WO2021147869A1 - Liraglutide derivative and preparation method therefor - Google Patents

Abstract

A method for preparing a Liraglutide product, in which Fmoc modification is performed on a Boc-modified Liraglutide backbone chain and sidechain addition of Liraglutide is implemented using Fmoc modification. A Fmoc- and Boc-modified Liraglutide backbone chain and a fusion protein containing the Liraglutide backbone chain involved in the process of the preparation method.

Description

Liraglutide derivative and preparation method thereof Technical field

The present invention relates to the field of biomedicine, and more specifically to a derivative of GLP-1 analogue liraglutide and a preparation method thereof.

Background technique

Diabetes is a major disease threatening human health worldwide. In China, as the people’s lifestyle changes and the aging process accelerates, the prevalence of diabetes is showing a rapid upward trend. The acute and chronic complications of diabetes, especially chronic complications that accumulate multiple organs, are disabling and have a high mortality rate, which seriously affects the physical and mental health of patients and brings a heavy burden to individuals, families and society.

Liraglutide is a glucagon-like peptide-1 (GLP-1) analogue, which promotes the glucose concentration-dependent secretion of insulin from pancreatic β cells by activating the GLP-1 receptor, and is clinically used to treat type 2 diabetes .

Natural GLP-1 is easily degraded by dipeptidyl peptidase IV (DPP-IV) in the body, and its plasma half-life is less than 2 minutes. It must be continuously injected intravenously or subcutaneously to produce curative effects. In order to overcome this clinical problem, a series of GLP-1 analogs have been developed. Among them, liraglutide, which was launched in the European Union and the United States in July 2009 and January 2010, has the most significant efficacy. Liraglutide can significantly reduce the fasting or post-prandial blood sugar of type 2 diabetic patients to adjust the blood glucose level in the body, and at the same time can reduce the weight of patients and reduce the risk of death in patients with cardiovascular diseases.

There are many reports on the preparation of liraglutide at home and abroad. For example, US patents US 6268343, 6458924 and 7235627 have invented a gene recombination technology to obtain the main chain (main chain) of liraglutide, which is the sequence Arg ³⁴ GLP-1(7- 37), and then connect Pal-Glu-(OSu)-OtBu substances by liquid phase synthesis method to obtain liraglutide. Since the main chain of liraglutide is in an unprotected state, the side chain is easily attached to the N-terminus during the connection process On the amino group, impurities are generated, resulting in difficulty in purification and low yield, and the process is more complicated.

Therefore, those skilled in the art are committed to developing new and longer-acting insulin derivatives.

Summary of the invention

The purpose of the present invention is to provide liraglutide derivatives and preparation methods thereof.

In the first aspect of the present invention, a Boc-modified liraglutide backbone is provided, the 20th position of the Boc-modified liraglutide backbone is a protected lysine, and the protection Lysine is Nε-(tert-butoxycarbonyl)-lysine.

In another preferred embodiment, the epsilon amino group of the protected lysine is modified with tert-butoxycarbonyl.

In another preferred embodiment, the N-terminus of the main chain of liraglutide is modified by Fmoc.

In another preferred embodiment, the Fmoc is fluorenylmethyloxycarbonyl.

In another preferred example, the amino acid sequence of the liraglutide backbone is shown in SEQ ID NO.: 7 ( HAEGTFTSDVSSYLEGQAA K EFIAWLVRGRG, where H is Fmoc modified histidine, K is Boc modified lysine acid).

In another preferred embodiment, the main chain of liraglutide is used in the synthesis of liraglutide.

In the second aspect of the present invention, a liraglutide backbone fusion protein is provided, and the liraglutide fusion protein has the structure shown in formula I from the N-terminus to the C-terminus:

FP-TEV-EK-GLP-1 (I)

Where

"-" represents a peptide bond;

FP is the green fluorescent protein folding unit;

TEV is the first restriction site, preferably TEV restriction site (as shown in the sequence ENLYFQG, SEQ ID NO.: 8);

EK is the second restriction site, preferably enterokinase restriction site (as shown in the sequence DDDDK, SEQ ID NO.: 9);

GLP-1 represents the Boc-modified liraglutide of the first aspect of the present invention.

In another preferred example, the GLP-1 does not contain Fmoc modification.

In another preferred embodiment, the green fluorescent protein folding unit is selected from the following group: u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, or a combination thereof, and

To Amino acid sequence u1 VPILVELDGDVNG(SEQ ID NO.:11) u2 HKFSVRGEGEGDAT(SEQ ID NO.:12) u3 KLTLKFICTT (SEQ ID NO.: 13) u4 YVQERTISFKD(SEQ ID NO.:14) u5 TYKTRAEVKFEGD (SEQ ID NO.: 15) u6 TLVNRIELKGIDF(SEQ ID NO.:16) u7 HNVYITADKQ(SEQ ID NO.:17) u8 GIKANFKIRHNVED (SEQ ID NO.: 18) u9 VQLADHYQQNTPIG(SEQ ID NO.:19) u10 HYLSTQSVLSKD(SEQ ID NO.:20) u11 HMVLLEFVTAAGI (SEQ ID NO.: 2).

In another preferred embodiment, the green fluorescent protein folding unit is u2-u3, u4-u5 or u4-u5-u6.

In another preferred embodiment, the N-terminus of the green fluorescent protein folding unit contains a signal peptide. Preferably, the signal peptide is shown in SEQ ID NO.: 10 (MVSKGEELFTGV).

In another preferred embodiment, the amino acid sequence of the liraglutide backbone fusion protein is shown in SEQ ID NO.: 1, 3, 4.

In another preferred embodiment, the liraglutide backbone fusion protein is used in the preparation of the liraglutide backbone.

In the third aspect of the present invention, there is provided a method for preparing liraglutide, the method comprising the steps:

(i) Provide a Boc-modified liraglutide backbone;

(ii) Fmoc modification is performed on the Boc-modified liraglutide backbone to obtain Fmoc and Boc-modified liraglutide backbones;

(iii) De-Boc treatment of the Fmoc and Boc-modified liraglutide main chain, and react it with the side chain of liraglutide, thereby preparing Fmoc-modified liraglutide; and

(iv) De-Fmoc treatment is performed on the Fmoc-modified liraglutide to obtain liraglutide.

In another preferred example, the 20th position of the Boc-modified liraglutide backbone is a protected lysine, and the protected lysine is Nε-(tert-butoxycarbonyl)-lysine .

In another preferred embodiment, the N-terminus of the main chain of liraglutide modified by Fmoc and Boc is modified by Fmoc.

In another preferred embodiment, the side chain of liraglutide is Na-palmitoyl-D-glutamic acid-γ-succinimidyl-A-tert-butyl ester (Pal-Glu-(OSu)-OtBu ).

In another preferred embodiment, the side chain of liraglutide is as follows:

In another preferred example, in step (ii), Fmoc-Osu, NaHCO ₃ and DMF/H ₂ O are added to perform Fmoc modification.

In another preferred example, the molar ratio of the added Fmoc-Osu, NaHCO ₃ and the Boc-modified liraglutide backbone is (0.8-1.5): (1.5-2.5): (0.8-1.2), preferably It is (1.0-1.2): (1.8-2.2): (0.8-1.2).

In another preferred embodiment, between step (ii) and step (iii), it also includes the step of purifying the prepared Fmoc and Boc modified liraglutide backbone. Preferably, a C8 preparation column is used. For purification, the mobile phase is an aqueous solution of TFA.

In another preferred example, in step (iii), the method further includes the following steps:

(a) Add the TFA solution, stir at low temperature, and perform de-Boc treatment to obtain a de-Boc product;

(b) Purifying the Boc-removed product, preferably C8 reversed-phase purification;

(c) Optionally, adding an organic solvent to the purified collection liquid obtained from the purification treatment to obtain a solid de-Boc product. Preferably, the organic solvent is a mixture of methyl tertiary ether and petroleum ether;

(d) Mix the de-Boc product with the side chain of liraglutide to prepare Fmoc-modified liraglutide.

In another preferred embodiment, in step (d), the solid de-Boc product and the side chain of liraglutide are mixed in NMP and reacted at room temperature.

In another preferred embodiment, in step (d), the reaction is terminated with an aqueous ethanol solution containing glycine.

In another preferred example, in step (d), the reaction system further contains EDPA.

In another preferred embodiment, in step (iv), a DMF solution containing piperidine is added to undergo de-Fmoc treatment, thereby preparing liraglutide.

In another preferred embodiment, step (iv) includes the step of purifying the obtained liraglutide.

In another preferred embodiment, the Boc-modified liraglutide backbone is prepared by gene recombination technology.

In another preferred example, in step (i), the steps are included:

(ia) Using recombinant bacteria to prepare the liraglutide backbone fusion protein of the second aspect of the present invention,

(ib) Using enterokinase to digest the liraglutide fusion protein to obtain a Boc-modified liraglutide backbone.

In another preferred example, in step (ia), liraglutide backbone fusion protein inclusion bodies are separated from the fermentation broth of the recombinant bacteria, and the inclusion bodies are denatured, renatured and digested. , Obtain the liraglutide backbone fusion protein.

In another preferred embodiment, before and after step (ib), a purification step is further included.

In another preferred example, in step (ib), the mass ratio of the liraglutide fusion protein to enterokinase is 1:3000-12000, preferably 1:5000-6000.

In another preferred embodiment, the recombinant bacteria contains or integrates an expression cassette for expressing liraglutide backbone fusion protein.

The fourth aspect of the present invention provides an isolated polynucleotide encoding the Boc-modified liraglutide backbone of the first aspect of the present invention or the fusion of the second aspect of the present invention protein.

In the fifth aspect of the present invention, a vector is provided, which includes the polynucleotide according to the fourth aspect of the present invention.

In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

The sixth aspect of the present invention provides a host cell containing the vector according to the fifth aspect of the present invention, or the polynucleotide according to the fourth aspect of the present invention integrated into the chromosome, Or express the fusion protein described in the second aspect of the present invention.

In another preferred example, the host cell is Escherichia coli, Bacillus subtilis, yeast cells, insect cells, mammalian cells or a combination thereof.

The seventh aspect of the present invention provides a Fmoc-modified liraglutide backbone, the N-terminus of the liraglutide backbone is Fmoc-modified histidine.

In another preferred embodiment, the amino acid sequence of the main chain of liraglutide is shown in SEQ ID NO: 7.

The eighth aspect of the present invention provides a Fmoc-modified liraglutide, the N-terminal of the liraglutide main chain is Fmoc-modified histidine, and the liraglutide main chain is connected Favorable laglutide side chain.

In another preferred embodiment, the side chain of liraglutide is connected to the lysine of the main chain of liraglutide.

In another preferred embodiment, the side chain of liraglutide is Na-palmitoyl-D-glutamic acid-γ-succinimidyl-A-tert-butyl ester (Pal-Glu-(OSu)- OtBu).

The ninth aspect of the present invention provides a formulation comprising the Boc-modified liraglutide backbone of the first aspect of the present invention and the liraglutide backbone of the second aspect of the present invention Fusion protein, the Fmoc modified liraglutide backbone of the seventh aspect of the present invention or the Fmoc modified liraglutide of the eighth aspect of the present invention.

In another preferred embodiment, the preparation also contains a pharmaceutically acceptable carrier.

The tenth aspect of the present invention provides a liraglutide preparation, which is prepared using the method described in the third aspect of the present invention.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.

Description of the drawings

Figure 1 shows the map of plasmid pBAD-FP-TEV-EK-GLP-1 (20).

Figure 2 shows the map of plasmid pEvol-pylRs-pylT.

Figure 3 shows the SDS-PAGE electrophoresis of the Boc-liraglutide backbone fusion protein after denaturation and renaturation of inclusion bodies.

Figure 4 shows the HPLC detection profile of Boc-liraglutide intermediate polypeptide.

Figure 5 shows the liraglutide preparation process of the present invention.

Detailed ways

After extensive and in-depth research, the inventor found a new method for preparing liraglutide products. Specifically, the method utilizes the Fmoc orthogonal protection method to carry out the side chain addition step in the preparation process of liraglutide, and optimizes the purification and synthesis conditions in the preparation process. The method of the invention does not require expensive solid-phase synthesis equipment, shortens the production cycle, simple production process, and improves product purity and yield.

Liraglutide

Liraglutide is developed by Novo Nordisk, English name Liraglutide, molecular formula: C172H265N43O51, molecular weight: 3751.2, CAS number: 204656-20-2, is a human glucagon-like peptide-1 (GLP-1) Analogue, the sequence is: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Nε( Nα-PAL-γ-Glu))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH has 97% sequence homology with human natural GLP-1.

The structure of liraglutide is that the 28th lysine of the natural GLP-1(7-37) molecule is replaced with arginine, and the epsilon amino group of the side chain of the 20th lysine is replaced by hexadecanoic acid. Glutamate acylation. Due to the existence of the fatty chain, the degradation of DPP-4 can be reduced, the half-life can be prolonged, and the frequency of administration can reach once a day. It can significantly reduce the fasting or post-meal blood sugar of type 2 diabetic patients to adjust the blood sugar level in the body, and at the same time can reduce the weight of patients and reduce the risk of death in patients with cardiovascular diseases.

Liraglutide fusion protein

The present invention provides a liraglutide fusion protein, which has the structure shown in formula I from the N-terminus to the C-terminus:

FP-TEV-EK-GLP-1 (I)

Where

"-" represents a peptide bond;

FP is the green fluorescent protein folding unit;

TEV is the first restriction site, preferably TEV restriction site (as shown in the sequence ENLYFQG, SEQ ID NO.: 8);

EK is the second restriction site, preferably enterokinase restriction site (as shown in the sequence DDDDK, SEQ ID NO.: 9);

GLP-1 represents the Boc-modified liraglutide described in the first aspect of the present invention.

In another preferred example, the green fluorescent protein folding unit FP can be selected from: u8, u9, u2-u3, u4-u5, u8-u9, u1-u2-u3, u2-u3-u4, u3- u4-u5, u5-u6-u7, u8-u9-u10, u9-u10-u11, u3-u5-u7, u3-u4-u6, u4-u7-u10, u6-u8-u10, u1-u2- u3-u4, u2-u3-u4-u5, u3-u4-u3-u4, u3-u5-u7-u9, u5-u6-u7-u8, u1-u3-u7-u9, u2-u2-u7- u8, u7-u2-u5-u11, u3-u4-u7-u10, u1-I-u2, u1-I-u5, u2-I-u4, u3-I-u8, u5-I-u6, or u10 -I-u11.

And, the units described have the sequence shown below:

To Amino acid sequence u1 VPILVELDGDVNG(SEQ ID NO.:11) u2 HKFSVRGEGEGDAT(SEQ ID NO.:12) u3 KLTLKFICTT (SEQ ID NO.: 13) u4 YVQERTISFKD(SEQ ID NO.:14) u5 TYKTRAEVKFEGD (SEQ ID NO.: 15) u6 TLVNRIELKGIDF(SEQ ID NO.:16) u7 HNVYITADKQ(SEQ ID NO.:17) u8 GIKANFKIRHNVED (SEQ ID NO.: 18) u9 VQLADHYQQNTPIG(SEQ ID NO.:19) u10 HYLSTQSVLSKD(SEQ ID NO.:20)

u11 HMVLLEFVTAAGI (SEQ ID NO.: 2).

In another preferred example, the sequence of the fusion protein of the present invention is as follows:

FP1-TEV-EK-GLP-1(20), the amino acid sequence is shown in SEQ ID NO.:1:

FP2-TEV-EK-GLP-1(20), the amino acid sequence is shown in SEQ ID NO.: 3:

FP3-TEV-EK-GLP-1(20), the amino acid sequence is shown in SEQ ID NO.: 4:

Where K is Boc modified lysine.

As used herein, the term "fusion protein" also includes variant forms having the above-mentioned activities. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions or additions at the C-terminus and/or N-terminus One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted. For example, in the field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes the polypeptide of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

The present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion protein. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogues of the present invention can be (i) one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fusion A polypeptide formed from this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence, or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.

A preferred type of active derivative means that compared with the amino acid sequence of the present invention, at most 3, preferably at most 2, and more preferably at most 1 amino acid are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.

Table A

Initial residues Representative substitution Preferred substitution

Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

The present invention also provides analogs of the fusion protein of the present invention. The difference between these analogs and the polypeptide of the present invention may be a difference in the amino acid sequence, a difference in the modified form that does not affect the sequence, or both. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

In addition, the fusion protein of the present invention can also be modified. Modified (usually not changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

The term "polynucleotide encoding the fusion protein of the present invention" may include a polynucleotide encoding the fusion protein of the present invention, or a polynucleotide that also includes additional coding and/or non-coding sequences.

The present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or fusion proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the fusion protein encoded by it. Function.

The present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%.

The fusion protein and polynucleotide of the present invention are preferably provided in an isolated form, and more preferably, are purified to homogeneity.

The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.

At present, the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.

The method of using PCR technology to amplify DNA/RNA is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods. The amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

Expression vector

The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the fusion protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.

Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce a recombinant fusion protein. Generally speaking, there are the following steps:

(1) Use the polynucleotide (or variant) of the present invention encoding the fusion protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;

(2). Host cells cultured in a suitable medium;

(3). Separate and purify protein from culture medium or cells.

In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast and plant cells (such as ginseng cells).

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.

Those of ordinary skill in the art know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture can be selected from various conventional mediums. The culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Construction of Liraglutide Expression Vector

The expression construct FP-TEV-EK-GLP1 contains the coding gene of GLP1, which is fused to the C-terminus of FP-TEV-EK. The sequence has been codon optimized, which can achieve high-level expression of functional proteins in E. coli. After expression, the expression vector "pBAD/His A(KanaR)" was cut open with restriction enzymes NcoⅠ and XhoⅠ, the digested products were separated by agarose electrophoresis, and then extracted with agarose gel DNA recovery kit, and finally Use T4 DNA ligase to join the two DNA fragments. The ligation product was chemically transformed into E. coli Top10 cells, and the transformed cells were cultured in LB agar medium (10g/L yeast peptone, 5g/L yeast extract powder, containing 50μg/mL kanamycin, 10g/L NaCl, 1.5% agar) overnight. Pick 3 live colonies and culture them overnight in 5mL liquid LB medium (10g/L yeast peptone, 5g/L yeast extract, 10g/L NaCl) containing 50μg/mL kanamycin, and use a small amount of plasmid to extract The kit is used for plasmid extraction. Then, the extracted plasmid was sequenced using the sequencing oligonucleotide primer 5'-ATGCCATAGCATTTTTATCC-3' to confirm the correct insertion. The resulting plasmid was named "pBAD-FP-TEV-EK-GLP1".

Fmoc modification

In the field of biomedicine, peptides are becoming more and more useful. Amino acids are the basic raw materials for synthetic peptide technology. All amino acids contain α-amino and carboxyl groups, and some also contain side-chain active groups, such as hydroxyl, amino, guanidine and Heterocycles, etc., therefore, amino groups and side chain active groups need to be protected in the peptide reaction. After the peptide is synthesized, the protective groups are removed, otherwise misconnection of amino acids and many side reactions will occur.

Fluorenylmethyloxycarbonyl (Fmoc) is an alkali-sensitive protecting group, which can be used in concentrated ammonia or dioxane-methanol-4N Na OH (30:9:1), piperidine, ethanolamine, cyclohexylamine, 1,4-di Oxane, pyrrolidone and other ammonia are removed in a 50% dichloromethane solution.

Under weak alkaline conditions such as sodium carbonate or sodium bicarbonate, Fmoc-Cl or Fmoc-OSu is generally used to introduce the Fmoc protecting group. Compared with Fmoc-Cl, Fmoc-OSu is easier to control the reaction conditions and has fewer side reactions. Under acidic conditions, the Fmoc protecting group is particularly stable, but very sensitive to alkaline conditions, so it is usually used together with the acid-sensitive protecting group Boc or Z to protect amino acids containing active side chain groups.

Fmoc has strong ultraviolet absorption, the maximum absorption wavelength is 267nm (ε18950), 290nm (ε5280), 301nm (ε6200), so it can be detected by ultraviolet absorption, which brings a lot of convenience to the instrument for automatic peptide synthesis. Furthermore, it is compatible with a wide range of solvents and reagents, has high mechanical stability, and can be used with multiple carriers and multiple activation methods. Therefore, the most commonly used in peptide synthesis today is the Fmoc protecting group.

Fmoc-OSu (fluorene methoxycarbonyl succinimide)

Liraglutide Side Chain

Pal-Glu-(OSu)-OtBu is Na-palmitoyl-D-glutamic acid-γ-succinimidyl-A-tert-butyl ester, abbreviated as D-type-liraglutide side chain.

Liraglutide is prepared by first using gene recombination technology to obtain the 20-position Boc protected lysine liraglutide main chain, that is, the sequence Arg ³⁴ GLP-1 (7-37), and then connect the liraglutide side chain Pal-Glu-(OSu)-OtBu to obtain liraglutide.

Preparation of Liraglutide

The present invention provides a synthetic route for liraglutide as shown in Figure 5. Fmoc-modified compound 2 is prepared from the Boc-liraglutide backbone (compound 1), compound 2 is de-Boc protected to obtain compound 3, compound 3 and The Pal-Glu-(OSu)-OtBu reaction of the side chain of liraglutide is activated to obtain compound 4, and then the Fmoc reaction is removed to obtain compound 5, and the tBu protecting group is removed from the side chain to obtain liraglutide.

Specifically, the present invention provides a method for preparing liraglutide, the method comprising the steps:

(i) Provide a Boc-modified liraglutide backbone;

(ii) Fmoc modification is performed on the Boc-modified liraglutide backbone to obtain Fmoc and Boc-modified liraglutide backbones;

(iii) De-Boc treatment of the Fmoc and Boc-modified liraglutide main chain, and react it with the side chain of liraglutide, thereby preparing Fmoc-modified liraglutide; and

(iv) The Fmoc-modified liraglutide is subjected to de-Fmoc and side chain de-tBu treatments to prepare liraglutide.

In another preferred example, in step (ii), Fmoc-Osu, NaHCO ₃ and DMF/H ₂ O are added to perform Fmoc modification.

In another preferred example, the molar ratio of the added Fmoc-Osu, NaHCO ₃ and the Boc-modified liraglutide backbone is (0.8-1.5): (1.5-2.5): (0.8-1.2), preferably It is (1.0-1.2): (1.8-2.2): (0.8-1.2).

In another preferred embodiment, between step (ii) and step (iii), it also includes the step of purifying the prepared Fmoc and Boc modified liraglutide backbone. Preferably, a C8 preparation column is used. For purification, the mobile phase is TFA in acetonitrile.

In another preferred example, in step (iii), the method further includes the following steps:

(a) Add the TFA solution, stir at low temperature, and perform de-Boc treatment to obtain a de-Boc product;

(b) Purifying the Boc-removed product, preferably C8 reversed-phase purification;

(c) Optionally, adding an organic solvent to the purified collection liquid obtained from the purification treatment to obtain a solid de-Boc product. Preferably, the organic solvent is a mixture of methyl tertiary ether and petroleum ether;

(d) Mix the de-Boc product with the side chain of liraglutide to prepare Fmoc-modified liraglutide.

In another preferred example, in step (i), the steps are included:

(ia) Using recombinant bacteria to prepare the liraglutide backbone fusion protein of the second aspect of the present invention,

(ib) Using enterokinase to digest the liraglutide fusion protein to obtain a Boc-modified liraglutide backbone.

In another preferred example, in step (ia), liraglutide backbone fusion protein inclusion bodies are separated from the fermentation broth of the recombinant bacteria, and the inclusion bodies are denatured, renatured and digested. , The Boc-liraglutide backbone fusion protein was obtained.

The main advantages of the present invention include:

(1) The present invention directly uses biosynthesis to produce the Boc-modified liraglutide backbone, and does not need to use methods such as dilution, ultrafiltration and liquid exchange to remove excess inorganic salts in the supernatant of the fermentation broth. In the method of the present invention, a chromatographic column is used to separate the Boc-liraglutide backbone or the precursor of the analog, and the one-step yield is more than 70%, which is 3 times higher than that of the conventional method. The yield is about 600-700mg. In addition, the method of the present invention can remove most of the pigments, and directly separate and purify in one step from the original multi-step process, which reduces the process time and equipment investment cost;

(2) Due to the protection of Boc-lysine at position 26, the present invention can directly utilize the orthogonal reaction with Fmoc protection to synthesize liraglutide.

(3) The liraglutide synthesized by the method of the present invention has no impurities caused by the acylation of N-terminal fatty acids, which facilitates downstream purification and reduces costs.

(4) Compared with solid-phase synthesis, the method of the present invention does not produce racemic impurity polypeptides, does not need to use a large amount of modified amino acids, does not use a large amount of organic reagents, and has little environmental pollution and lower cost;

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

Example 1 Construction of Liraglutide-Expressing Strain

For the construction of the liraglutide expression vector, refer to the description of the example in the patent application number 201910210102.9. The DNA fragment of the fusion protein FP1-TEV-EK-GLP-1 (20) (the expression cassette for expressing the liraglutide backbone fusion protein) was cloned into the expression vector plasmid pBAD/His A (purchased from NTCC, Cana The NcoI-XhoI site downstream of the araBAD promoter of araBAD, and the plasmid pBAD-FP-TEV-EK-GLP-1 (20) was obtained. The plasmid map is shown in Figure 1.

Then the DNA sequence of pyrrolysyl-tRNA synthetase (pylRs) was cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (purchased from NTCC, chloramphenicol resistant), and at the same time Downstream of the proK promoter, the DNA sequence (SEQ ID NO.: 6) of the tRNA (pylTcua) of lysyl-tRNA synthetase was inserted by PCR. This plasmid was named pEvol-pylRs-pylT. The plasmid map is shown in Figure 2.

The amino acid sequence of pylRs (SEQ ID NO.: 5):

DNA sequence of tRNA (pylTcua) (SEQ ID NO.: 6):

The constructed plasmids pBAD-FP-TEV-EK-GLP-1(20) and pEvol-pylRs-pylT were co-transformed into E. coli TOP10 strain, and the liraglutide backbone fusion protein FP1-TEV-EK-GLP was screened and expressed -1 (20) recombinant E. coli strain.

Example 2 Expression of Boc-liraglutide backbone

Connect the recombinant E. coli to the E. coli seed solution (incubated by the company) at a 5% inoculum amount (volume ratio), 37°C, pH 7.0, feed in batches until the pH rises to 7.05, and then start the separation of carbon and nitrogen sources Feeding, according to the constant pH method for carbon source flow addition. Feeding 11h-fermentation is over, the mass ratio of carbon to nitrogen source is 1:1.0. After feeding, 7.5M ammonia is added through feeding and automatic flow to control the pH at 7.0-7.2. After culturing for about 4-6 hours, start to add 2.5g/L L-arabinose for induction, and the induction lasts for 14 hours until the end of fermentation. The fermentation broth containing the liraglutide backbone fusion protein is obtained.

Example 3 Preparation of Boc-liraglutide backbone inclusion body

After centrifuging the fermentation broth obtained in Example 2, the wet bacteria were mixed with the bactericidal buffer at a volume of 1:1 and suspended for 3 hours. The suspension was bactericidalized three times with a high-pressure homogenizer. After the bacteriostasis, the inclusion bodies were collected by centrifugation. Wash twice, the buffer composition is: 0.5% T-80, 1mm EDTA-2Na, 100mm NaCl, pH 7.5. After washing, the yield of the inclusion bodies weighed is 41-45g/L.

The results of SDS-PAGE electrophoresis are shown in Figure 3. The results showed that the fusion protein was expressed, and Boc-liraglutide main chain inclusion bodies were obtained after bacterial cell disruption, washing, and centrifugation.

Example 4 Denaturation and restriction digestion of Boc-liraglutide backbone inclusion bodies

Add 7.5mol/L urea solubilization buffer to the inclusion bodies obtained in Example 3 at a weight-volume ratio of 1:10, stir and dissolve at room temperature, measure the protein concentration by Bradford method, and control the total protein concentration of the inclusion body solubilization solution at 25mg/ml Around, NaOH adjusts the pH to 9.0±0.1. Add the inclusion body dissolving solution dropwise to the refolding buffer containing 5-10mmol/L Tris, 10mmol/L NaCl, 10mmol/L Na ₂ CO ₃ , 0.3-0.5mmol/L EDTA-2Na, to dissolve the inclusion bodies The solution is diluted 5-10 times for renaturation, and the pH value of the fusion protein renaturation solution is maintained at 9.0-10.0, the temperature is controlled at 4-8℃, and the renaturation time is 10-20h.

The results showed that after dissolution, the fusion protein accounted for about 33%.

Example 5 Preliminary purification of Boc-liraglutide fusion protein

Take the fusion protein refolding solution obtained in Example 4 and filter it through a 0.45 μm filter membrane to remove undissolved substances; according to the difference in protein isoelectric point, the fusion protein is preliminarily purified using an anion exchange column with Q Sepharose FF packing.

The experimental results showed that the purity of the Boc-liraglutide backbone fusion protein reached more than 65% after anion exchange chromatography, the load was about 18 mg/mL, and the yield was greater than 80%.

Example 6 Restriction digestion of Boc-liraglutide backbone fusion protein

The sample of the Boc-liraglutide backbone fusion protein preliminarily purified in Example 5 was desalted through a hydrophobic column and eluted with pure water. The elution volume was about 5 times the column volume. Adjust the pH of the fusion protein solution to 7.5-8.5, control the temperature at 25°C, add enterokinase digestion, and the digestion time is 5-16h to obtain Boc-liraglutide backbone and Boc-liraglutide backbone About 600mg/L, digestion efficiency ≥85%.

Example 7 Reversed phase chromatography of Boc-liraglutide backbone

According to the difference in hydrophobicity of peptides and proteins, polymer reversed-phase chromatography is used to purify the main chain of Boc-liraglutide to remove some impurities.

The enzyme digestion solution of the Boc-liraglutide backbone fusion protein obtained in Example 6 was clarified by filtration, and then subjected to reversed-phase chromatography for separation and purification. An aqueous solution containing 0.065% trifluoroacetic acid was used as mobile phase A; an acetonitrile solution containing 0.065% trifluoroacetic acid was used as mobile phase B. The Boc-liraglutide main chain is combined with the filler to control the loading amount of the Boc-liraglutide main chain to be less than 10mg/ml, and then gradient elution is performed to collect the Boc-liraglutide main chain. The experimental results show that the purity of the Boc-liraglutide backbone collected by polymer reversed phase chromatography is ≥90%, and the yield is greater than 60%. The HPLC detection chart of the collected Boc-liraglutide backbone is shown in Figure 4.

Example 8 Preparation of liraglutide using Boc-liraglutide backbone

Take the Boc-liraglutide backbone compound 1 obtained in Example 7, add Fmoc-Osu, NaHCO ₃ and DMF/H ₂ O according to the molar ratio in Table 1, and react for 8-12 hours to prepare Fmoc and Boc protected GLP-1. Purification was performed using a C8 column, the aqueous solution containing 0.065% (v/v) TFA was used as mobile phase A, and the acetonitrile containing 0.065% (v/v) TFA was used as mobile phase B, and gradient elution was performed. Methyl tertiary ether was added to the purified collection solution, precipitation was centrifuged, and the precipitate was washed with methyl tertiary ether 2 to 3 times to obtain Fmoc-protected compound 2: DiFmoc-GLP-1 (Lys ²⁰ Boc).

Table 1 The molar ratio of the feed

To Boc-liraglutide Fmoc-OSu NaHCO3 DMF/H ₂ O Equivalent or volume 1.0eq 1.1eq 2.0eq 30V/30V

Take the purified compound 2, add TFA solution, stir at low temperature for 10-20 minutes, and purify the deprotection reactant by C8 reverse phase. Add 20 times the volume of methyl tertiary ether: petroleum ether mixture (3:1) to the purified collection solution, centrifuge the precipitate, wash the precipitate with the mixture for 2 to 3 times, and finally obtain the Boc-removed solid compound 3: DiFmoc-GLP-1 (Lys ²⁰ NH ₂ ).

Take compound 3 after Boc removal, add 30 eq. EDPA, NMP and water mixture (2:1), and stir gently at room temperature for 5 min. The equivalent Pal-Glu-(OSu)-OtBu (23.7 μmol) was dissolved in NMP (303 μL) and added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 2 hours. To this was added 625 μL of a 50% ethanol aqueous solution containing glycine (6.5 mg, 86.9 μmol) to terminate the reaction and obtain compound 4: DiFmoc-GLP-1-(Pal-Glu-(Lys ²⁰ NH ₂ )-OtBu).

Take the purified compound 4 (300 mg), add 20% piperidine-containing DMF solution, and react at room temperature for 30 minutes. Add 10 times the volume of a mixed solvent of methyl tertiary ether and petroleum ether into the reaction system, precipitate and centrifuge, and wash the solid with a mixed solvent of methyl tertiary ether and petroleum ether for 3 to 5 times to obtain compound 5 (270 mg) after Fmoc removal: GLP -1-(Pal-Glu-(Lys ²⁰ NH ₂ )-OtBu).

GLP-1 Take compound 5 (270 mg), add _{10 mL of a mixed solution of (TFA:TIS:H 2} O =95:2.5:2.5):DCM (v:v=1:1), shake at room temperature for 3 to 4 hours to remove Side chain tBu protective group, 10 times volume of methyl tertiary ether and petroleum ether mixed solvent is added to the reaction system, precipitation is centrifuged, and the solid is washed 3 times with methyl tertiary ether and petroleum ether mixed solvent to obtain 250 mg of the final product. After HPLC purification, 120 mg of liraglutide with a purity greater than 98% was obtained.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (14)

A liraglutide backbone fusion protein, characterized in that, the liraglutide fusion protein has a structure shown in formula I from N-terminus to C-terminus: FP-TEV-EK-GLP-1 (I) Where "-" represents a peptide bond; FP is the green fluorescent protein folding unit; TEV is the first restriction site, preferably TEV restriction site (as shown in the sequence ENLYFQG, SEQ ID NO.: 8); EK is the second restriction site, preferably enterokinase restriction site (as shown in the sequence DDDDK, SEQ ID NO.: 9); GLP-1 represents a Boc-modified liraglutide backbone, and the 20th position of the Boc-modified liraglutide backbone is a protected lysine. The fusion protein of claim 1, wherein the green fluorescent protein folding unit is selected from the group consisting of u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, or Its combination, and To Amino acid sequence u1 VPILVELDGDVNG(SEQ ID NO.:11) u2 HKFSVRGEGEGDAT(SEQ ID NO.:12) u3 KLTLKFICTT (SEQ ID NO.: 13) u4 YVQERTISFKD(SEQ ID NO.:14) u5 TYKTRAEVKFEGD (SEQ ID NO.: 15) u6 TLVNRIELKGIDF(SEQ ID NO.:16) u7 HNVYITADKQ(SEQ ID NO.:17) u8 GIKANFKIRHNVED (SEQ ID NO.: 18) u9 VQLADHYQQNTPIG(SEQ ID NO.:19) u10 HYLSTQSVLSKD(SEQ ID NO.:20) u11 HMVLLEFVTAAGI (SEQ ID NO.: 2). The fusion protein of claim 1, wherein the green fluorescent protein folding unit is u2-u3, u4-u5 or u4-u5-u6, wherein: To Amino acid sequence u2 HKFSVRGEGEGDAT(SEQ ID NO.:12) u3 KLTLKFICTT (SEQ ID NO.: 13) u4 YVQERTISFKD(SEQ ID NO.:14) u5 TYKTRAEVKFEGD (SEQ ID NO.: 15) u6 TLVNRIELKGIDF (SEQ ID NO.: 16). The fusion protein of claim 1, wherein the protected lysine is Nε-(tert-butoxycarbonyl)-lysine. The fusion protein of claim 1, wherein the amino acid sequence of the liraglutide backbone fusion protein is shown in SEQ ID NO.: 1, 3, 4. A method for preparing liraglutide, the method comprising the steps: (i) Provide a Boc-modified liraglutide backbone; (ii) Fmoc modification is performed on the Boc-modified liraglutide backbone to obtain Fmoc and Boc-modified liraglutide backbones; (iii) De-Boc treatment of the Fmoc and Boc-modified liraglutide main chain, and react it with the side chain of liraglutide, thereby preparing Fmoc-modified liraglutide; and (iv) The Fmoc-modified liraglutide is subjected to de-Fmoc and side chain de-tBu treatments to prepare liraglutide. The method of claim 6, wherein in step (ii), Fmoc-Osu, NaHCO ₃ and DMF/H ₂ O are added to perform Fmoc modification. Preferably, the added Fmoc-Osu, NaHCO ₃ The molar ratio with Boc-modified liraglutide backbone is (0.8-1.5): (1.5-2.5): (0.8-1.2). The method according to claim 6, characterized in that, in step (iii), it further comprises the step of: (a) Add the TFA solution, stir at low temperature, and perform de-Boc treatment to obtain a de-Boc product; (b) Purifying the Boc-removed product, preferably C8 reversed-phase purification; (c) Optionally, adding an organic solvent to the purified collection liquid obtained from the purification treatment to obtain a solid de-Boc product. Preferably, the organic solvent is a mixture of methyl tertiary ether and petroleum ether; (d) Mix the de-Boc product with the side chain of liraglutide to prepare Fmoc-modified liraglutide. The method according to claim 6, characterized in that, in step (iv), a DMF solution containing piperidine is added to undergo de-Fmoc treatment, thereby preparing liraglutide. The method according to claim 6, characterized in that, in step (i), it comprises the steps of: (ia) Using recombinant bacteria to prepare the liraglutide backbone fusion protein of claim 1, (ib) Using enterokinase to digest the liraglutide fusion protein to obtain a Boc-modified liraglutide backbone. A Boc-modified liraglutide backbone, characterized in that the 20th position of the Boc-modified liraglutide backbone is a protected lysine, and the protected lysine is Nε- (Tert-Butoxycarbonyl)-Lysine. The liraglutide backbone of claim 11, wherein the N-terminus of the liraglutide backbone is modified by Fmoc. An Fmoc-modified liraglutide backbone is characterized in that the N-terminus of the liraglutide backbone is Fmoc-modified histidine. A Fmoc-modified liraglutide, wherein the N-terminal of the liraglutide main chain is Fmoc-modified histidine, and the liraglutide main chain is connected to the side of the liraglutide chain.

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