CN103980358B - A kind of method preparing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] - Google Patents

Abstract

The invention relates to a technology for synthesizing liraglutide, which solves the problems of long synthesis period, high cost, low yield and many impurities in the prior art. The specific steps of the present invention are: A) synthesizing the fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH through the liquid phase; Gly‑OH coupling to obtain Fmoc‑Gly‑resin; C) by solid-phase synthesis, sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the peptide sequence of liraglutide main chain, in which lysine tripeptide The fragment was cleaved with Fmoc‑Lys‑(Glu(N ^α ‑Palmitoyl)‑OtBu)‑OH; D), purified and lyophilized to obtain liraglutide. The invention provides a synthesis process of liraglutide with short synthesis period, low cost, high yield and suitable for large-scale production.

Description

A kind of method for preparing liraglutide

technical field

The present invention relates to a preparation method of polypeptide drugs, which is a synthetic long-acting type II diabetes drug-liraglutide with glucagon-like peptide-1 (GLP-1) receptor agonist method of preparation.

Background technique

Liraglutide, British name: Liraglutide, structural formula is as follows:

The peptide sequence is:

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-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH

Liraglutide is the first and currently only long-acting GLP-1 analog developed by Novo Nordisk, Denmark. Immunogenicity and other aspects are similar to GLP-1. The molecular structure of liraglutide is 97% homologous to GLP-1(7-37). The structural difference is that Lys ³⁴ is replaced by Arg, Lys ²⁶ is palmitoylated through glutamic acid, and the fatty acid side chain It can reversibly combine liraglutide with albumin in blood, prolong the action time of liraglutide, and enhance the resistance to DPP-4 enzyme degradation. The fatty acid side chain can also make liraglutide molecule in The injection site is self-cross-linked into a heptamer, thereby delaying its subcutaneous absorption, so that its action time can be as long as nearly 24 hours. It can be injected once a day and at any time, and the risk of hypoglycemia is small. In addition, this product can also reduce the secretion of glucagon in a glucose-dependent manner and delay gastric emptying.

Novo Nordisk's liraglutide is prepared by biological methods such as genetic engineering, which is technically difficult and high in production costs, which is not conducive to the large-scale production of liraglutide. US6268343B1 and US6458924B2 reported the solid-liquid synthesis of liraglutide, the intermediate GLP-1(7-37)-OH needs to be purified by reverse phase HPLC, and then combined with N ^α -Palmitoyl-Glu(OSu) under liquid phase conditions -OtBu reaction, this method requires two purifications, long synthesis period, many waste liquids, high cost, and is not conducive to large-scale production.

WO2013037266A1 discloses a preparation method of liraglutide, the specific steps are: through the Fmoc solid-phase synthesis method, sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the peptide sequence of the main chain of liraglutide, wherein Amino acid uses Fmoc-Lys (Alloc)-OH, removes Alloc, and couples Palmitoyl-Glu-Offiu to the amino group of the side chain of lysine by solid-phase synthesis, and the product is obtained after cleavage. Because this method uses tetrakis (triphenylphosphine) palladium to remove Alloc, not only the cost is relatively high, which is not conducive to large-scale production, but also the metal residues will cause the heavy metal content to exceed the standard, resulting in low product quality and content.

To sum up, the existing solid-phase synthesis process of liraglutide is not suitable for industrial production due to long synthesis period, high cost, low yield and many impurities.

The present inventors used the existing synthetic method to prepare liraglutide, and found that there were many synthetic steps, a long synthetic cycle, low purity and yield, and it was not suitable for industrial scale production. For this reason, the inventors studied the synthesis method of liraglutide, thereby obtaining the technical solution of the present invention.

Contents of the invention

The purpose of the present invention is to provide a solid phase synthesis method of liraglutide. The technical problems to be solved in the present invention are: long synthesis period, high cost, low yield, many impurities, and unsuitable for industrialized production.

The synthetic route of the present invention is as shown in Figure 1: at first by liquid phase synthesis lysine tripeptide fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH, secondly under the existence of activator system, The Fmoc-Gly-resin is obtained by coupling the resin solid-phase carrier and Fmoc-Gly-OH, and then by solid-phase synthesis, the amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled according to the peptide sequence of the main chain of liraglutide , wherein the lysine tripeptide fragment is Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH, finally cleaved, purified, and lyophilized to obtain liraglutide.

Some commonly used abbreviations in the present invention have the following meanings;

Fmoc: fluorenylmethoxycarbonyl

Fmoc-AA: fluorenylmethoxycarbonyl protected amino acid

DIC: N,N′-diisopropylcarbodiimide

DCC: N, N'-Dicyclohexylcarbodiimide

PyBOP : Benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate

HATU : 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

HOBt: 1-Hydroxybenzotriazole

HOSu: N-Hydroxysuccinimide

tBu: tert-butyl

Trt: Trityl

Boc : tert-butoxycarbonyl

Palmitoyl: Palmitoyl

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

Tyr: Tyrosine

Ile: Isoleucine

Gln: Glutamine

Asn: asparagine

Cys: cysteine

Pro: Proline

Leu: Leucine

Gly: Glycine

Arg: arginine

Val: Valine

Trp: Tryptophan

Ala : alanine

Phe: Phenylalanine

Glu: glutamic acid

Lys: lysine

Ser: serine

Asp: aspartic acid

Thr : Threonine

His: histidine

DMF: N,N'-Dimethylformamide

MeOH: Methanol

DCM: dichloromethane

NMP : N-Methylpyrrolidone

DMSO: dimethyl sulfoxide

TFA: Trifluoroacetic acid

EDT: Ethylenedithiol

Piperidine: Hexahydropyridine

DMAP: 4-Dimethylaminopyridine

DIEA : N,N′-Diisopropylethylamine

TMP: 2,4,6-collidine.

For this reason, the present invention provides a synthetic method of liraglutide, the steps of which are as follows:

Step 1, synthesizing the lysine tripeptide fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH by a liquid phase method;

Step 2, in the presence of an activator system, the Fmoc-Gly-resin is obtained by coupling the resin solid phase carrier and Fmoc-Gly-OH;

Step 3, by solid-phase synthesis, sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the peptide sequence of the main chain of liraglutide, wherein the lysine tripeptide fragment uses Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH;

Step 4, cleavage, purification, and freeze-drying to obtain liraglutide.

Wherein, in the solid phase synthesis method described in step 1, the liquid phase synthesis of the fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH is obtained by coupling n-hexadecanoic acid, HOSu, and DCC Palmitoyl-OSu activated lipid, then reacted with H-Glu-OtBu to obtain the dipeptide fragment Palmitoyl-Glu-OtBu; Palmitoyl-Glu-OtBu, HOSu, DCC were coupled to obtain Palmitoyl-Glu(OSu)-OtBu activated lipid, and then combined with Fmoc -Lys-OH reaction to obtain the lysine tripeptide fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH.

Wherein, in the solid-phase synthesis method described in step 2, the resin solid carrier adopts 2-CTC resin, the activator system is selected from DIEA, TMP or NMM, and the Fmoc-Gly-resin is 0.10 ~ 0.35mmol/g Degree of substitution of Fmoc-Gly-CTC resin.

Wherein, in the solid-phase synthesis method described in step 2, the resin solid carrier adopts Wang resin, the activator system is composed of DIC, HOBt and DMAP, and the Fmoc-Gly-King resin is 0.10 ~ 0.35mmol/g replacement Degree of Fmoc-Gly-King Resin.

Wherein, the solid-phase synthesis method described in step 3,

1) Remove the Fmoc protecting group on the Fmoc-Gly-resin with a deprotection solution composed of piperidine and DMF with a volume ratio of 1:4 to obtain H-Gly-resin;

2) In the presence of a coupling agent system, H-Gly-resin is coupled with Fmoc-protected and side-chain-protected arginine to obtain Fmoc-Arg(Pbf)-Gly-resin;

3) Repeat steps 1) and 2) to carry out coupling of amino acids in sequence according to the peptide sequence of the main chain of ^liraglutide . The amino acid sequence 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, Fmoc-Glu(OtBu)-OH, Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH, Fmoc-Ala-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-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, Fmoc- Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His(Trt)-OH;

The coupling agent system includes a condensing agent and a reaction solvent, the condensing agent is selected from DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA; the reaction solvent is selected from DMF, DCM, NMP, DMSO or their any combination in between.

The method of the present invention is obtained through screening, and the screening process is as follows:

1) Selection of molar ratio: H-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-resin: Fmoc-Lys-( The molar ratios of Glu(N ^α -Palmitoyl)-OtBu)-OH:HATU:HOBt:DIEA are: 1:3:3:3:3 and 1:5:5:5:5;

2) Selection of reaction temperature:

25 ^o C and 35 ^o C

3) Choice of reaction time:

The reaction times are: 2 hours and 3 hours.

Eight experimental conditions were proposed for this purpose:

Experimental condition 1: take 3.43g (1.0mmol) H-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-resin, 2.16 Add g (3.0 mmol) Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH, 0.41g (3.0 mmol) HOBt and 1.14g (3.0 mmol) HATU into 20ml DMF and stir to dissolve, cool to 0 ^o C , add 0.5ml (3.0 mmol) DIEA to the above solution, react at 25 ^o C for 2 hours, and then sequentially couple the remaining amino acids, the coupling amino acid sequence is: Fmoc-Ala-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-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, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His(Trt)-OH, cracked, purified, and lyophilized to obtain liraglutide refined peptide;

Experimental conditions 2-8, experimental operation as shown in experimental condition 1, different experimental conditions and their experimental results are as follows: Experimental conditions The molar ratio of temperature time total yield purity Experimental Condition 1 1:3:3:3 25°C 2 hours twenty three% 99.10% Experimental condition 2 1:5:5:5 25°C 2 hours 27% 99.11% Experimental condition 3 1:3:3:3 35°C 2 hours 27% 99.23% Experimental Condition 4 1:5:5:5 35°C 2 hours 31% 99.75% Experimental Condition 5 1:3:3:3 25°C 3 hours 28% 99.54% Experimental Condition 6 1:5:5:5 25°C 3 hours 29% 99.64% Experimental Condition 7 1:3:3:3 35°C 3 hours 28% 99.13% Experimental conditions 8 1:5:5:5 35°C 3 hours 26% 99.26%

The above results indicated that experimental condition 4 had the best purification effect.

Compared with the prior art, the method of the present invention has obvious advantages, and relevant comparative experiments are as follows: patent total yield purity The technology of the present invention 31% 99.75% WO2013037266A1 15.87% 99.17% US6458924B2 twenty two% NA

The beneficial effects of the present invention are: select fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH to directly synthesize liraglutide in solid phase, which solves the problems of long synthesis period, high cost and high purity in the prior art. Low, many impurities, not suitable for industrial production; the present invention provides a synthesis process of liraglutide with short synthesis period, low cost and high yield, which is suitable for large-scale production.

Description of drawings

Fig. 1 synthetic route of the present invention;

Fig. 2 HPLC spectrogram of lysine tripeptide fragment;

The HPLC spectrogram of Fig. 3 liraglutide crude peptide;

The HPLC spectrogram of Fig. 4 liraglutide fine peptide;

Fig. 5 Example 12 Liraglutide fine peptide mass spectrogram.

detailed description

The present invention is further illustrated by the following examples.

Embodiment one: the synthesis of Palmitoyl-OSu activated lipid

Weigh 256.42g n-hexadecanoic acid (1.0mol), add 138.10g HOSu (1.2mol) into 2000ml THF, add 247.56g DCC (1.2mol) under ice-water bath, react for 1 hour, warm up to room temperature for 3 hours, and react Liquid filtration, mother liquor spin-dried, dissolved in DCM, filtered, washed 3 times with saturated sodium bicarbonate, 2 times with pure water, back-extracted 2 times, combined organic phase, dried with anhydrous sodium carbonate, spin-dried, 0-5°C ice ethanol Recrystallize 3 times, filter, and dry the solid oil pump to obtain 314.62g Palmitoyl-OSu activated lipid, yield 89%.

Embodiment two: the synthesis of Palmitoyl-Glu-OtBu

Weigh 101.62g H-Glu-OtBu (0.5mol) and 79.50g Na ₂ CO ₃ (0.75mol) into the mixed solution of 500ml water and 500ml THF to dissolve, weigh 176.75g Palmitoyl-OSu (0.5mol) into 500ml THF, dissolved and added dropwise to the above mixed solution, reacted overnight at room temperature, adjusted the pH to 7 with 10% dilute hydrochloric acid, removed THF by rotary evaporation, and then adjusted the pH to 3. A large white precipitate was obtained which was filtered. The resulting white precipitate was recrystallized from 0-5°C ice-cold ethanol. The solid oil pump was dried to obtain 192.11g Palmitoyl-Glu-OtBu, yield 87%.

Example 3: Synthesis of Palmitoyl-Glu(OSu)-OtBu

Weigh 88.33g Palmitoyl-Glu-OtBu (0.2mol), add 27.62g HOSu (0.24mol) into 1000ml THF, add 49.51g DCC (0.24mol) under ice-water bath, react for 1 hour, warm up to room temperature for 3 hours, react Liquid filtration, mother liquor spin-dried, dissolved in DCM, filtered, washed 3 times with saturated sodium bicarbonate, 2 times with pure water, back-extracted 2 times, combined organic phase, dried with anhydrous sodium carbonate, spin-dried, 0-5°C ice ethanol Recrystallize 3 times, filter, and dry the solid oil pump to obtain 94.81g Palmitoyl-Glu(OSu)-OtBu activated fat, with a yield of 88%.

Example 4: Synthesis of Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH

Weigh 36.74g Fmoc-Lys-OH (0.1mol) and 15.90g Na ₂ CO ₃ (0.15mol) into the mixed solution of 100ml water and 100ml THF to dissolve, weigh 53.87g Palmitoyl-Glu(OSu)-OtBu ( 0.1mol) into 100ml THF, dissolved and added dropwise to the above mixed solution, reacted overnight at room temperature, adjusted the pH to 7 with 10% dilute hydrochloric acid, removed THF by rotary evaporation, and then adjusted the pH to 3. A large white precipitate was obtained which was filtered. The resulting white precipitate was recrystallized from 0-5°C ice-cold ethanol. The solid oil was pumped dry to obtain 67.24 g of Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH, the HPLC purity was 97.40%, and the yield was 85%.

Embodiment five: the synthesis of the Fmoc-Gly-CTC resin that the degree of substitution is 0.10mmol/g

Weigh 20g of 2-CTC resin with a degree of substitution of 0.4mmol/g, add it to the solid phase reaction column, wash it once with DMF, and swell the resin with DMF for 30 minutes, then take 13.37g Fmoc -Gly-OH was dissolved in DMF, activated by adding 7.5ml DIEA in an ice-water bath, and then added to the above-mentioned reaction column equipped with resin. After 2 hours of reaction, 100ml of anhydrous methanol was added to block for 1 hour. Wash 3 times with DMF and 3 times with DCM, block with anhydrous methanol for 30 minutes, shrink and dry with methanol to obtain 22.34 g of Fmoc-Gly-CTC resin, the detection degree of substitution is 0.10 mmol/g.

Embodiment six: the synthesis of the Fmoc-Gly-CTC resin that the degree of substitution is 0.25mmol/g

Weigh 10 g of 2-CTC resin with a degree of substitution of 0.95 mmol/g, add it to a solid phase reaction column, wash it once with DMF, and swell the resin with DMF for 30 minutes, then take 14.11 g of Fmoc -Gly-OH was dissolved in DMF, activated by adding 8.0ml DIEA in an ice-water bath, then added to the above-mentioned reaction column equipped with resin, reacted for 2 hours, and blocked by adding 100ml of anhydrous methanol for 1 hour. Wash 3 times with DMF and 3 times with DCM, block with anhydrous methanol for 30 minutes, shrink and dry with methanol to obtain Fmoc-Gly-CTC resin, the detection substitution degree is 0.25mmol/g.

Embodiment seven: the degree of substitution is the synthesis of Fmoc-Gly-king resin of 0.10mmol/g

Weigh 20 g of Wang resin with a degree of substitution of 0.45 mmol/g, add it to a solid phase reaction column, wash it once with DMF, and swell the resin with DMF for 30 minutes, then take 13.37 g of Fmoc-Gly -OH, 6.01g of HOBt were dissolved in DMF, activated by adding 6.0ml of DIC in an ice-water bath, then added to the above-mentioned reaction column equipped with resin, after 5 minutes, 2.75g of DMAP was added, reacted for 2 hours, washed 3 times with DMF, DCM Washed 3 times, capped with 100ml acetic anhydride/pyridine overnight, shrunk and dried with methanol to obtain Fmoc-Gly-King resin, and the detected substitution degree was 0.10mmol/g.

Embodiment eight: the degree of substitution is the synthesis of Fmoc-Gly-king resin of 0.25mmol/g

Weigh 20 g of Wang resin with a degree of substitution of 0.75 mmol/g, add it to a solid phase reaction column, wash it once with DMF, and swell the resin with DMF for 30 minutes, then take 22.28 g of Fmoc-Gly -OH, 10.13g HOBt were dissolved in DMF, activated by adding 8.0ml DIC in an ice-water bath, then added to the above-mentioned reaction column with resin, added 4.5g DMAP after 5 minutes, reacted for 2 hours, washed 3 times with DMF, DCM Washed 3 times, capped with 100ml acetic anhydride/pyridine overnight, shrunk and dried with methanol to obtain 22.54g Fmoc-Gly-King resin, the detection degree of substitution was 0.25mmol/g.

Example 9: Preparation of liraglutide CTC resin

Weigh 4.46g (1mmol) of Fmoc-Gly-CTC resin with a substitution degree of 0.10mmol/g, add it to a solid-phase reaction column, wash it once with DMF, and swell the Fmoc-Gly-CTC resin with DMF for 30 minutes, then wash it with DMF : The mixed solution of pyridine with a volume ratio of 4:1 was deprotected by Fmoc, and then washed 6 times with DMF, and 3.24g Fmoc-Arg(Pbf)-OH (5mmol) and 0.68g HOBt (5mmol) were added in a volume ratio of 1 : 1 mixed solution of DCM and DMF, add 0.8ml DIC (10mmol) under ice-water bath to activate, then add to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin method to detect and judge the reaction end point, if If the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, it means that the reaction is incomplete and needs to be reacted for another 1 hour. This judgment standard is applicable to the detection and judgment of the end point of the reaction by the ninhydrin method in the subsequent amino acid coupling. Repeat the above steps of removing Fmoc protection and adding corresponding amino acid coupling, and complete 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-(Glu(N ^α - Palmitoyl)-OtBu)-OH, Fmoc-Ala-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-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, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His( Trt)-OH coupling. When Fmoc-Leu-OH and Fmoc-Phe-OH are coupled, the solvent is replaced by: a mixed solution of DMSO and DMF with a volume ratio of 1:4 is selected; when Fmoc-Asp(OtBu)-OH is coupled, the coupling reagent is replaced by: PyBOP/HOBt/DIEA; when coupling with Boc-His(Trt)-OH, the coupling reagent is changed to: HATU/HOBt/DIEA. After the coupling is completed, the liraglutide CTC resin is washed 3 times with DMF and 3 times with DCM , washed 3 times with MeOH, 3 times with DCM, 3 times with MeOH, and dried to obtain 9.67g liraglutide CTC resin.

Example 10: Preparation of Liraglutide King Resin

Weigh 4.57g (1mmol) of Fmoc-Gly-King resin with a substitution degree of 0.10mmol/g, add it to a solid-phase reaction column, wash it once with DMF, swell the Fmoc-Gly-King resin with DMF for 30 minutes, and wash it with DMF : The mixed solution of pyridine with a volume ratio of 4:1 was deprotected by Fmoc, and then washed 6 times with DMF, and 3.24g Fmoc-Arg(Pbf)-OH (5mmol) and 0.68g HOBt (5mmol) were added in a volume ratio of 1 : 1 mixed solution of DCM and DMF, add 0.8ml DIC (10mmol) under ice-water bath to activate, then add to the above-mentioned reaction column equipped with resin, react at room temperature for 2 hours, use ninhydrin method to detect and judge the reaction end point, if If the resin is colorless and transparent, it means that the reaction is complete; if the resin develops color, it means that the reaction is incomplete and needs to be reacted for another 1 hour. This judgment standard is applicable to the detection and judgment of the end point of the reaction by the ninhydrin method in the subsequent amino acid coupling. Repeat the above steps of removing Fmoc protection and adding corresponding amino acid coupling, and complete 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-(Glu(N ^α - Palmitoyl)-OtBu)-OH, Fmoc-Ala-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-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, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His( Trt)-OH coupling.

When Fmoc-Leu-OH and Fmoc-Phe-OH are coupled, the solvent is replaced by: a mixed solution of DMSO and DMF with a volume ratio of 1:4 is selected; when Fmoc-Asp(OtBu)-OH is coupled, the coupling reagent is replaced by: PyBOP/HOBt/DIEA; when coupling with Boc-His(Trt)-OH, the coupling reagent was changed to: HATU/HOBt/DIEA. After coupling, the liraglutide CTC resin was washed 3 times with DMF and 3 times with DCM , washed 3 times with MeOH, 3 times with DCM, 3 times with MeOH, and dried to obtain 9.78 g of liraglutide king resin.

Example 11: Large-scale preparation of liraglutide king resin

Weigh 4570g (1mol) of Fmoc-Gly-King resin with a substitution degree of 0.10mmol/g, add it to a solid-phase reaction column, wash it once with DMF, and swell the Fmoc-Gly-King resin with DMF for 30 minutes, then use DMF: The mixed solution with a volume ratio of pyridine of 4:1 was deprotected by Fmoc, and then washed 6 times with DMF, and 3240g of Fmoc-Arg(Pbf)-OH (5mol) and 682g of HOBt (5mol) were weighed and added into a solution with a volume ratio of 1:1. The mixed solution of DCM and DMF is activated by adding 800ml DIC (5mol) in an ice-water bath, and then added to the above-mentioned reaction column equipped with resin. After reacting at room temperature for 2 hours, use the ninhydrin method to detect and judge the reaction end point. If the resin is colorless and transparent , it means that the reaction is complete; if the resin develops color, it means that the reaction is incomplete and needs to be reacted for another 1 hour. This judgment standard is applicable to the detection and judgment of the reaction end point by ninhydrin method in the subsequent amino acid coupling. Repeat the above steps of removing Fmoc protection and adding corresponding amino acid coupling, and complete 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-(Glu(N ^α - Palmitoyl)-OtBu)-OH, Fmoc-Ala-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-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, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His( Trt)-OH coupling. When Fmoc-Leu-OH and Fmoc-Phe-OH are coupled, the solvent is replaced by: a mixed solution of DMSO and DMF with a volume ratio of 1:4 is selected; when Fmoc-Asp(OtBu)-OH is coupled, the coupling reagent is replaced by: PyBOP/HOBt/DIEA; when coupling with Boc-His(Trt)-OH, the coupling reagent is changed to: HATU/HOBt/DIEA. After the coupling is completed, the liraglutide CTC resin is washed 3 times with DMF and 3 times with DCM , washed 3 times with MeOH, 3 times with DCM, 3 times with MeOH, and dried to obtain 9795g liraglutide king resin.

Example 12: Preparation of Liraglutide Crude Peptide

Weigh 100g of fully protected liraglutide CTC resin or liraglutide king resin, and add it to a 1000mL three-neck round bottom flask, according to TFA: thioanisole: anisole: EDT=90: 5: 3: Prepare 10L of lysate with a volume ratio of 2, add the lysate to the above resin, react at room temperature for 2 hours, filter, wash the cleaved resin with a small amount of TFA for 3 times, combine the filtrate, concentrate, and add the concentrated liquid to ice ether Precipitate for 1 hour, centrifuge, wash with anhydrous ether for 6 times, and dry in vacuo to obtain 34.13 g of crude liraglutide peptide with an HPLC purity of 83.03% and a crude peptide yield of 78%.

Example 13: Preparation of liraglutide fine peptide acetate

Weighed 3413g of liraglutide crude peptide and dissolved it in 30L of a mixed solution of 50% acetonitrile + 50% water, purified twice through C18 or C8 column, converted to salt, and freeze-dried to obtain the target product. Purification conditions for the first time: mobile phase: phase A: 0.1% TFA; phase B: acetonitrile, detection wavelength 220nm, collect the target peak fraction. Conditions for the second purification: mobile phase: phase A: 0.3% acetic acid; phase B: acetonitrile. The detection wavelength is 220nm, and the target peak fraction is collected. Salt conversion conditions: mobile phase: phase A: 20mM ammonium acetate-water solution; phase B: acetonitrile; detection wavelength 220nm. The target peak fractions were collected, concentrated by rotary evaporation, and lyophilized to obtain 11.24 g of liraglutide acetate refined peptide, with an HPLC purity of 99.75%, a total purification yield of 40%, and a total yield of 31%.

The above content is a further detailed description of the present invention in conjunction with specific modified embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, they can also make some simple deduction or replacement, which should be regarded as belonging to the insurance scope of the present invention.

Claims (6)

1. A synthetic method of liraglutide, the method steps are as follows: Step 1, synthesizing the lysine tripeptide fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH by a liquid phase method; Step 2, in the presence of an activator system, the resin solid phase carrier and Fmoc-Gly-OH are coupled by a solid phase synthesis method to obtain Fmoc-Gly-resin; Step 3, by solid-phase synthesis, sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the peptide sequence of the main chain of liraglutide, wherein the lysine tripeptide fragment uses Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH; Step 4, cleavage, purification, and freeze-drying to obtain liraglutide. 2. The method according to claim 1, characterized in that: Wherein, in the liquid phase synthesis method described in step 1, the liquid phase synthesis of the fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH is obtained by coupling n-hexadecanoic acid, HOSu and DCC Palmitoyl-OSu, then Palmitoyl-OSu and H-Glu-OtBu were reacted to obtain the dipeptide fragment Palmitoyl-Glu-OtBu; Palmitoyl-Glu-OtBu, HOSu and DCC were coupled to obtain Palmitoyl-Glu(OSu)-OtBu, then Palmitoyl-Glu (OSu)-OtBu reacted with Fmoc-Lys-OH to obtain the lysine tripeptide fragment Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)-OH. 3. The method according to claim 1, characterized in that: Wherein, in the solid-phase synthesis method described in step 2, the resin solid carrier adopts 2-CTC resin, the activator system is selected from DIEA, TMP or NMM, and the Fmoc-Gly-resin is 0.10 ~ 0.35mmol/g Degree of substitution of Fmoc-Gly-CTC resin. 4. The method according to claim 1, characterized in that: Wherein, in the solid-phase synthesis method described in step 2, the resin solid carrier adopts Wang resin, the activator system is composed of DIC, HOBt and DMAP, and the Fmoc-Gly-resin is 0.10 ~ 0.35mmol/g substitution degree Fmoc-Gly-King Resin. 5. The method according to claim 1, characterized in that: Among them, in the solid-phase synthesis method described in step 3, 1) use a deprotection solution composed of piperidine and DMF with a volume ratio of 1:4 to remove the Fmoc protecting group on the Fmoc-Gly-resin to obtain H-Gly- resin; 2) In the presence of a coupling agent system, H-Gly-resin is coupled with Fmoc-protected and side-chain-protected arginine to obtain Fmoc-Arg(Pbf)-Gly-resin; 3) Repeat steps 1) and 2), and perform amino acid coupling in sequence according to the peptide sequence of the main chain of liraglutide, in which the lysine tripeptide fragment uses Fmoc-Lys-(Glu(N ^α -Palmitoyl)-OtBu)- Oh. 6. The method according to claim 5, characterized in that: The coupling agent system includes a condensing agent and a reaction solvent, the condensing agent is selected from DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA; the reaction solvent is selected from DMF, DCM, NMP, DMSO or their any combination in between.