CN106699871B - Preparation method of liraglutide - Google Patents

Abstract

The invention discloses a preparation method of liraglutide, belonging to the technical field of medicines. The method comprises the following steps: 1) solid phase synthesis of 4 peptide fragments of liraglutide. Wherein the peptide fragment 1 is the amino acids 1-9 in the backbone sequence of liraglutide; the peptide fragment 2 is 10 th to 14 th amino acids in the backbone sequence of liraglutide; the peptide fragment 3 is amino acids 15-23 in the backbone sequence of liraglutide; the peptide fragment 4 is the 24 th to 31 th amino acids in the backbone sequence of liraglutide. 2) Stepwise coupling of the peptide fragments gives a fully protected backbone peptide of liraglutide. 3) And removing the protecting group of the lysine at the 20 th position, and completing the connection of a side chain to obtain the fully-protected liraglutide peptide resin. 4) And cracking, purifying and freeze-drying to obtain the liraglutide. The preparation method has the advantages of high production efficiency, stable process, high product purity and high yield, and is suitable for industrial production.

Description

Preparation method of liraglutide

Technical Field

The invention relates to a preparation method of liraglutide, belonging to the technical field of medicines.

Background

The liraglutide is developed by Novonide company, is an analogue of human glucagon-like polypeptide-1 (GLP-1), has 97 percent of homology with human natural GLP-1, and has GLP-1 receptor agonism. Has multiple effects of reducing blood sugar, reducing body mass, promoting islet cell regeneration and protecting cardiovascular system, and can be used in combination with other antidiabetic drugs for treating type II diabetes. Liraglutide was developed over 10 years, approved for marketing in the european union and united states respectively in 2009, month 7 and 2010, month 1, and in china in 2011, month 3. Norshanodle obtains liraglutide by replacing lysine 34 of the native GLP-1 molecule with arginine and adding a 16-carbon fatty acid side chain to lysine 26 based on its unique platform for fatty acid chain modification. Due to the existence of the fatty acid side chain, the reversible non-covalent bond combination of the drug and plasma albumin is increased, so that the drug can temporarily avoid the degradation effect of dipeptidyl peptidase-4 (DPP-4), and the drug is slowly released due to the formation of a reversible complex with albumin, so that the absorption and distribution processes in vivo are slowed, the half-life period is obviously prolonged compared with that of natural GLP-1, and the administration frequency reaches once a day. The latest LEADER study showed that liraglutide reduced the risk of cardiovascular death by 22%, which was the first GLP-1 drug to benefit from cardiovascular events.

The molecular formula of the liraglutide is C₁₇₂H₂₆₅N₄₃O₅₁Molecular weight is 3751.20, CAS number is 204656-20-2, and its structure is shown as follows:

NH₂-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-γ-Glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH

the production process developed by Novonide is to obtain liraglutide by gene recombination technology and yeast fermentation. Patent CN104592381 discloses a process for obtaining liraglutide by fermentation using escherichia coli as a carrier through a gene recombination technology. Generally, the technical difficulty of gene recombination is high, the process is complex, and the production cost is high. And the side chain of GLP-1(7-37) -OH obtained by fermentation is in an unprotected state, so impurities are easily generated in the reaction process of connecting the side chain, thereby leading to more difficult purification and more complex process.

With the maturation of the technology for preparing polypeptides by solid-phase synthesis methods, more and more liraglutide synthesis processes are developed. Patents CN102286092, CN103145828 and CN103980358, etc. report the preparation of liraglutide by solid phase synthesis method using Fmoc protection strategy and stepwise coupling. Because the sequence of liraglutide has 15 hydrophobic amino acids, the shrinkage of resin is serious in the coupling process, and deletion peptide is easy to generate, so that the yield is reduced. Meanwhile, the crude peptide has more impurities with similar properties to the product, and the purification is very difficult. Therefore, the method for preparing liraglutide with high yield and short period has important practical significance by controlling the generation of impurities in the synthesis process, reducing the times of product purification and providing the liraglutide with high yield.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a preparation method of liraglutide. The method is a fragment condensation method, has the advantages of high yield, short synthesis period, good stability and the like, and is suitable for large-scale industrial production.

The invention aims to provide a preparation method of liraglutide, which comprises the following steps:

1) solid phase synthesis of 4 peptide fragments of liraglutide. Wherein the peptide fragment 1 is the amino acids 1-9 in the backbone sequence of liraglutide; the peptide fragment 2 is 10 th to 14 th amino acids in the backbone sequence of liraglutide; the peptide fragment 3 is amino acids 15-23 in the backbone sequence of liraglutide; the peptide fragment 4 is the 24 th to 31 th amino acids in the backbone sequence of liraglutide.

2) Stepwise coupling of the peptide fragments gives a fully protected backbone peptide of liraglutide.

3) And removing the protecting group of the lysine at the 20 th position, and completing the connection of a side chain to obtain the fully-protected liraglutide peptide resin.

4) And cracking, purifying and freeze-drying to obtain the liraglutide.

The method specifically comprises the following steps:

1) adopting a solid phase synthesis method, taking resin as a carrier, and respectively synthesizing the resin of peptide fragments 1,2, 3 and 4 of the liraglutide under the action of a condensing agent and an activating agent; wherein the peptide fragment 1 is the amino acids 1-9 in the backbone sequence of liraglutide; the peptide fragment 2 is 10 th to 14 th amino acids in the backbone sequence of liraglutide; the peptide fragment 3 is amino acids 15-23 in the backbone sequence of liraglutide; the peptide fragment 4 is amino acids 24-31 in the backbone sequence of liraglutide;

2) respectively cracking the peptide fragments 1-3 from the resin under the action of a cracking solution to obtain fully protected peptide fragments 1-3; removing the amino protecting group on the peptide fragment 4 resin, and coupling the peptide fragment 3 with the peptide fragment 4 resin under the action of a condensing agent and an activating agent to obtain a peptide fragment 5 resin; removing an amino protecting group on the peptide fragment 5 resin, and coupling the peptide fragment 2 with the peptide fragment 5 resin under the action of a condensing agent and an activating agent to obtain a peptide fragment 6 resin; and removing the amino protecting group on the peptide fragment 6 resin, and coupling the peptide fragment 1 with the peptide fragment 6 resin under the action of a condensing agent and an activating agent to obtain the liraglutide main chain peptide resin.

3) And removing the lysine side chain protection of the 20 th site of the liraglutide main chain peptide resin, and connecting the side chains to obtain the liraglutide peptide resin.

4) Cracking the liraglutide peptide resin under the action of a cracking solution to obtain crude liraglutide; and purifying and freeze-drying the crude liraglutide to obtain the liraglutide.

Preferably, the resin used in the solid phase synthesis of the peptide fragment in step 1) is a Wang resin or a 2-chlorotrityl chloride resin, and more preferably a 2-chlorotrityl chloride resin. Wherein the substitution degree of the resin used is preferably 0.3 to 0.8mmol/g, more preferably 0.4 to 0.6 mmol/g.

Preferably, the combination of condensing agent and activating agent used in the solid phase synthesis of the peptide fragment in step 1) is a mixture of DIC and HOBt, a mixture of DIEA and TBTU or a mixture of DIEA and HATU, more preferably a mixture of DIEA and TBTU. The mol ratio of the condensing agent to the activating agent is 1.5-2.0: 1, the used solvent is DMF, and the Fmoc removal protective solution is a DMF solution with 20% piperidine by volume percentage.

Preferably, the lysis solution in step 2) is 1% by volume of TFA in DCM.

Preferably, the combination of the condensing agent and the activating agent selected in the coupling step 2) is a mixture of DIC and HOBt, or a mixture of DIEA and TBTU, more preferably a mixture of DIC and HOBt, and the molar ratio of the condensing agent to the activating agent is 1.0-1.5: 1.

Preferably, the reagent for removing the Dde protection of the lysine side chain in the step 3) is a mixture of hydrazine hydrate and DMF, and the volume ratio of the hydrazine hydrate to the DMF is 1 (10-13).

Preferably, the cleavage reagent in step 4) is TFA, EDT, PhSMe, TIS and H₂A mixture of O; TFA, EDT, PhSMe, TIS and H as described above₂The volume ratio of O is (80-85): (3-6): (3-5): 1-2): 2-4).

Preferably, the purification step in the step 4) comprises reversed phase high performance liquid chromatography purification and isoelectric point adjustment, precipitation and sedimentation.

Some preferred embodiments of the above method specifically comprise the steps of:

1) coupling Fmoc-Asp (Otbu) -OH and 2-chlorotrityl chloride resin with substitution degree of 0.4-0.6 mmol/g in the presence of a coupling reagent, and removing Fmoc protecting groups to obtain the Gly-resin carrier. Then DIEA/TBTU was added, coupling the following amino acids in sequence: 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 to give peptide fragment 1 resin BOC-His (trt) -Ala-Glu (Otbu) -Gly-Thr (tbu) -Phe-Thr (tbu) -Ser (tbu) -Asp (Otbu) -resin; coupling Fmoc-Leu-OH with 2-chlorotrityl chloride resin with the substitution degree of 0.4-0.6 mmol/g in the presence of a coupling reagent, and removing the Fmoc protecting group to obtain the Leu-resin carrier. Then DIEA/TBTU was added, coupling the following amino acids in sequence: Fmoc-Tyr (tbu) -OH, Fmoc-Ser (tbu) -OH, Fmoc-Val-OH to obtain peptide fragment 2 resin Fmoc-Val-Ser (tbu) -Tyr (tbu) -Leu-resin; coupling Fmoc-Ile-OH with 2-chlorotrityl chloride resin with the substitution degree of 0.4-0.6 mmol/g in the presence of a coupling reagent, and removing the Fmoc protecting group to obtain the Ile-resin carrier. Then DIEA/TBTU was added, coupling the following amino acids in sequence: Fmoc-Phe-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Lys (Dde) -OH, Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (Otbu) -OH to obtain peptide fragment 3 resin Fmoc-Glu (Otbu) -Gly-Gln (trt) -Ala-Ala-Lys (Dde) -Glu (Otbu) -Phe-Ile-resin; coupling Fmoc-Gly-OH with 2-chlorotrityl chloride resin with the substitution degree of 0.4-0.6 mmol/g in the presence of a coupling reagent, and removing the Fmoc protecting group to obtain the Gly-resin carrier. Then DIEA/TBTU was added, coupling the following amino acids in sequence: Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (BOC) -OH, Fmoc-Ala-OH to obtain peptide fragment 4 resin Fmoc-Ala-Trp (BOC) -Leu-Val-Arg (Pbf) -Gly-resin;

2) cleaving the peptide fragment 1,2, 3 resin using 1% by volume TFA in DCM as a cleaving reagent to obtain a fully protected peptide fragment 1, peptide fragment 2, peptide fragment 3, respectively; removing amino protecting group on the peptide fragment 4 resin using DMF solution with 20% piperidine by volume percentage, activating the peptide fragment 3 with mixture of DIC and HOBt, adding into DMF solution of the above resin, reacting to obtain peptide fragment 5 resin; removing amino protecting group on the peptide fragment 5 resin using DMF solution of piperidine 20% by volume, activating the peptide fragment 2 with mixture of DIC and HOBt, adding into the above resin in DMF solution, reacting to obtain peptide fragment 6 resin; removing amino protecting group on the peptide fragment 6 resin by using 20% piperidine by volume in DMF solution, activating the peptide fragment 1 by using a mixture of DIC and HOBt, adding the activated peptide fragment 1 into the DMF solution of the resin, and reacting to obtain liraglutide main chain peptide resin;

3) and removing Dde protection of lysine at the 20 th position of the liraglutide main chain peptide resin by using a mixed solution of hydrazine hydrate and DMF at the volume ratio of 1: 13. And coupling Fmoc-Glu-OtBu with lysine at the position 20, removing Fmoc protection, and adding palmitoyl chloride to complete side chain connection.

4) Cracking the liraglutide peptide resin by using a mixed solution of TFA, EDT, PhSMe, TIS and H2O in a volume ratio of 85:5:4:2:4 as a lysate to obtain crude liraglutide; and (3) after the crude liraglutide is purified by reversed-phase high performance liquid chromatography, adjusting the pH value of the solution to an isoelectric point, separating out a product, performing centrifugal sedimentation, and freeze-drying to obtain the liraglutide.

The English abbreviation used in this patent explains:

abbreviations and English	Explaining the meaning
		Fmoc	9-fluorene methoxycarbonyl
Boc	Tert-butyloxycarbonyl radical
		tBu	Tert-butyl radical
trt	Trityl radical
		Pbf	2,2,4,6, 7-pentamethylbenzofuran-5-sulfonyl
Dde	1- (4, 4-dimethyl-2, 6-dioxocyclohexylmethylene) -3-methylbutyl
		DMF	N, N-dimethylformamide
DIEA	N, N-diisopropylethylamine
		DIC	N, N-diisopropylcarbodiimide
DCM	Methylene dichloride
		TBTU	O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate
HOBt	1-hydroxybenzotriazoles
		HATU	2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate
TFA	Trifluoroacetic acid
		PhSMe	Phenylmethyl sulfide
EDT	1, 2-ethanedithiol
		PhOH	Phenol and its preparation
TIS	Tri-isopropyl silane

The invention has the beneficial effects that:

the invention provides a preparation method of liraglutide, which has high efficiency, low cost and higher yield and is suitable for industrial production by technical optimization, and the advantages of the preparation method are mainly reflected in that: 1) the process is stable, the purity of the prepared liraglutide can reach more than 99.5 percent, and the total yield can reach more than 40 percent; 2) the process of the invention can synthesize 4 polypeptide fragments simultaneously, effectively shorten the synthesis period and improve the production efficiency. 3) Because the peptide chain of the liraglutide is overlong and the hydrophobic amino acid is more, the peptide is easy to generate deletion peptide. The multi-fragment synthesis method adopted by the invention can reduce the generation of deletion peptide, the main impurities of the method are not deletion peptide but are uncoupled fragments, and the difference between the amino acid sequences of the impurities and the main components is more, so that the product is easier to purify. 4) The buffer solution after the crude peptide purification is desalted by adopting a method of adjusting the pH value to isoelectric point precipitation, compared with a method of desalting by adopting a chromatographic column, the method disclosed by the invention avoids the problem of silica gel adsorption, reduces the product loss and has higher yield.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

Example 1:

the embodiment provides a preparation method of liraglutide, which comprises the following steps:

1. peptide fragment preparation

1.1 preparation of peptide fragment 1

300g (0.36mol) of resin with a degree of substitution of 1.2mmol/g was weighed into a polypeptide synthesis reactor, 2LDCM was added to swell the resin for 30 minutes, and washed once with DCM. 119.3g of Fmoc-Asp (Otbu) -OH (0.29mol) and 69.8g of DIEA (0.54mol) were weighed out and dissolved in DCM, added to the above reactor, reacted for 1.5 hours, then drained and washed 1 time with DCM. DCM and methanol solution in a volume ratio of 10:1 were added, and the mixture was stirred and blocked for 30 minutes. The solvent was drained, washed 3 times with DCM, the resin was shrunk 3 times with methanol and dried under vacuum to give Fmoc-Asp (Otbu) -2-chloro-trityl resin, which was tested to have a degree of resin substitution of 0.45 mmol/g.

360g of Fmoc-Asp (Otbu) -2-chloro-trityl resin was weighed, and the Fmoc protecting group was removed with 20% piperidine in DMF for 10 minutes each time for a total of two times, followed by 6 washes with DMF to give H-Asp (Otbu) -2-chloro-trityl resin.

Weighing Fmoc-Ser (tbu) -OH (2eq), TBTU (2eq) and DIEA (2eq), dissolving in DMF, stirring and activating for 5 min, adding into a reactor, adding DIEA (1eq) into the reactor, and reacting for 2 h at room temperature (the reaction end point is detected by the indetrione method). The solvent was drained and washed twice with DMF to give Fmoc-Fmoc-Ser (tbu) -Asp (Otbu) -2-chloro-trityl resin.

The above steps of 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 were repeated in this order, washed 3 times with DMF, washed 3 times with DCM, contracted 3 times with methanol, and dried in vacuo to give a resin of the fully protected peptide fragment 1.

Cleavage of peptide fragment 1 resin with 1% by volume TFA in DCM for 1 hour, resin removal by filtration, rotary evaporation of the filtrate to 1/4 of the original volume, followed by addition to 10L of ether to precipitate a white solid, collection of the precipitate by filtration, washing 5 times with anhydrous ether, and drying in vacuo to give 256.3g of peptide fragment 1: BOC-His (trt) -Ala-Glu (Otbu) -Gly-Thr (tbu) -Phe-Thr (tbu) -Ser (tbu) -Asp (Otbu), purity 95.2% and yield 94.8%.

1.2 preparation of peptide fragment 2

300g (0.36mol) of resin with a degree of substitution of 1.2mmol/g was weighed into a polypeptide synthesis reactor, 2LDCM was added to swell the resin for 30 minutes, and washed once with DCM. 102.5g of Fmoc-Leu-OH (0.29mol) and 69.8g of DIEA (0.54mol) were weighed out and dissolved in DCM, added to the above reactor, reacted for 1.5 hours, drained and washed 1 time with DCM. DCM and methanol solution in a volume ratio of 10:1 were added, and the mixture was stirred and blocked for 30 minutes. The solvent was drained, washed with DCM 3 times, the resin was shrunk with methanol 3 times, and dried under vacuum to give Fmoc-Leu-2-chloro-trityl resin, which was found to have a degree of substitution of 0.50 mmol/g.

360g of Fmoc-Leu-2-chloro-trityl resin was weighed, and the Fmoc protecting group was removed with 20% piperidine in DMF for 10 minutes each time, followed by 6 washes with DMF to give Leu-2-chloro-trityl resin.

Fmoc-Tyr (tbu) -OH (2eq), TBTU (2eq) and DIEA (2eq) are weighed, dissolved in an appropriate amount of DMF, stirred and activated for 5 minutes, added into a reactor, and then DIEA (1eq) is added into the reactor and reacted for 2 hours at room temperature (the reaction endpoint is detected by the indetrione method). The solvent was drained and washed twice with DMF to give Fmoc-Tyr (tbu) -Leu-2-chloro-trityl resin.

Repeating the steps of Fmoc removal and amino acid coupling, sequentially coupling Fmoc-Ser (tbu) -OH, Fmoc-Ser (tbu) -OH and Fmoc-Val-OH, washing with DMF 3 times, washing with DCM 3 times, shrinking with methanol 3 times, and vacuum drying to obtain the fully protected peptide fragment 2 resin.

Cleavage of peptide fragment 2 resin with 1% by volume TFA in DCM for 1 h, resin removal by filtration, rotary evaporation of the filtrate to 1/4 of the original volume, followed by addition to 5L of ether to precipitate a white solid, collection of the precipitate by filtration, washing 5 times with anhydrous ether, and drying in vacuo to give 131.8g of peptide fragment 2: Fmoc-Val-Ser (tbu) -Tyr (tbu) -Leu with purity of 96.8% and yield of 95.9%.

1.3 preparation of peptide fragment 3

300g (0.36mol) of resin with a degree of substitution of 1.2mmol/g was weighed into a polypeptide synthesis reactor, 2LDCM was added to swell the resin for 30 minutes, and washed once with DCM. 102.5g of Fmoc-Ile-OH (0.29mol) and 69.8g of DIEA (0.54mol) were weighed out and dissolved in DCM, added to the above reactor, reacted for 1.5 hours, then drained and washed 1 time with DCM. DCM and methanol solution in a volume ratio of 10:1 were added, and the mixture was stirred and blocked for 30 minutes. The solvent was drained, washed with DCM 3 times, the resin was shrunk with methanol 3 times, and dried under vacuum to give Fmoc-Ile-2-chloro-trityl resin, which was tested to have a degree of substitution of 0.49 mmol/g.

360g of Fmoc-Ile-2-chloro-trityl resin was weighed, and the Fmoc protecting group was removed with 20% piperidine in DMF for 10 minutes each time, followed by 6 washes with DMF to give Ile-2-chloro-trityl resin.

Fmoc-Phe-OH (2eq), TBTU (2eq) and DIEA (2eq) were weighed, dissolved in an appropriate amount of DMF, stirred and activated for 5 minutes, added to the reactor, followed by addition of DIEA (1eq) to the reactor and reaction at room temperature for 2 hours (end of reaction was detected by the ninhydrin method). The solvent was drained and washed twice with DMF to give Fmoc-Phe-Ile-2-chloro-trityl resin.

The above steps of Fmoc-Glu (otbu) -OH, Fmoc-Lys (Dde) -OH, Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (otbu) -OH were repeated in this order, followed by 3-time DMF washing, 3-time DCM washing, 3-time methanol contraction, and vacuum drying to obtain a fully protected peptide fragment 3 resin.

Cleavage of peptide fragment 3 resin with 1% by volume TFA in DCM for 1 h, resin removal by filtration, rotary evaporation of the filtrate to 1/4 of the original volume, followed by addition to 10L of ether to precipitate a white solid, collection of the precipitate by filtration, washing 5 times with anhydrous ether, and drying in vacuo to give 304.6g of peptide fragment 3: Fmoc-Glu (Otbu) -Gly-Gln (trt) -Ala-Ala-Lys (Dde) -Glu (Otbu) -Phe-Ile, purity 95.7%, yield 95.3%.

1.4 preparation of peptide fragment 4

300g (0.36mol) of resin with a degree of substitution of 1.2mmol/g was weighed into a polypeptide synthesis reactor, 2LDCM was added to swell the resin for 30 minutes, and washed once with DCM. 86.2g Fmoc-Gly-OH (0.29mol) and 69.8g DIEA (0.54mol) were weighed out and dissolved in DCM, added to the above reactor, reacted for 1.5 h, drained and washed 1 time with DCM. DCM and methanol solution in a volume ratio of 10:1 were added, and the mixture was stirred and blocked for 30 minutes. The solvent was drained, washed with DCM 3 times, the resin was shrunk with methanol 3 times, and dried under vacuum to give Fmoc-Gly-2-chloro-trityl resin, which was found to have a degree of substitution of 0.54 mmol/g.

350g of Fmoc-Gly-2-chloro-trityl resin was weighed, and the Fmoc protecting group was removed with 20% by volume of piperidine in DMF for 10 minutes twice, followed by washing with DMF 6 times to obtain Gly-2-chloro-trityl resin.

Fmoc-Arg (Pbf) -OH (2eq), TBTU (2eq) and DIEA (2eq) were weighed, dissolved in DMF and stirred for 5 min, and then added to the reactor, followed by addition of DIEA (1eq) to the reactor and reaction at room temperature for 2 h (end point of reaction was detected by the ninhydrin method). The solvent was drained and washed twice with DMF to give Fmoc-Arg (Pbf) -Gly-2-chloro-trityl resin.

Repeating the steps of Fmoc-removing and amino acid coupling, sequentially completing the coupling of Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (BOC) -OH and Fmoc-Ala-OH, washing 3 times with DMF, washing 3 times with DCM, shrinking 3 times with methanol, and vacuum drying to obtain the fully protected peptide fragment 4 resin.

2. Coupling of peptide fragments

Taking the fully protected peptide fragment 4 resin, adding 20% by volume piperidine in DMF to remove Fmoc protecting group for 10 minutes each time twice, followed by washing 6 times with DMF to obtain NH₂-Ala-Trp (BOC) -Leu-Val-Arg (Pbf) -Gly-resin. 98.8g of the peptide fragment 3(57mmol), 10.4g of HOBt (68mmol) were weighed out, dissolved in an appropriate amount of DMF, added with 8.6g of DIC (68mmol), stirred for activation for 5 minutes, and charged into a reactor to react at room temperature for 4 hours (end point of reaction detected by the ninhydrin method). The solvent was drained and washed twice with DMF to give the fully protected peptide fragment 5 resin.

The Fmoc protecting group was removed by addition of 20% by volume piperidine in DMF for 10 min twice, followed by 6 washes with DMF to give NH₂-Glu (Otbu) -Gly-Gln (trt) -Ala-Ala-Lys (Dde) -Glu (Otbu) -Phe-Ile-Ala-Trp (BOC) -Leu-Val-Arg (Pbf) -Gly-resin. 42.1g of the peptide fragment 2(57mmol), 10.4g of HOBt (68mmol) were weighed out, dissolved in an appropriate amount of DMF, added with 8.6g of DIC (68mmol), stirred and activated for 5 minutes, and then charged into a reactor to react at room temperature for 3.5 hours (end point of reaction detected by the ninhydrin method). The solvent was drained and washed twice with DMF to give the fully protected peptide fragment 6 resin.

The Fmoc protecting group was removed by addition of 20% by volume piperidine in DMF for 10 min twice, followed by 6 washes with DMF to give NH₂-Val-Ser (tbu) -Tyr (tbu) -Leu-Glu (Otbu) -Gly-Gln (trt) -Ala-Ala-Lys (Dde) -Glu (Otbu) -Phe-Ile-Ala-Trp (BOC) -Leu-Val-Arg (Pbf) -Gly-resin. 90.6g of the peptide fragment 1(57mmol), 10.4g of HOBt (68mmol) were weighed out, dissolved in an appropriate amount of DMF,8.6g DIC (68mmol) was added, stirred and activated for 5 minutes, and the mixture was charged into a reactor and reacted at room temperature for 3.5 hours (end point of reaction was detected by the ninhydrin method). And (3) pumping the solvent, and washing twice with DMF to obtain the fully-protected liraglutide main chain peptide resin.

3. Deprotection of the linkage to the side chain

Adding 1L of hydrazine hydrate and DMF mixed solution (volume ratio is 1:13) into the fully protected liraglutide main chain peptide resin, stirring for 15 minutes at room temperature, removing the Dde protection of the lysine at the 20 th position of the liraglutide main chain peptide resin, and then washing 6 times by using DMF.

48.5g of Fmoc-Glu-OtBu (114mmol), 36.6g of TBTU (114mmol) and 14.7g of DIEA (114mmol) were weighed out, dissolved in an appropriate amount of DMF, stirred and activated for 5 minutes, and then added to the reactor, followed by addition of 7.4g of DIEA (57mmol) to the reactor and reaction at room temperature for 2 hours (end point of reaction was detected by the ninhydrin method). The solvent was drained, washed twice with DMF, and the Fmoc protecting group was removed by addition of 20% by volume piperidine in DMF for 10 minutes twice, followed by 6 washes with DMF.

29.2g of palmitic acid (114mmol), 36.6g of TBTU (114mmol) and 22.1g of DIEA (171mmol) were weighed out, dissolved in an appropriate amount of DMF, activated with stirring for 5 minutes, added to the reactor and reacted at room temperature for 2 hours (the end point of the reaction was determined by the ninhydrin method). The solvent was pumped dry, washed 4 times with DMF, 4 times with DCM, 4 times with methanol shrinkage, and dried under vacuum to give 241g of liraglutide peptide resin.

4. Cracking and purification

241g of the liraglutide peptide resin was added with 2.5L of a lysis solution (TFA: EDT: PhSMe: TIS: H)₂The volume ratio of O is 85:5:4:2:4), stirring and reacting for 2.5 hours at room temperature, filtering, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, adding into 20L of precooled anhydrous ether, standing and settling for 2 hours. Filtering, washing the filter cake with a proper amount of anhydrous ether for 3 times, and drying in vacuum to obtain the crude liraglutide peptide with the purity of 101.6g, the purity of 73.4 percent and the yield of 82.2 percent.

And (3) adding 20L of purified water into the crude liraglutide, stirring, adjusting the pH value of the solution to 8-8.5 by using ammonia water, and stirring until the product is completely dissolved. Then filtered through a 0.45 μm filter for purification. And (3) purification conditions: the method comprises the following steps of (1) using a 50 x 250mm chromatographic column, using octaalkylsilane bonded silica as a stationary phase, detecting the wavelength of 220nm, using a mobile phase A as a 0.1% TFA/water solution, using a mobile phase B as a 0.1% TFA/acetonitrile solution, performing flow rate of 50-60 ml/min, performing gradient of 35% B-75% B, performing circulating sample injection purification, and collecting a main peak.

And combining the qualified purified products, adding a proper amount of diluted ammonia water to adjust the pH value of the solution to 4.9, separating out a white solid, stirring for 30 minutes, centrifuging to remove a supernatant, washing the solid with precooled purified water for 3 times, dispersing into a proper amount of purified water, and freeze-drying to obtain 37.9g of a pure liraglutide product with the purity of 99.6 percent, the purification yield of 50.6 percent and the total yield of 41.6 percent.

Example 2

The difference from example 1 is that: the degree of substitution of Fmoc-Asp (Otbu) -2-chloro-trityl resin in step 1 was 0.32 mmol/g; the substitution degree of Fmoc-Leu-2-chloro-trityl resin is 0.31 mmol/g; the substitution degree of Fmoc-Ile-2-chloro-trityl resin is 0.35 mmol/g; the degree of substitution of Fmoc-Gly-2-chloro-trityl resin was 0.31 mmol/g; the combination of condensing agent and activating agent in step 1 is DIEA/HATU, and the combination of condensing agent and activating agent in step 2 is DIEA/TBTU.

And 4, vacuum drying is carried out to obtain 92.8g of crude liraglutide, the purity is 72.8%, and the yield of the crude liraglutide is 74.5%.

After purification in step 4, 33.8g of the liraglutide pure product is finally obtained, the purity is 99.5%, the purification yield is 49.8%, and the total yield is 37.1%.

Example 3

The difference from example 2 is that: the degree of substitution of Fmoc-Asp (Otbu) -2-chloro-trityl resin in step 1 was 0.78 mmol/g; the substitution degree of Fmoc-Leu-2-chloro-trityl resin is 0.72 mmol/g; the substitution degree of Fmoc-Ile-2-chloro-trityl resin is 0.75 mmol/g; the degree of substitution of Fmoc-Gly-2-chloro-trityl resin was 0.78 mmol/g; the combination of condensing agent and activating agent in step 1 is DIC/HOBt.

And 4, vacuum drying is carried out to obtain 118.5g of crude liraglutide peptide with the purity of 57.6 percent and the yield of the crude peptide of 75.2 percent.

And 4, after purification in the step 4, finally obtaining the liraglutide pure product 32.5g with the purity of 99.3 percent, the purification yield of 47.3 percent and the total yield of 35.6 percent.

Example 4

The difference from example 2 is that: the carrier adopted in the step 1 is the queen resin with the substitution degree of 1.02 mmol/g. The substitution degree of Fmoc-Asp (Otbu) -queen resin is 0.48 mmol/g; the substitution degree of Fmoc-Leu-Wang resin is 0.52 mmol/g; the substitution degree of Fmoc-Ile-Wang resin is 0.50 mmol/g; the substitution degree of Fmoc-Gly-Wang resin is 0.55 mmol/g; the combination of condensing agent and activating agent in step 2 is DIEA/TBTU.

And 4, vacuum drying is carried out to obtain 95.8g of crude liraglutide peptide with the purity of 56.7 percent and the yield of the crude peptide of 59.9 percent.

After purification in step 4, the liraglutide pure product 25.5g is finally obtained, the purity is 99.5%, the purification yield is 46.8%, and the total yield is 28.0%.

Comparative example 1

The difference from example 1 is that: and (4) desalting and purifying the qualified product obtained in the step (4) by using a chromatographic column. The specific purification conditions were as follows: a 50 x 250mm chromatographic column, using octaalkylsilane chemically bonded silica as a stationary phase, detecting wavelength of 220nm, mobile phase A of 0.05% ammonia water/water solution, mobile phase B of acetonitrile, flow rate of 50-60 ml/min, gradient of 30% B-60% B. And (3) removing the organic solvent by rotary evaporation, and freeze-drying to obtain a liraglutide pure product 30.7g, wherein the purity is 99.6%, and the purification yield is 41.0%.

Comparative example 2

1. Synthesis of backbone peptide resin

100g (0.12mol) of a substitution degree of 1.2mmol/g resin was weighed into a polypeptide synthesis reactor, 700mL of DCM was added to swell the resin for 30 minutes, and washed once with DCM. 29.7g Fmoc-Gly-OH (0.10mol) and 23.3g DIEA (0.27mol) were dissolved in DCM and added to the above reactor, reacted for 1.5 hours, then drained and washed 1 time with DCM. DCM and methanol solution in a volume ratio of 10:1 were added, and the mixture was stirred and blocked for 30 minutes. The solvent was drained, washed with DCM 3 times, the resin was shrunk with methanol 3 times, and dried under vacuum to give Fmoc-Gly-2-chloro-trityl resin, which was found to have a degree of substitution of 0.56 mmol/g.

102g of Fmoc-Gly-2-chloro-trityl resin was weighed, and the Fmoc protecting group was removed with 20% by volume of piperidine in DMF for 10 minutes twice, followed by washing with DMF 6 times to obtain Gly-2-chloro-trityl resin.

Fmoc-Arg (Pbf) -OH (2eq), TBTU (2eq) and DIEA (2eq) were weighed, dissolved in DMF and stirred for 5 min, and then added to the reactor, followed by addition of DIEA (1eq) to the reactor and reaction at room temperature for 2 h (end point of reaction was detected by the ninhydrin method). The solvent was drained and washed twice with DMF to give Fmoc-Arg (Pbf) -Gly-2-chloro-trityl resin.

Repeating the above steps of Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (BOC) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (otbu) -OH, Fmoc-Lys (Dde) -OH, Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (otbu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tbu) -OH, Fmoc-Ser (tbu) -OH, (Fmoc-Val-OH, Fmoc-Asp (otbu) -OH, Fmoc-Asp (Ou) -OH, Coupling of 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, 3 washes with DMF, 3 washes with DCM, 3 methanol contractions, vacuum drying to give the fully protected liraglutide backbone peptide resin.

2. Deprotection of the linkage to the side chain

Adding 1L of hydrazine hydrate and DMF mixed solution (volume ratio is 1:13) into the fully protected liraglutide main chain peptide resin, stirring for 15 minutes at room temperature, removing the Dde protection of the lysine at the 20 th position of the liraglutide main chain peptide resin, and then washing 6 times by using DMF.

48.5g of Fmoc-Glu-OtBu (114mmol), 36.6g of TBTU (114mmol) and 14.7g of DIEA (114mmol) were weighed out, dissolved in an appropriate amount of DMF, stirred and activated for 5 minutes, and then added to the reactor, followed by addition of 7.4g of DIEA (57mmol) to the reactor and reaction at room temperature for 2 hours (end point of reaction was detected by the ninhydrin method). The solvent was drained, washed twice with DMF, and the Fmoc protecting group was removed by addition of 20% by volume piperidine in DMF for 10 minutes twice, followed by 6 washes with DMF.

29.2g of palmitic acid (114mmol), 36.6g of TBTU (114mmol) and 22.1g of DIEA (171mmol) were weighed out, dissolved in an appropriate amount of DMF, activated with stirring for 5 minutes, added to the reactor and reacted at room temperature for 2 hours (the end point of the reaction was determined by the ninhydrin method). The solvent was pumped dry, washed 4 times with DMF, 4 times with DCM, 4 times with methanol shrinkage, and dried under vacuum to give 232g of liraglutide resin.

4. Cracking and purification

232g of the liraglutide peptide resin was added with 2.4L of a lysis solution (TFA: EDT: PhSMe: TIS: H)₂The volume ratio of O is 85:5:4:2:4), stirring and reacting for 2.5 hours at room temperature, filtering, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, adding into 20L of precooled anhydrous ether, standing and settling for 2 hours. Filtering, washing the filter cake with a proper amount of anhydrous ether for 3 times, and drying in vacuum to obtain the crude liraglutide peptide with the purity of 96.9g, the purity of 47.8 percent and the yield of 51.1 percent.

And (3) adding 20L of purified water into the crude liraglutide, stirring, adjusting the pH value of the solution to 8-8.5 by using ammonia water, and stirring until the product is completely dissolved. Then filtered through a 0.45 μm filter for purification. And (3) purification conditions: the method comprises the following steps of (1) using a 50 x 250mm chromatographic column, using octaalkylsilane bonded silica as a stationary phase, detecting the wavelength of 220nm, using a mobile phase A as a 0.1% TFA/water solution, using a mobile phase B as a 0.1% TFA/acetonitrile solution, performing flow rate of 50-60 ml/min, performing gradient of 35% B-75% B, performing circulating sample injection purification, and collecting a main peak.

And combining the qualified purified products, adding a proper amount of diluted ammonia water to adjust the pH value of the solution to 4.9, separating out a white solid, stirring for 30 minutes, centrifuging to remove a supernatant, washing the solid with precooled purified water for 3 times, dispersing into a proper amount of purified water, and freeze-drying to obtain a pure liraglutide product 22.5g, wherein the purity is 99.4%, the purification yield is 48.3%, and the total yield is 24.7%.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (9)

1. A preparation method of liraglutide is characterized by comprising the following steps:

1) solid phase synthesis of 4 peptide fragments of liraglutide, wherein the peptide fragment 1 is the 1 st to 9 th amino acid in the backbone sequence of liraglutide; the peptide fragment 2 is 10 th to 14 th amino acids in the backbone sequence of liraglutide; the peptide fragment 3 is amino acids 15-23 in the backbone sequence of liraglutide; the peptide fragment 4 is amino acids 24-31 in the backbone sequence of liraglutide;

2) gradually coupling each peptide fragment to obtain a fully-protected liraglutide main chain peptide resin;

3) removing the protecting group of the 20 th lysine, and completing the connection of a side chain to obtain the fully-protected liraglutide peptide resin;

4) obtaining liraglutide by cracking, purifying and freeze-drying;

the method specifically comprises the following steps:

1) adopting a solid phase synthesis method, taking resin as a carrier, and respectively synthesizing the resin of peptide fragments 1,2, 3 and 4 of the liraglutide under the action of a condensing agent and an activating agent; wherein the peptide fragment 1 is the amino acids 1-9 in the backbone sequence of liraglutide; the peptide fragment 2 is 10 th to 14 th amino acids in the backbone sequence of liraglutide; the peptide fragment 3 is amino acids 15-23 in the backbone sequence of liraglutide; the peptide fragment 4 is amino acids 24-31 in the backbone sequence of liraglutide;

2) respectively cracking the peptide fragments 1-3 from the resin under the action of a cracking solution to obtain fully protected peptide fragments 1-3; removing the amino protecting group on the peptide fragment 4 resin, and coupling the peptide fragment 3 with the peptide fragment 4 resin under the action of a condensing agent and an activating agent to obtain a peptide fragment 5 resin; removing an amino protecting group on the peptide fragment 5 resin, and coupling the peptide fragment 2 with the peptide fragment 5 resin under the action of a condensing agent and an activating agent to obtain a peptide fragment 6 resin; removing an amino protecting group on the peptide fragment 6 resin, and coupling the peptide fragment 1 with the peptide fragment 6 resin under the action of a condensing agent and an activating agent to obtain the liraglutide main chain peptide resin;

3) removing the lysine side chain protection of the 20 th site of the liraglutide main chain peptide resin, and connecting the side chain to obtain the liraglutide peptide resin;

4) cracking the liraglutide peptide resin under the action of a cracking solution to obtain crude liraglutide; purifying and freeze-drying the crude liraglutide to obtain liraglutide;

wherein, the resin used for solid phase synthesis of the peptide fragment in step 1) is 2-chlorotrityl chloride resin.

2. The method according to claim 1, wherein the substitution degree of the resin used in step 1) is 0.3 to 0.8 mmol/g.

3. The method as claimed in claim 1, wherein the combination of the condensing agent and the activating agent selected in the step 1) solid phase synthesis of the peptide fragment is a mixture of DIC and HOBt, a mixture of DIEA and TBTU or a mixture of DIEA and HATU, the molar ratio of the condensing agent to the activating agent is 1.5-2.0: 1, the solvent used is DMF, and the Fmoc-removing protective solution is a DMF solution of 20% piperidine by volume.

4. The method of claim 1, wherein the lysis solution in step 2) is 1% by volume of TFA in DCM.

5. The method of claim 1, wherein the combination of the condensing agent and the activating agent selected in the coupling in the step 2) is a mixture of DIC and HOBt, a mixture of DIEA and TBTU, and the molar ratio of the condensing agent to the activating agent is 1.0-1.5: 1.

6. The method as claimed in claim 1, wherein the reagent for removing the Dde protection of the lysine side chain in step 3) is a mixture of hydrazine hydrate and DMF, and the volume ratio of the hydrazine hydrate to the DMF is 1 (10-13).

7. The method of claim 1, wherein the cleavage reagent of step 4) is a mixture of TFA, EDT, PhSMe, TIS and H2O; the volume ratio of TFA, EDT, PhSMe, TIS and H2O is (80-85): (3-6): 3-5): 1-2): 2-4.

8. The method as claimed in claim 1, wherein the purification step of step 4) comprises reversed phase high performance liquid chromatography purification and isoelectric point precipitation and sedimentation adjustment.

9. The method according to claim 1, characterized in that it comprises in particular the steps of:

1) coupling Fmoc-Asp (Otbu) -OH with 2-chlorotrityl chloride resin with substitution degree of 0.4-0.6 mmol/g in the presence of a coupling reagent, removing Fmoc protecting group to obtain Gly-resin carrier, adding DIEA/TBTU, and sequentially coupling the following amino acids: 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 to give peptide fragment 1 resin BOC-His (trt) -Ala-Glu (Otbu) -Gly-Thr (tbu) -Phe-Thr (tbu) -Ser (tbu) -Asp (Otbu) -resin; coupling Fmoc-Leu-OH with 2-chlorotrityl chloride resin with the substitution degree of 0.4-0.6 mmol/g in the presence of a coupling reagent, removing the Fmoc protecting group to obtain a Leu-resin carrier, adding DIEA/TBTU, and sequentially coupling the following amino acids: Fmoc-Tyr (tbu) -OH, Fmoc-Ser (tbu) -OH, Fmoc-Val-OH to obtain peptide fragment 2 resin Fmoc-Val-Ser (tbu) -Tyr (tbu) -Leu-resin; coupling Fmoc-Ile-OH with 2-chlorotrityl chloride resin with the substitution degree of 0.4-0.6 mmol/g in the presence of a coupling reagent, removing an Fmoc protecting group to obtain an Ile-resin carrier, adding DIEA/TBTU, and sequentially coupling the following amino acids: Fmoc-Phe-OH, Fmoc-Glu (Otbu) -OH, Fmoc-Lys (Dde) -OH, Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (Otbu) -OH to obtain peptide fragment 3 resin Fmoc-Glu (Otbu) -Gly-Gln (trt) -Ala-Ala-Lys (Dde) -Glu (Otbu) -Phe-Ile-resin; coupling Fmoc-Gly-OH with 2-chlorotrityl chloride resin with the substitution degree of 0.4-0.6 mmol/g in the presence of a coupling reagent, removing Fmoc protecting groups to obtain a Gly-resin carrier, adding DIEA/TBTU, and sequentially coupling the following amino acids: Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (BOC) -OH, Fmoc-Ala-OH to obtain peptide fragment 4 resin Fmoc-Ala-Trp (BOC) -Leu-Val-Arg (Pbf) -Gly-resin;

2) cleaving the peptide fragment 1,2, 3 resin using 1% by volume TFA in DCM as a cleaving reagent to obtain a fully protected peptide fragment 1, peptide fragment 2, peptide fragment 3, respectively; removing amino protecting group on the peptide fragment 4 resin using DMF solution with 20% piperidine by volume percentage, activating the peptide fragment 3 with mixture of DIC and HOBt, adding into DMF solution of the above resin, reacting to obtain peptide fragment 5 resin; removing amino protecting group on the peptide fragment 5 resin using DMF solution of piperidine 20% by volume, activating the peptide fragment 2 with mixture of DIC and HOBt, adding into the above resin in DMF solution, reacting to obtain peptide fragment 6 resin; removing amino protecting group on the peptide fragment 6 resin by using 20% piperidine by volume in DMF solution, activating the peptide fragment 1 by using a mixture of DIC and HOBt, adding the activated peptide fragment 1 into the DMF solution of the resin, and reacting to obtain liraglutide main chain peptide resin;

3) removing Dde protection of lysine at the 20 th site of the liraglutide main chain peptide resin by using a mixed solution of hydrazine hydrate and DMF (dimethyl formamide) with the volume ratio of 1:13, then coupling Fmoc-Glu-OtBu and the lysine at the 20 th site, removing the Fmoc protection, and then adding palmitoyl chloride to complete side chain connection;

4) cracking the liraglutide peptide resin by using a mixed solution of TFA, EDT, PhSMe, TIS and H2O in a volume ratio of 85:5:4:2:4 as a lysate to obtain crude liraglutide; and (3) after the crude liraglutide is purified by reversed-phase high performance liquid chromatography, adjusting the pH value of the solution to an isoelectric point, separating out a product, performing centrifugal sedimentation, and freeze-drying to obtain the liraglutide.