylene dithiol: the water is 90:1:2:2:5.
The purification process comprises the following steps: the crude liraglutide product cleaved from the resin is subjected to repeated washing with a solvent (preferably diethyl ether: acetonitrile=2:1) to perform preliminary purification, and part of the hetero peptide is removed.
Therefore, the invention firstly splits 31 peptides of the liraglutide main chain into 5 fragments, and in the presence of an activator system, the 5 fragments are synthesized by taking Wang-resin as an initial part and adopting a solid phase synthesis method one by one, and the sequences of the 5 fragments are respectively as follows: his-Ala-Glu-Gly, thr-Phe-Thr-Ser-Asp-Val-Ser, ser-Tyr-Leu-Glu-Gly, gln-Ala-Ala-Lys-Glu, phe-Ile-Ala-Trp-Leu-Val-Arg-Gly; then, the 5 fragments are sequentially connected to form a liraglutide main peptide chain structure. Wherein the 4 th fragment Lys side chain needs to be modified, fmoc-Glu-OtBu, palmitoyl chloride and carboxyl are sequentially coupled for activation, and the synthesis is carried out according to a liquid phase method.
Compared with the prior art, the invention has the beneficial effects that: in the synthesis process, a fragment method is adopted for synthesis, so that the loss of the hetero peptide caused by overlong peptide chain of individual amino acid is avoided; meanwhile, a plurality of fragments can be simultaneously carried out, so that the period of synthesizing liraglutide is shortened. In the purification process, diethyl ether/acetonitrile mixed solvent is used for pulping and purifying the crude liraglutide, so that part of hetero peptide in the crude liraglutide is effectively removed; greatly shortens the subsequent preparation and purification period of the liraglutide and reduces the production cost.
Detailed Description
The invention is further described below with reference to the following examples:
examples: the reagents and raw materials involved in the invention are as follows:
wang resin, 1-hydroxybenzotriazole (HOBt), N '-Diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), N-hydroxysuccinimide (HOSu), phenylsulfide, anisole, N' -Dimethylformamide (DMF), piperidine (PIP), phenol and ninhydrin, fmoc-amino acid (Fmoc-His (Boc) -OH, fmoc-Ala-OH, fmoc-Glu (OtBu) -OH, fmoc-Gly-OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH, fmoc-Ser (tBu) -OH, glu-Asp (OtBu) -OH, fmoc-Val-OH, fmoc-Tyr (tBu) -OH, fmoc-Leu-OH, fmoc-Gln (Trt) -OH, fmoc-Lys (Mtt) -OH, fmoc-LOH, fmoc-OH, fmoc-Phe-OH, fmoc-Val-OH, fmoc-Tyr (Trt) -OH, fmoc-LbOH.
(1) Synthesis of palmitoyl benzotriazole (R5): 10mmol of benzotriazole (R3) is dissolved in 100ml of methyl tertiary butyl ether at 20 ℃, 11mmol of triethylamine is added, the mixture is cooled in an ice bath after complete dissolution, 11mmol of palmitoyl chloride (R4) is slowly dripped into the mixture, the ice bath is removed after dripping, and the mixture is naturally warmed to room temperature and stirred for 2 hours. Filtering, evaporating the filtrate at 60 ℃ under reduced pressure, adding 50ml of acetone into the residual white solid, dissolving in water bath at 60 ℃, crystallizing in a refrigerator at 4 ℃ for 12 hours, filtering, washing the filter cake with 15ml of cold acetone x 2, and drying at room temperature under reduced pressure to obtain white solid R5.TLC [ developing reagent: petroleum ether: ethyl acetate (2:1) ] appears as a single spot.
The chemical equation of the above reaction is:
(2) Synthesis of palmitoyl-L-glutamic acid tert-butyl ester (R6): 12mmol of H-Glu-OtBu is dissolved in 200ml of DMF, 15mmol of triethylamine and 10mmol of R5 are added, stirring reaction is carried out for 16H, 0.2mol/L of 150ml of hydrochloric acid is added, the mixture is refrigerated and placed for 3H in a refrigerator, filtration is carried out, a filter cake is washed by 100ml of water, and the filter cake is dried under reduced pressure in a vacuum drying oven at 35 ℃ to obtain white solid R6.TLC [ developing reagent: dichloromethane: methanol (10:1) ] appears as a single spot.
The chemical equation of the above reaction is:
(3) Synthesis of N-palmitoyl-L-glutamic acid-5-succinimidyl ester tert-butyl ester (R7): 5mmol of R6 is dissolved in 300ml of THF, 6mmol of HOSu and 6mmol of DCC are added, the mixture is stirred magnetically for 12h after complete dissolution, the mixture is filtered after the reaction is finished, and the filtrate is evaporated to dryness under reduced pressure at 40 ℃ to obtain a white solid R7 crude product. TLC [ developing reagent: n-hexane: ethyl acetate (1:1) ] appears as a mixed spot.
The chemical equation of the above reaction is:
(4) Purification of N-palmitoyl-L-glutamic acid-5-succinimidyl ester tert-butyl ester (R7): the crude R7 product was crystallized from an acetone-n-hexane (1:4) mixture, filtered, and the filter cake was washed with n-hexane 15ml of 3 and dried under reduced pressure at room temperature to give R7 as a white solid (yield 92.03%). TLC [ developing reagent: n-hexane: ethyl acetate (1:1) ] appears as a single spot.
(5)Fmoc-Lys-(Glu(N α Synthesis of Palmitoyl) -OtBu) -OH: weighing 36.74g 0.1mol Fmoc-Lys-OH and 15.90g 0.15mol Na 2 CO 3 Dissolving in a mixed solution of 100ml water and 100ml THF, weighing 53.87g 0.1mol Palmitoyl-Glu (OSu) -OtBu, adding into 100ml THF, dropwise adding into the mixed solution, reacting at room temperature overnight, adjusting pH to 7 with 10% diluted hydrochloric acid, removing THF by rotary evaporation, adjusting pH to 2 to obtain a large amount of white precipitate, filtering, and vacuum drying at 35deg.C under reduced pressure to obtain Fmoc-Lys- (Glu (N) α -Palmitoyl) -OtBu) -OH with HPLC purity 92.14% in 87% yield.
(6) Synthesis of Fmoc-Gly-Wang resin: 10g of Wang resin having a substitution degree of 0.5mmol/g was weighed into a solid phase synthesis tube, washed 2 times with DMF and the resin was swollen with DMF for 30 minutes. 10mmol Fmoc-Gly-OH and 12.5mmol HOBt were weighed, dissolved in 50ml DMF, activated for 3 min by adding 12.5mmol DIC and added to the solid phase synthesis tube, and after 1h reaction, detection and judgment were made by ninhydrin (if the resin was colorless and transparent, the reaction was complete). After completion of the reaction, the resin was repeatedly washed with DMF for 6 times to obtain Fmoc-Gly-Wang resin, and the degree of substitution was determined to be 0.32mmol/g.
(7) Fmoc-Gly-Wang resin deprotection: 9.37g of 3mmol Fmoc-GIy-Wang resin was taken and placed in a 250ml polypeptide synthesis reactor, 150ml DMF was added, and after mixing, the resin was fully swollen by soaking for 30 min. Adding 20ml of a deprotection reagent PIP, and introducing nitrogen to fully mix for 45min; the mixture was washed with DMF 100 ml.times.9, a small amount of the resin was taken out and placed in a 0.5ml EP tube, and 20. Mu.l of 5% ninhydrin in absolute ethanol and 80. Mu.l of 80% phenol in absolute ethanol were added as ninhydrin detection reagent and heated in a boiling water bath for 5min. If the resin is mauve, the Fmoc protecting group is completely removed, and the next coupling reaction is carried out; if the resin is colorless or pale yellow, it is necessary to extend the deprotection time.
(8) Peptide chain extension: adding 12mmol Fmoc-Arg (Pbd) -OH and two condensing reagents HOBt and DIC (12 mmol each) into the mixing reactor, reacting at room temperature, detecting the reaction progress by using ninhydrin reagent, and if the condensation is complete, obtaining the resin which is colorless or pale yellow; in the case of mauve, it is necessary to lengthen the condensation time or repeat the condensation. After the condensation is complete, the resin is washed with DMF 100ml x 6 to perform Fmoc deprotection as per step (7), the deprotected complete and washed resin is subjected to the condensation of the next amino acid as per step (8), and the cycle is repeated until the coupling of the last amino acid is completed. After the peptide chain synthesis was completed, the resin was washed with absolute ethanol 8 times and then blow-dried with nitrogen.
Repeating the steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing the coupling of Fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH and Fmoc-Phe-OH according to the 5 th segment sequence of the liraglutide.
(9) Synthesis of Fmoc-Glu (OtBu) -Wang resin: 10g of Wang resin having a substitution degree of 0.5mmol/g was weighed into a solid phase synthesis tube, washed 2 times with DMF and the resin was swollen with DMF for 30 minutes. 10mmol Fmoc-Glu (OtBu) -OH and 12.5mmol HOBt were weighed, dissolved in 50ml DMF, activated for 3 min by adding 12.5mmol DIC and added to the solid phase synthesis tube, and after 1h reaction was judged by ninhydrin detection (complete reaction if the resin was colorless and transparent). After completion of the reaction, the resin was repeatedly washed with DMF for 6 times to obtain Fmoc-Glu (OtBu) -Wang resin, which was measured to have a substitution degree of 0.31mmol/g.
(10) Fmoc-Glu (OtBu) -Wang resin deprotection: 9.68g of 3mmol Fmoc-Glu (OtBu) -Wang resin was placed in a 250ml polypeptide synthesis reactor, 150ml DMF was added, and after mixing, the resin was fully swollen by soaking for 30 min. Adding 20ml of a deprotection reagent PIP, and introducing nitrogen to fully mix for 45min; the mixture was washed with DMF 100 ml.times.9, a small amount of the resin was taken out and placed in a 0.5ml EP tube, and 20. Mu.l of 5% ninhydrin in absolute ethanol and 80. Mu.l of 80% phenol in absolute ethanol were added as ninhydrin detection reagent and heated in a boiling water bath for 5min. If the resin is mauve, the Fmoc protecting group is completely removed, and the next coupling reaction is carried out; if the resin is colorless or pale yellow, it is necessary to extend the deprotection time.
(11) Peptide chain extension: to the above mixing reactor was added 12mmol Fmoc-Lys- (Glu (N) α -Palmitoyl) -OtBu) -OH and two condensing reagents HOBt, DIC (12 mmol each), at room temperature, detecting the progress of the reaction with ninhydrin reagent, if the condensation is complete, the resin is colorless or pale yellow; in the case of mauve, it is necessary to lengthen the condensation time or repeat the condensation. After the condensation is complete, the resin is subjected to Fmoc deprotection as per step (10) by washing with DMF 100ml x 6, the deprotected complete and washed resin is subjected to the condensation of the next amino acid as per step (11), and the cycle is repeated until the coupling of the last amino acid is completed. After the peptide chain synthesis was completed, the resin was washed with absolute ethanol 8 times and then blow-dried with nitrogen.
Repeating the steps of removing Fmoc protection and adding corresponding amino acid for coupling, and sequentially completing Fmoc-Glu (OtBu) -OH, fmoc-Lys- (Glu (N) according to the 4 th segment sequence of liraglutide α Coupling of-Palmitonyl) -OtBu-OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH.
(12) And (3) completing the sequence of the 3 rd segment according to the methods of (6), (7) and (8), and completing the coupling of Fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH and Fmoc-Ser (tBu) -OH.
(13) Synthesis of Fmoc-Ser (tBu) -Wang resin: 10g of Wang resin having a substitution degree of 0.5mmol/g was weighed into a solid phase synthesis tube, washed 2 times with DMF and the resin was swollen with DMF for 30 minutes. 10mmol Fmoc-Ser (tBu) -OH and 12.5mmol HOBt were weighed, dissolved in 50ml DMF, activated for 3 min by adding 12.5mmol DIC and added to the solid phase synthesis tube, and after 1h reaction was judged by ninhydrin detection (complete reaction if the resin was colorless and transparent). After completion of the reaction, the resin was repeatedly washed with DMF for 6 times to obtain Fmoc-Ser (tBu) -Wang resin, and the degree of substitution was determined to be 0.31mmol/g.
(14) Fmoc-Ser (tBu) -Wang resin deprotection: 9.68g of 3mmol Fmoc-Ser (tBu) -Wang resin was placed in a 250ml polypeptide synthesis reactor, 150ml DMF was added, and after mixing, the resin was fully swollen by soaking for 30 min. Adding 20ml of a deprotection reagent PIP, and introducing nitrogen to fully mix for 45min; the mixture was washed with DMF 100 ml.times.9, a small amount of the resin was taken out and placed in a 0.5ml EP tube, and 20. Mu.l of 5% ninhydrin in absolute ethanol and 80. Mu.l of 80% phenol in absolute ethanol were added as ninhydrin detection reagent and heated in a boiling water bath for 5min. If the resin is mauve, the Fmoc protecting group is completely removed, and the next coupling reaction is carried out; if the resin is colorless or pale yellow, it is necessary to extend the deprotection time.
(15) Peptide chain extension: adding 12mmol Fmoc-Val-OH and two condensing reagents HOBt and DIC (12 mmol each) into the mixing reactor, reacting at room temperature, detecting the reaction progress by using ninhydrin reagent, and if the condensation is complete, obtaining colorless or pale yellow resin; in the case of mauve, it is necessary to lengthen the condensation time or repeat the condensation. After the condensation is complete, the resin is subjected to Fmoc deprotection as per step (14) by washing with DMF 100ml x 6, the deprotected complete and washed resin is subjected to the condensation of the next amino acid as per step (15), and the cycle is repeated until the coupling of the last amino acid is completed. After the peptide chain synthesis was completed, the resin was washed with absolute ethanol 8 times and then blow-dried with nitrogen.
Repeating the steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing the coupling of Fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH and Fmoc-Thr (tBu) -OH according to the sequence of the 2 nd section of liraglutide.
(16) The sequence of paragraph 1 was completed according to the methods of (6), (7) and (8), whereby the coupling of Fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH and Fmoc-His (Boc) -OH was completed.
(17) 100g of fully protected 4 th fragment Wang resin is weighed, added into a 2L three-neck round bottom flask, 1L of split resin is reacted for 2 hours at room temperature by using a DCM solution with volume ratio of 1% TFA, filtered, the filtrate is concentrated under reduced pressure, the concentrated liquid is added into glacial diethyl ether to be precipitated for 1 hour, centrifuged and washed for 6 times, 300ml of diethyl ether and acetonitrile (2:1) solution are stirred for 2 hours and fully stirred and washed, suction filtration is carried out, and the filter cake is dried under reduced pressure in a vacuum drying oven at 35 ℃ to obtain 15.36g of 4 th fragment with purity of 96.3% and yield of 95.5%.
(18) 80g of the fully protected 5 th fragment resin is taken, 150ml of DMF is added, and after mixing, the resin is soaked for 30min to fully swell. Adding 20ml of a deprotection reagent PIP, and introducing nitrogen to fully mix for 45min; the mixture was washed with DMF 100 ml.times.9, a small amount of the resin was taken out and placed in a 0.5ml EP tube, and 20. Mu.l of 5% ninhydrin in absolute ethanol and 80. Mu.l of 80% phenol in absolute ethanol were added as ninhydrin detection reagent and heated in a boiling water bath for 5min. If the resin is mauve, the Fmoc protecting group is completely removed, and the next coupling reaction is carried out; if the resin is colorless or pale yellow, it is necessary to extend the deprotection time. Adding 12mmol of fragment 4 and two condensing reagents HOBt and DIC (12 mmol each) into a reactor, reacting at room temperature, detecting the reaction progress by using ninhydrin reagent, and if the condensation is complete, obtaining colorless or pale yellow resin; in the case of mauve, it is necessary to lengthen the condensation time or repeat the condensation.
(19) Repeating the cleavage-coupling operation, and sequentially connecting 5 fragments to obtain the full-protection liraglutide Wang resin.
(20) Preparation and purification of crude liraglutide peptides
100g of fully protected liraglutide Wang resin was weighed into a 2L three-necked round bottom flask and TFA: phSMe: TES: EDT: h 2 The volume ratio of O (90:1:2:2:5) is prepared into 1L of lysate, the lysate is added into the resin, the reaction is carried out for 2 hours at room temperature, filtration is carried out, the resin after the cleavage is washed with a small amount of TPA for 3 times, the filtrates are combined and concentrated, the concentrated liquid is added into glacial diethyl ether for precipitation for 1 hour, centrifugation and washing with anhydrous diethyl ether are carried out for 6 times, vacuum drying is carried out, 34.13g of liraglutide crude peptide is obtained, the HPLC purity is 65.26%, and the crude peptide yield is 52%.
30g of liraglutide crude peptide is stirred with 300ml of diethyl ether and acetonitrile (2:1) solution for 2h, fully stirred and washed, suction filtered, and a filter cake is dried under reduced pressure in a vacuum drying oven at 35 ℃ to obtain 22.87g of liraglutide crude peptide with HPLC purity of 88.23% and crude peptide yield of 57%.
(21) Refining the liraglutide crude peptide: weighing 22.87g of crude liraglutide, adding water, stirring, adjusting pH to 8.5 with ammonia water to dissolve completely, filtering the solution with 0.45 μm mixed microporous membrane, and purifying for use. Purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is reverse phase C18 with the size of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column with the size of 77mm and 250mm is 90ml/min, eluting by a gradient system, circularly sampling and purifying, sampling the crude product solution into the chromatographic column, starting mobile phase eluting, collecting main peaks, evaporating acetonitrile, and obtaining the liraglutide purified intermediate concentrated solution.
Filtering the liraglutide purified intermediate concentrated solution with 0.45 μm filter membrane for standby, changing salt with high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic column flow rate of chromatographic packing for purification is 10 μm reversed phase C18, 77mm x 250mm is 90ml/min (corresponding flow rate can be adjusted according to chromatographic columns of different specifications); adopting a gradient elution and cyclic loading method, loading in a chromatographic column, starting mobile phase elution, collecting a spectrum, observing the change of absorbance, collecting a salt-exchange main peak, analyzing the liquid phase to detect the purity, combining the salt-exchange main peak solutions, concentrating under reduced pressure to obtain an aqueous solution of liraglutide acetic acid, and freeze-drying to obtain Lu Taichun g of liraglutide with the total yield of 41%.
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by this patent.
Claims (8)
1. A solid phase synthesis method of liraglutide is characterized in that: the method comprises the following steps:
step one, preparing a lysine tripeptide fragment Fmoc-Lys- (Glu (N) by a liquid phase synthesis method α -Palmitoyl)-OtBu)-OH；
Step two, under the action of an activator environment, fmoc-Gly-Wang resin, fmoc-Ser (tBu) -Wang resin and Fmoc-Glu (OtBu) -Wang resin are prepared by respectively coupling Fmoc-Gly-OH, fmoc-Ser (tBu) -OH and Fmoc-Glu (OtBu) -OH from Wang resin serving as starting ends;
coupling the amino acids with N-end Fmoc protection and side chain protection in sequence according to the sequence of 5 fragments of liraglutide by a solid phase synthesis method, wherein the lysine tripeptide fragment adopts Fmoc-Lys- (Glu (N) α -Palmitoyl)-OtBu)-OH；
And step four, sequentially connecting 5 fragments according to the main chain sequence of the liraglutide.
And fifthly, cracking, purifying and freeze-drying to obtain the liraglutide.
2. The solid phase synthesis method of liraglutide according to claim 1, characterized in that: in step one, the fragment Fmoc-Lys- (Glu (N) α The liquid phase synthesis method of-Palmitoyl) -OtBu) -OH is as follows: coupling n-hexadecanoic acid, HOSu and DCC to obtain Palmitoyl-OSu activated lipid, and then reacting with H-Glu-OtBu to obtain a dipeptide fragment Palmitoyl-Glu-OtBu; palmitoyl-Glu-OtBu, HOSu and DCC are coupled to obtain Palmitoyl-Glu (OSu) -OtBu activated lipid, and then reacted with Fmoc-Lys-OH to obtain lysine tripeptide fragment Fmoc-Lys- (Glu (N) α -Palmitoyl)-OtBu)-OH。
3. The solid phase synthesis method of liraglutide according to claim 1, characterized in that: in the second step, the resin solid carrier adopts Wang resin, the activator system is selected from DIC, HOBt and DMF, and the Wang resin has a substitution degree of 0.2-0.4 mmol/g.
4. The solid phase synthesis method of liraglutide according to claim 1, characterized in that: in step three, the solid phase synthesis method comprises the steps of:
1) Under the action of an activator environment, fmoc-Gly-Wang resin, fmoc-Ser (tBu) -Wang resin and Fmoc-Glu (OtBu) -Wang resin are prepared by respectively coupling Fmoc-Gly-OH, fmoc-Ser (tBu) -OH and Fmoc-Glu (OtBu) -OH from Wang resin as starting ends;
2) The volume ratio of the adopted components is 1:3, removing Fmoc protecting groups on the Fmoc-Gly-Wang resin, the Fmoc-Ser (tBu) -Wang resin and the Fmoc-Glu (OtBu) -Wang resin respectively by the deprotected solution composed of PIP and DMF to obtain H-Gly-Wang resin, H-Ser (tBu) -Wang resin and H-Glu (OtBu) -Wang resin;
3) In the presence of a coupling agent system, coupling the H-Gly-Wang resin, the H-Ser (tBu) -Wang resin and the H-Glu (OtBu) -Wang resin with Fmoc-protected and side chain-protected amino acids to obtain Fmoc-amino acid-Wang resin;
4) Repeating the above steps, and respectively coupling amino acids according to the peptide sequences of 5 fragments of liraglutide, wherein the lysine tripeptide fragment adopts Fmoc-Lys- (Glu (N) α -Palmitoyl)-OtBu)-OH。
5. The solid phase synthesis method of liraglutide according to claim 1, characterized in that: in step four, the cleavage agent was 1% TFA in DCM, and the cleaved fragments were cleaved in diethyl ether: acetonitrile = 2:1 to give a solid which is then ligated with another fragment.
6. The solid phase synthesis method of liraglutide according to claim 1, characterized in that: in step four, the cleavage-ligation procedure was repeated until 5 fragments were sequentially ligated to obtain liraglutide-Wang resin.
7. The solid phase synthesis method of liraglutide according to claim 1, characterized in that: in step five, the cleavage agent is TFA: phSMe: TES: EDT: h 2 O＝90:1:2:2:5。
8. The solid phase synthesis method of liraglutide according to claim 1, characterized in that: in the fifth step, the crude liraglutide product obtained by cleavage from the resin is purified by diethyl ether: acetonitrile=2:1, and preliminary purification was performed to remove a part of the hetero peptide.
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