CN113135990A - Liraglutide derivatives and preparation method thereof - Google Patents

Liraglutide derivatives and preparation method thereof Download PDF

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CN113135990A
CN113135990A CN202010066293.9A CN202010066293A CN113135990A CN 113135990 A CN113135990 A CN 113135990A CN 202010066293 A CN202010066293 A CN 202010066293A CN 113135990 A CN113135990 A CN 113135990A
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liraglutide
boc
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查若鹏
刘慧玲
唐亚连
陈卫
杨接运
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Ningbo Kunpeng Biotech Co Ltd
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Abstract

The invention provides a method for preparing a liraglutide product. Specifically, the method comprises the steps of carrying out Fmoc modification on a Boc modified liraglutide main chain and carrying out side chain addition of the liraglutide by utilizing the Fmoc modification. The invention also provides the Fmoc and Boc modified liraglutide main chains involved in the preparation method and fusion proteins containing the liraglutide main chains.

Description

Liraglutide derivatives and preparation method thereof
Technical Field
The invention relates to the field of biological medicines, and particularly relates to a GLP-1 analogue liraglutide derivative and a preparation method thereof.
Background
Diabetes is a major disease threatening human health worldwide. In China, the prevalence rate of diabetes is on a rapid rising trend along with the change of life styles and the accelerated aging process of people. Acute and chronic complications of diabetes, especially chronic complications, accumulate a plurality of organs, are disabled, have high fatality rate, seriously affect physical and psychological health of patients and bring heavy burden to individuals, families and society.
Liraglutide is a glucagon-like peptide-1 (GLP-1) analogue, promotes glucose concentration-dependent insulin secretion of pancreatic beta cells by activating GLP-1 receptor, and is clinically used for treating type 2 diabetes.
Native GLP-1 is highly susceptible to degradation by dipeptidyl peptidase IV (DPP-IV) in vivo, has a plasma half-life of less than 2 minutes, and must be continuously intravenously or subcutaneously injected to produce therapeutic effects. To overcome this clinical problem, a series of GLP-1 analogs were developed, with the most significant therapeutic effects of liraglutide marketed in the european union and the us in months 7 and 1 in 2010, respectively. The liraglutide can remarkably reduce fasting blood sugar or postprandial blood sugar of a type 2 diabetes patient to regulate the blood sugar level in the body, and simultaneously can reduce the weight of the patient and the death risk of the patient with cardiovascular disease.
There are many reports on the preparation of liraglutide at home and abroad, for example, U.S. Pat. nos. US 6268343, 6458924 and 7235627 disclose a gene recombination technique to obtain the backbone (main chain) of liraglutide, i.e., the sequence Arg34GLP-1(7-37), and then the Pal-Glu- (OSu) -OtBu substance is connected by a liquid phase synthesis method to obtain the liraglutide, because the main chain of the liraglutide is in an unprotected state, the side chain is easily connected on the amino group at the N terminal in the connection process, so that impurities are generated, the problems of difficult purification and low yield are caused, and the process is complex.
Accordingly, the skilled person is working to develop new, more long-acting insulin derivatives.
Disclosure of Invention
The invention aims to provide liraglutide derivatives and a preparation method thereof.
In a first aspect of the invention, a Boc-modified liraglutide backbone is provided, wherein the 20 th position of the Boc-modified liraglutide backbone is a protected lysine, and the protected lysine is N ∈ - (tert-butoxycarbonyl) -lysine.
In another preferred embodiment, the epsilon amino group of the protected lysine is modified with t-butyloxycarbonyl.
In another preferred embodiment, the N-terminus of the backbone of the liraglutide is Fmoc modified.
In another preferred embodiment, Fmoc is fluorenylmethyloxycarbonyl.
In another preferred embodiment, the amino acid sequence of the backbone of liraglutide is shown in SEQ ID No. 7 (HAEGTFTSDVSSYLEGQAA)KEFIAWLVRGRG, wherein H is Fmoc-modified histidine,Kboc modified lysine).
In another preferred embodiment, the backbone of liraglutide is used for the synthesis of liraglutide.
In a second aspect of the present invention, there is provided a liraglutide backbone fusion protein having a structure represented by formula I from N-terminus to C-terminus:
FP-TEV-EK-GLP-1 (I)
in the formula (I), the compound is shown in the specification,
"-" represents a peptide bond;
FP is a green fluorescent protein folding unit;
TEV is a first enzyme cutting site, preferably a TEV enzyme cutting site (shown as a sequence ENLYFQG, SEQ ID NO: 8);
EK is a second enzyme cutting site, preferably enterokinase enzyme cutting site (shown as a sequence DDDDDDK, SEQ ID NO. 9);
GLP-1 represents said Boc-modified liraglutide of the first aspect of the invention.
In another preferred embodiment, said GLP-1 does not comprise an Fmoc modification.
In another preferred embodiment, 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 a combination thereof, and
Figure BDA0002376054740000021
Figure BDA0002376054740000031
in another preferred embodiment, the green fluorescent protein folding unit is u2-u3, u4-u5 or u4-u5-u 6.
In another preferred embodiment, the N-terminus of the green fluorescent protein folding unit comprises a signal peptide, preferably the signal peptide is as 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 and 4.
In another preferred embodiment, the liraglutide backbone fusion protein is used for preparing the liraglutide backbone.
In a third aspect of the invention, there is provided a method of preparing liraglutide, the method comprising the steps of:
(i) providing a Boc-modified liraglutide backbone;
(ii) carrying out Fmoc modification on the Boc modified liraglutide main chain to prepare Fmoc and Boc modified liraglutide main chains;
(iii) carrying out Boc removal treatment on the Fmoc and Boc modified liraglutide main chain, and reacting the Fmoc and Boc modified liraglutide main chain with a liraglutide side chain to prepare Fmoc modified liraglutide; and
(iv) and (3) carrying out Fmoc removal treatment on the Fmoc modified liraglutide to prepare the liraglutide.
In another preferred embodiment, the 20 th position of the Boc-modified liraglutide backbone is a protected lysine, and the protected lysine is N ∈ - (tert-butoxycarbonyl) -lysine.
In another preferred embodiment, the Fmoc and Boc modified liraglutide backbone is Fmoc modified at the N-terminus.
In another preferred embodiment, the liraglutide side chain is N α -palmitoyl-D-glutamic acid- γ -succinimidyl-a-tert-butyl ester (Pal-Glu- (OSu) -OtBu).
In another preferred embodiment, the liraglutide side chains are as follows:
Figure BDA0002376054740000032
in another preferred embodiment, Fmoc-Osu and NaHCO are added in step (ii)3And DMF/H2O, thereby performing Fmoc modification.
In another preferred embodiment, Fmoc-Osu and NaHCO are added3The molar ratio to Boc-modified liraglutide backbone was (0.8-1.5): (1.5-2.5): (0.8-1.2), preferably (1.0-1.2): (1.8-2.2): (0.8-1.2).
In another preferred embodiment, between step (ii) and step (iii), the step of purifying the Fmoc and Boc modified liraglutide backbone obtained is further included, preferably using a C8 preparation column, with the mobile phase being TFA in water.
In another preferred embodiment, in step (iii), the method further comprises the steps of:
(a) adding TFA solution, stirring at low temperature, and removing Boc to obtain a Boc-removed product;
(b) purifying the de-Boc product, preferably performing C8 reversed phase purification;
(c) optionally, adding an organic solvent to the purified collection from the purification treatment, preferably a tert-methyl ether: petroleum ether mixed liquor;
(d) and mixing the de-Boc product with the side chain of liraglutide to prepare the Fmoc modified liraglutide.
In another preferred embodiment, in step (d), said solid Boc-removed product is mixed with the liraglutide side chain 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 embodiment, in step (d), the reaction system further comprises EDPA.
In another preferred embodiment, in step (iv), a piperidine-containing DMF solution is added to perform Fmoc removal treatment, thereby preparing liraglutide.
In another preferred embodiment, in step (iv), the purification step of the produced liraglutide is included.
In another preferred embodiment, said Boc-modified liraglutide backbone is prepared using genetic recombination techniques.
In another preferred example, in the step (i), the method comprises the steps of:
(ia) preparing the liraglutide backbone fusion protein of the second aspect of the present invention using the recombinant bacteria,
(ib) carrying out enzyme digestion treatment on the liraglutide fusion protein by using enterokinase, thereby obtaining the Boc modified liraglutide main chain.
In another preferred example, in the step (ia), the liraglutide backbone fusion protein inclusion body is obtained by separating from the fermentation broth of the recombinant bacterium, and the liraglutide backbone fusion protein is obtained after the inclusion body is renatured and enzyme-cut.
In another preferred embodiment, a purification step is further included before and after step (ib).
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 bacterium comprises or integrates an expression cassette for expressing the liraglutide backbone fusion protein.
In a fourth aspect of the invention, there is provided an isolated polynucleotide encoding the Boc modified liraglutide backbone of the first aspect of the invention or the fusion protein of the second aspect of the invention.
In a fifth aspect of the invention, there is provided a vector comprising a polynucleotide according to the fourth aspect of the invention.
In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.
According to a sixth aspect of the present invention, there is provided a host cell comprising a vector according to the fifth aspect of the present invention, or having integrated into its chromosome an exogenous polynucleotide according to the fourth aspect of the present invention, or expressing a fusion protein according to the second aspect of the present invention.
In another preferred embodiment, the host cell is Escherichia coli, Bacillus subtilis, a yeast cell, an insect cell, a mammalian cell, or a combination thereof.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
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FIG. 1 shows a map of plasmid pBAD-FP-TEV-EK-GLP-1 (20).
FIG. 2 shows a map of the plasmid pEvol-pylRs-pylT.
FIG. 3 shows a SDS-PAGE electrophoresis of Boc-liraglutide backbone fusion proteins after renaturation of inclusion bodies.
FIG. 4 shows the HPLC detection profile of the Boc-liraglutide intermediate polypeptide.
Fig. 5 shows a process for preparing liraglutide of the present invention.
Detailed Description
The present inventors have extensively and intensively studied and found a novel method for preparing liraglutide products. Specifically, the method utilizes an Fmoc orthogonal protection method to perform a side chain addition step in the preparation process of the liraglutide, and optimizes the purification and synthesis conditions in the preparation process. The method of the invention does not need expensive solid phase synthesis instruments, shortens the production period, has simple production process and improves the purity and the yield of the product.
Liraglutide
Liraglutide was developed by noh and knode corporation, having the 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, and has the sequence: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (N epsilon (N alpha-PAL-gamma-Glu)) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, with a sequence homology of 97% with the human native GLP-1.
The structure of liraglutide is that 28 th lysine of natural GLP-1(7-37) molecule is replaced by arginine, and epsilon amino group of 20 th lysine side chain is acylated by hexadecanoic acid glutamic acid. Due to the existence of the aliphatic chain, the degradation effect of DPP-4 can be reduced, the half-life period is prolonged, and the administration frequency reaches once a day. Can significantly reduce fasting or postprandial blood sugar of type 2 diabetes patients to achieve the regulation of blood sugar level in vivo, and simultaneously can reduce the weight of the patients and the death risk of the patients with cardiovascular diseases.
Liraglutide fusion proteins
The invention provides a liraglutide fusion protein which has a structure shown in a formula I from an N end to a C end:
FP-TEV-EK-GLP-1 (I)
in the formula (I), the compound is shown in the specification,
"-" represents a peptide bond;
FP is a green fluorescent protein folding unit;
TEV is a first enzyme cutting site, preferably a TEV enzyme cutting site (shown as a sequence ENLYFQG, SEQ ID NO: 8);
EK is a second enzyme cutting site, preferably enterokinase enzyme cutting site (shown as a sequence DDDDDDK, SEQ ID NO. 9);
GLP-1 represents the Boc modified liraglutide according to the first aspect of the invention.
In another preferred embodiment, 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-u 9-u 9-u 9, u9-u 9-u 9-u 9, u 9-36u 9, u 9-9, u 36u 9-36u 9, u 36u 9-9, u 9-36u 9-9, u 9-36u 9-9, u 9-9, u 9-36u 9-9, u 9-36u 9-9, u-36u-9, u 36u 9, u 9-36u 9, u 36u 9-9, u 9-36u 9, u-9, u 9-9, u-9, u 9-36u-9, u-9, u 9-9, u 9-9, u 36u-9, u 9-36u 9, u 36u-9, u-36u-9, u-9-, u1-I-u5, u2-I-u4, u3-I-u8, u5-I-u6, or u10-I-u 11.
And, the units have the sequence shown below:
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 sequence of the fusion protein of the invention is as follows:
FP1-TEV-EK-GLP-1(20), the amino acid sequence is shown in SEQ ID NO. 1:
MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFISTTENLYFQGDDDDKHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG*
FP2-TEV-EK-GLP-1(20), the amino acid sequence is shown in SEQ ID NO. 3:
MVSKGEELFTGVYVQERTISFKDTYKTRAEVKFEGDVNGHKFSVRGEGEGDATNGKLTLKFISTTENLYFGDDDDKHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG*
FP3-TEV-EK-GLP-1(20), the amino acid sequence is shown in SEQ ID NO. 4:
MVSKGEELFTGVYVQERTISFKDTYKTRAEVKFEGDTLVNRIELKGIDFVNGHKFSVRGEGEGDATNGKLTLKFISTTENLYFQGDDDDKHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG*
whereinKIs Boc modified lysine.
The term "fusion protein" as used herein also includes variants having the above-described activities. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).
The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which a polypeptide is fused with another compound (such as a compound for increasing the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused with the polypeptide sequence (a fusion protein in which a tag sequence such as a leader sequence, a secretory sequence or 6His is fused). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 3, preferably up to 2, more preferably up to 1 amino acid with an amino acid of similar or analogous nature compared to the amino acid sequence of the present invention. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Figure BDA0002376054740000081
Figure BDA0002376054740000091
The invention also provides analogs of the fusion proteins of the invention. These analogs may differ from the polypeptides of the invention by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
In addition, modifications may be made to the fusion proteins of the invention. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.
The term "polynucleotide encoding a fusion protein of the present invention" may include a polynucleotide encoding a fusion protein of the present invention, and may also include polynucleotides that additionally include coding and/or non-coding sequences.
The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the 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 is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the fusion protein encoded thereby.
The present invention also relates to polynucleotides which hybridize to the sequences described above and which 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 hybridizable under stringent conditions (or stringent conditions) with the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more.
The fusion proteins and polynucleotides of the invention are preferably provided in isolated form, and more preferably, purified to homogeneity.
The full-length sequence of the polynucleotide of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.
Methods for amplifying DNA/RNA using PCR techniques are preferably used to obtain the polynucleotides of the invention. Particularly, when it is difficult to obtain a full-length cDNA from a library, it is preferable to use the RACE method (RACE-cDNA terminal rapid amplification method), and primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
Expression vector
The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells transformed with the vectors of the invention or the coding sequences of the fusion proteins of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.
The polynucleotide sequences of the present invention may be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques. Generally, the following steps are performed:
(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a fusion protein of the invention, or with a recombinant expression vector comprising the polynucleotide;
(2) a host cell cultured in a suitable medium;
(3) isolating and purifying the protein from the culture medium or the cells.
In the present invention, the polynucleotide sequence encoding the fusion protein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain 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 expression vectors containing the DNA sequences encoding the fusion proteins of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling 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.
Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.
Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast, plant cells (e.g., ginseng cells).
When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer at the late side of the replication origin at 100 to 270 bp, the polyoma enhancer at the late side of the replication origin, and adenovirus enhancers.
It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
Construction of Liraglutide expression vector
The expression construct FP-TEV-EK-GLP1 contains the gene encoding GLP1 fused to the C-terminus of FP-TEV-EK. The sequence is subjected to codon optimization, and can realize high-level expression of functional protein in escherichia coli. After expression, the expression vector "pBAD/His A (Kanar)" was cut with restriction enzymes Nco I and Xho I, the cut products were separated by agarose electrophoresis, extracted with agarose gel DNA recovery kit, and finally the two DNA fragments were ligated with T4 DNA ligase. The ligation product was chemically transformed into E.coli Top10 cells, and the transformed cells were cultured overnight on LB agar medium (10g/L yeast peptone, 5g/L yeast extract powder, 10g/L NaCl, 1.5% agar) containing 50. mu.g/mL kanamycin. 3 viable colonies were picked, cultured overnight in 5mL of liquid LB medium (10g/L yeast peptone, 5g/L yeast extract powder, 10g/L NaCl) containing 50. mu.g/mL kanamycin, and plasmid extraction was performed using a plasmid miniprep kit. The extracted plasmid was then sequenced using sequencing oligonucleotide primer 5'-ATGCCATAGCATTTTTATCC-3' to confirm correct insertion. The resulting plasmid was named "pBAD-FP-TEV-EK-GLP 1".
Fmoc modification
In the field of biological medicine, the use of polypeptides is increasing, amino acids are basic raw materials for polypeptide synthesis technology, and all amino acids contain alpha-amino and carboxyl, and some also contain side chain active groups, such as: hydroxyl, amino, guanidyl, heterocycle and the like, therefore, amino and side chain active groups need to be protected in a peptide grafting reaction, and the protecting groups are removed after the polypeptide is synthesized, so that amino acid misconnection and a plurality of side reactions can occur.
Fmoc is a base-sensitive protecting group and can be removed in 50% dichloromethane solution of ammonia such as concentrated ammonia water or dioxane-methanol-4N Na OH (30: 9: 1), piperidine, ethanolamine, cyclohexylamine, 1, 4-dioxane, pyrrolidone, etc.
Fmoc-protecting groups are generally introduced by Fmoc-Cl or Fmoc-OSu under weakly basic conditions such as sodium carbonate or sodium bicarbonate. Fmoc-OSu allows easier control of reaction conditions and fewer side reactions than Fmoc-Cl. The Fmoc protecting group is particularly stable under acidic conditions and very sensitive to basic conditions, so amino acids containing a reactive side chain group are usually protected with the acid sensitive protecting group Boc or Z.
Fmoc has strong ultraviolet absorption with maximum absorption wavelengths of 267nm (. epsilon.18950), 290nm (. epsilon.5280) and 301nm (. epsilon.6200), so that the detection can be realized by using the ultraviolet absorption, and a plurality of convenience is brought to the automatic polypeptide synthesis of an instrument. Moreover, the method is compatible with a wide range of solvents and reagents, has high mechanical stability, and can be used for various carriers and various activation modes, and the like. Therefore, the Fmoc protecting group is most commonly used in polypeptide synthesis today.
Fmoc-OSu (fluorenylmethoxycarbonylsuccinimides)
Figure BDA0002376054740000131
Liraglutide side chain
Pal-Glu- (OSu) -OtBu is N alpha-palmitoyl-D-glutamic acid-gamma-succinimidyl-A-tert-butyl ester, which is referred to as a D-type-liraglutide side chain for short.
Figure BDA0002376054740000132
The preparation of liraglutide is that firstly utilizing gene recombination technology to obtain the liraglutide main chain of 20-bit Boc protected lysine, namely sequence Arg34GLP-1(7-37), and then liraglutide side chain Pal-Glu- (OSu) -OtBu is connected, so that liraglutide is obtained.
Preparation of liraglutide
The invention provides a liraglutide synthesis route which is shown in figure 5, Fmoc modified compound 2 is prepared from Boc-liraglutide main chain (compound 1), compound 2 is subjected to Boc protection removal to obtain compound 3, compound 3 is reacted with activated liraglutide side chain Pal-Glu- (OSu) -OtBu to obtain compound 4, then compound 5 is obtained through Fmoc removal reaction, tBu protecting group is removed from the side chain, and finally liraglutide is obtained.
Specifically, the present invention provides a method for preparing liraglutide, comprising the steps of:
(i) providing a Boc-modified liraglutide backbone;
(ii) carrying out Fmoc modification on the Boc modified liraglutide main chain to prepare Fmoc and Boc modified liraglutide main chains;
(iii) carrying out Boc removal treatment on the Fmoc and Boc modified liraglutide main chain, and reacting the Fmoc and Boc modified liraglutide main chain with a liraglutide side chain to prepare Fmoc modified liraglutide; and
(iv) and (3) carrying out Fmoc removal and side chain tBu removal treatment on the Fmoc modified liraglutide to prepare the liraglutide.
In another preferred embodiment, Fmoc-Osu and NaHCO are added in step (ii)3And DMF/H2O, thereby performing Fmoc modification.
In another preferred embodiment, Fmoc-Osu and NaHCO are added3The molar ratio to Boc-modified liraglutide backbone was (0.8-1.5): (1.5-2.5): (0.8-1.2), preferably (1.0)-1.2)：(1.8-2.2)：(0.8-1.2)。
In another preferred embodiment, between step (ii) and step (iii), the step of purifying the Fmoc and Boc modified liraglutide backbone prepared is further included, preferably using a C8 preparation column with a mobile phase of TFA in acetonitrile.
In another preferred embodiment, in step (iii), the method further comprises the steps of:
(a) adding TFA solution, stirring at low temperature, and removing Boc to obtain a Boc-removed product;
(b) purifying the de-Boc product, preferably performing C8 reversed phase purification;
(c) optionally, adding an organic solvent to the purified collection from the purification treatment, preferably a tert-methyl ether: petroleum ether mixed liquor;
(d) and mixing the de-Boc product with the side chain of liraglutide to prepare the Fmoc modified liraglutide.
In another preferred example, in the step (i), the method comprises the steps of:
(ia) preparing the liraglutide backbone fusion protein of the second aspect of the present invention using the recombinant bacteria,
(ib) carrying out enzyme digestion treatment on the liraglutide fusion protein by using enterokinase, thereby obtaining the Boc modified liraglutide main chain.
In another preferred example, in the step (ia), liraglutide backbone fusion protein inclusion bodies are obtained by separating from the fermentation broth of the recombinant bacteria, and Boc-liraglutide backbone fusion protein is obtained after denaturation and enzyme digestion of the inclusion bodies.
The main advantages of the invention include:
(1) the invention directly utilizes a biosynthesis mode to produce the Boc modified liraglutide main chain without adopting methods such as dilution, ultrafiltration liquid exchange and the like to remove excessive inorganic salt in the supernatant of the fermentation liquid. In the method of the present invention, the Boc-liraglutide backbone or analog precursor is separated using a chromatography column at a yield of over 70% in one step, 3-fold higher than that of the conventional method, and the Boc-liraglutide backbone yield is about 600-700 mg. Moreover, the method can remove most of pigments, and the original multi-step process is directly separated and purified in one step, so that the process time and the equipment investment cost are reduced;
(2) due to the protection of the Boc-lysine at the 26 th position, the invention can directly utilize orthogonal reaction with Fmoc protection to synthesize the liraglutide.
(3) The liraglutide synthesized by the method disclosed by the invention has no impurities acylated by the fatty acid at the N-terminal, is beneficial to downstream purification and reduces the cost.
(4) Compared with solid phase synthesis, the method of the invention does not produce racemized impurity polypeptide, does not need to use a large amount of modified amino acid, does not use a large amount of organic reagent, has small environmental pollution and lower cost;
the invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1 construction of liraglutide-expressing strains
The construction of the liraglutide expression vector is described in the examples in patent application No. 201910210102.9. The DNA fragment of fusion protein FP1-TEV-EK-GLP-1(20) (expression cassette expressing liraglutide backbone fusion protein) was cloned into the NcoI-XhoI site downstream of the araBAD promoter of expression vector plasmid pBAD/His A (purchased from NTCC, kanamycin resistance) to give plasmid pBAD-FP-TEV-EK-GLP-1 (20). The plasmid map is shown in FIG. 1.
The DNA sequence of pylRs was then cloned into the SpeI-SalI site downstream of the araBAD promoter of the expression vector plasmid pEvol-pBpF (available from NTCC for chloramphenicol resistance), while the DNA sequence of the tRNA (pylTcua) of lysyl-tRNA synthetase (SEQ ID NO: 6) was inserted downstream of the proK promoter by PCR. This plasmid was designated pEvol-pylRs-pylT. The plasmid map is shown in FIG. 2.
Amino acid sequence of pylRs (SEQ ID No.: 5):
MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL*
DNA sequence of tRNA (pylTcua) (SEQ ID NO: 6):
GGAAACCTGATCATGTAGATCGAATGGACTCTAAATCCGTTCAGCCGGGTTAGATTCCCGGGGTTTCCGCCA
the constructed plasmid pBAD-FP-TEV-EK-GLP-1(20) and pEvol-pylRs-pylT are jointly transformed into an escherichia coli TOP10 strain, and a recombinant escherichia coli strain expressing liraglutide backbone fusion protein FP1-TEV-EK-GLP-1(20) is obtained through screening.
Example 2 expression of Boc-liraglutide backbone
Recombinant Escherichia coli was inoculated into an Escherichia coli seed solution (cultured by Co.) at 37 ℃ and pH7.0 in an inoculum size (volume ratio) of 5%, fed in batch until pH increased to 7.05, and then carbon-nitrogen source separate feeding was started, and carbon source feeding was performed according to the constant pH method. Feeding for 11h, and finishing fermentation, wherein the mass ratio of carbon to nitrogen is 1: 1.0. After the feed supplement, the pH value is controlled to be 7.0-7.2 by feed supplement and automatic fed-batch addition of 7.5M ammonia water. Culturing for 4-6 hr, adding 2.5g/L L-arabinose for induction, and continuing for 14 hr until fermentation is finished. Obtaining a fermentation broth comprising the liraglutide backbone fusion protein.
Example 3 preparation of Boc-liraglutide backbone inclusion bodies
After centrifuging the fermentation broth obtained in example 2, the wet biomass was centrifuged at a ratio of 1:1 volume and broken fungus buffer solution mix, and the suspension is 3h, and the suspension uses the broken fungus of high pressure homogenizer three times, and the inclusion body is collected in the centrifugation after breaking the fungus, washs it twice, and the buffer solution composition is: 0.5% T-80, 1mm EDTA-2Na, 100mm NaCl, pH 7.5. The yield of the inclusion bodies weighed after cleaning is 41-45 g/L.
The SDS-PAGE electrophoresis result is shown in figure 3, and the result shows that the fusion protein is expressed, and the Boc-liraglutide main chain inclusion body is obtained after the thalli are crushed, washed and centrifuged.
Example 4 denaturation and cleavage of Boc-liraglutide backbone inclusion bodies
7.5mol/L urea-dissolving buffer was added to the inclusion bodies obtained in example 3 at a weight/volume ratio of 1:10, the mixture was dissolved with stirring at room temperature, the protein concentration was measured by the Bradford method, the total protein concentration of the inclusion body-dissolving solution was controlled to about 25mg/ml, and the pH was adjusted to 9.0. + -. 0.1 with NaOH. Dropping the inclusion body solution into a solution containing 5-10mmol/L Tris, 10mmol/L NaCl and 10mmol/L Na2CO30.3-0.5mmol/L EDTA-2Na in renaturation buffer solution, diluting the inclusion body dissolving solution by 5-10 times for renaturation, maintaining the pH value of the fusion protein renaturation solution at 9.0-10.0, controlling the temperature at 4-8 ℃, and controlling the renaturation time at 10-20 h.
The results showed that the fusion protein was present in about 33% after solubilization.
Example 5 Primary purification of Boc-liraglutide fusion proteins
Filtering the fusion protein renaturation solution obtained in the embodiment 4 by a filter membrane of 0.45 mu m to remove undissolved substances; according to the difference of isoelectric points of the proteins, the fusion protein is primarily purified by adopting an anion exchange column filled with Q Sepharose FF.
Experimental results show that after anion exchange chromatography, the purity of the Boc-liraglutide main chain fusion protein reaches more than 65%, the loading capacity is about 18mg/mL, and the yield is more than 80%.
Example 6 enzymatic cleavage of Boc-liraglutide backbone fusion proteins
The Boc-liraglutide backbone fusion protein sample initially purified in example 5 was desalted through a hydrophobic column and eluted with pure water in a volume of about 5 times the column volume. Adjusting the pH value of the fusion protein solution to 7.5-8.5, controlling the temperature to 25 ℃, adding enterokinase for enzyme digestion for 5-16h to obtain a Boc-liraglutide main chain, wherein the Boc-liraglutide main chain is about 600mg/L, and the enzyme digestion efficiency is more than or equal to 85%.
Example 7 reverse phase chromatography of Boc-liraglutide backbone
And purifying the Boc-liraglutide main chain by adopting a polymer reverse phase chromatography technology according to the hydrophobicity difference of the polypeptide and the protein to remove a part of impurities.
The enzyme-digested solution of Boc-liraglutide backbone fusion protein obtained in example 6 was filtered, clarified, and purified by reverse-phase chromatography. Taking an aqueous solution containing 0.065% of trifluoroacetic acid as a mobile phase A; acetonitrile solution containing 0.065% trifluoroacetic acid was used as mobile phase B. And combining the Boc-liraglutide main chain with a filler, controlling the loading amount of the Boc-liraglutide main chain to be less than 10mg/ml, then carrying out gradient elution, and collecting the Boc-liraglutide main chain. The experimental result shows that the purity of the Boc-liraglutide main chain collected by the polymer reverse phase chromatography is more than or equal to 90 percent, the yield is more than 60 percent, and an HPLC detection map of the Boc-liraglutide main chain is shown in figure 4.
Example 8 preparation of liraglutide Using Boc-liraglutide backbone
Boc-liraglutide backbone compound 1 obtained in example 7 was added Fmoc-Osu and NaHCO at the molar ratio shown in Table 13And DMF/H2And O, reacting for 8-12 hours to obtain the GLP-1 protected by Fmoc and Boc. The purification was carried out using a C8 column, eluting with a gradient of 0.065% (v/v) TFA in water as mobile phase A and 0.065% (v/v) TFA in acetonitrile as mobile phase B. And (3) adding methyl tert-ether into the purified and collected solution, precipitating and centrifuging, and washing the precipitate for 2-3 times by using methyl tert-ether to obtain an Fmoc protected compound 2: DiFmoc-GLP-1 (Lys)20Boc)。
TABLE 1 molar ratio of the feeds
Boc-liraglutide Fmoc-OSu NaHCO3 DMF/H2O
Equivalent weight or volume 1.0eq 1.1eq 2.0eq 30V/30V
And (3) adding a TFA solution into the purified compound 2, stirring at low temperature for 10-20 min, and performing reverse phase purification on the deprotection reaction product by C8. To the purified pool was added 20 volumes of methyl tert-ether: and (3) petroleum ether mixed liquor (3:1), precipitating and centrifuging, washing the precipitate for 2-3 times by using the mixed liquor, and finally obtaining a solid compound 3 without Boc: DiFmoc-GLP-1 (Lys)20NH2)。
The Boc-removed compound 3 was added with 30eq. EDPA, NMP and water mixture (2:1) and stirred gently at room temperature for 5 min. An equivalent amount of Pal-Glu- (OSu) -OtBu (23.7. mu. mol) dissolved in NMP (303. mu.L) was added to the resulting mixture, and the reaction mixture was gently shaken at room temperature for 2 hours. To this was added 625 μ L of 50% aqueous ethanol solution containing glycine (6.5mg, 86.9 μmol), thereby terminating the reaction to obtain compound 4: DiFmoc-GLP-1- (Pal-Glu- (Lys)20NH2)-OtBu)。
After purification, Compound 4(300mg) was added to a DMF solution containing 20% piperidine and reacted at room temperature for 30 minutes. Adding a mixed solvent of methyl tert-ether and petroleum ether with the volume 10 times that of the reaction system, precipitating and centrifuging, washing the solid with the mixed solvent of methyl tert-ether and petroleum ether for 3-5 times to obtain a compound 5(270mg) after Fmoc removal: GLP-1- (Pal-Glu- (Lys)20NH2)-OtBu)。
GLP-1 Compound 5(270mg) was taken in and added to (TFA: TIS: H)2O95: 2.5:2.5) and 10mL of DCM (v: 1) mixed solution, carrying out shake reaction at room temperature for 3-4 hours to remove a side chain tBu protecting group, adding a 10-fold volume of mixed solvent of methyl tert-ether and petroleum ether into the reaction system, precipitating and centrifuging, washing the solid with the mixed solvent of methyl tert-ether and petroleum ether for 3 times to obtain 250mg of the solidThe final product of (a). After HPLC purification, 120mg liraglutide with a purity greater than 98% was obtained.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Asp Asp Asp Asp Lys
1 5
<210> 10
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val
1 5 10
<210> 11
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
1 5 10
<210> 12
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly Asp Ala Thr
1 5 10
<210> 13
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr
1 5 10
<210> 14
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Tyr Val Gln Glu Arg Thr Ile Ser Phe Lys Asp
1 5 10
<210> 15
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
1 5 10
<210> 16
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
1 5 10
<210> 17
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
His Asn Val Tyr Ile Thr Ala Asp Lys Gln
1 5 10
<210> 18
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp
1 5 10
<210> 19
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
1 5 10
<210> 20
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
His Tyr Leu Ser Thr Gln Ser Val Leu Ser Lys Asp
1 5 10

Claims (10)

1. A Boc modified liraglutide backbone, wherein the 20 th position of the Boc modified liraglutide backbone is a protected lysine, and the protected lysine is N epsilon- (tert-butyloxycarbonyl) -lysine.
2. The liraglutide backbone of claim 1, wherein the N-terminus of the liraglutide backbone is Fmoc modified.
3. A liraglutide backbone fusion protein, wherein the liraglutide backbone fusion protein has a structure represented by formula I from N-terminus to C-terminus:
FP-TEV-EK-GLP-1 (I)
in the formula (I), the compound is shown in the specification,
"-" represents a peptide bond;
FP is a green fluorescent protein folding unit;
TEV is a first enzyme cutting site, preferably a TEV enzyme cutting site (shown as a sequence ENLYFQG, SEQ ID NO: 8);
EK is a second enzyme cutting site, preferably enterokinase enzyme cutting site (shown as a sequence DDDDDDK, SEQ ID NO. 9);
GLP-1 represents the Boc modified liraglutide of claim 1.
4. The fusion protein of claim 3, wherein the green fluorescent protein folding unit is u2-u3, u4-u5, or u4-u5-u6, wherein,
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)。
5. The fusion protein of claim 3, wherein the amino acid sequence of the liraglutide backbone fusion protein is set forth in SEQ ID No. 1, 3, 4.
6. A method of preparing liraglutide, the method comprising the steps of:
(i) providing a Boc-modified liraglutide backbone;
(ii) carrying out Fmoc modification on the Boc modified liraglutide main chain to prepare Fmoc and Boc modified liraglutide main chains;
(iii) carrying out Boc removal treatment on the Fmoc and Boc modified liraglutide main chain, and reacting the Fmoc and Boc modified liraglutide main chain with a liraglutide side chain to prepare Fmoc modified liraglutide; and
(iv) and (3) carrying out Fmoc removal and side chain tBu removal treatment on the Fmoc modified liraglutide to prepare the liraglutide.
7. The method of claim 6, wherein in step (ii), Fmoc-Osu and NaHCO are added3And DMF/H2O, thereby carrying out Fmoc modification, preferably, added Fmoc-Osu, NaHCO3The molar ratio to Boc-modified liraglutide backbone was (0.8-1.5): (1.5-2.5): (0.8-1.2).
8. The method of claim 6, wherein in step (iii), further comprising the step of:
(a) adding TFA solution, stirring at low temperature, and removing Boc to obtain a Boc-removed product;
(b) purifying the de-Boc product, preferably performing C8 reversed phase purification;
(c) optionally, adding an organic solvent to the purified collection from the purification treatment, preferably a tert-methyl ether: petroleum ether mixed liquor;
(d) and mixing the de-Boc product with the side chain of liraglutide to prepare the Fmoc modified liraglutide.
9. The method of claim 6, wherein in step (iv), the liraglutide is prepared by performing Fmoc removal treatment by adding piperidine-containing DMF.
10. The method of claim 6, wherein in step (i), comprising the steps of:
(ia) preparing the liraglutide backbone fusion protein of claim 2 using a recombinant bacterium,
(ib) carrying out enzyme digestion treatment on the liraglutide fusion protein by using enterokinase, thereby obtaining the Boc modified liraglutide main chain.
CN202010066293.9A 2020-01-20 2020-01-20 Liraglutide derivatives and preparation method thereof Pending CN113135990A (en)

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