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CN116554299A - Long-acting GLP-1 polypeptide analogue, and preparation method and application thereof - Google Patents

Long-acting GLP-1 polypeptide analogue, and preparation method and application thereof Download PDF

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CN116554299A
CN116554299A CN202211555764.8A CN202211555764A CN116554299A CN 116554299 A CN116554299 A CN 116554299A CN 202211555764 A CN202211555764 A CN 202211555764A CN 116554299 A CN116554299 A CN 116554299A
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ser
glu
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孟静
蒋秀苹
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Qingdao Borui Jingchuang Technology Co ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • A—HUMAN NECESSITIES
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Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a long-acting GLP-1 polypeptide analogue for treating or preventing diabetes or obesity, and a preparation method and application thereof. Solves the problems of short half-life period and insufficient metabolic stability of complex polypeptide. The amino acid sequence structure of the long-acting GLP-1 polypeptide analogue is as follows: the Lys at the 20 th position is connected with a side arm short peptide chain in the following connection mode: the amino group of the 'side arm' short peptide chain of the Lys at the 20 th position forms an amide bond with the carboxyl group of the glycine of the 'side arm' short peptide chain; the "side arm" short peptide chain end "Z" amino acid is linked to a fatty acid substituent. The long-acting GLP-1 polypeptide analogue has the advantages of long half-life, high synthesis yield, good stability, easy scale-up production and low cost, and has good drug effect of treating diabetes and reducing weight.

Description

Long-acting GLP-1 polypeptide analogue, and preparation method and application thereof
The application is a divisional application of an invention patent application with the application number 202110815583.3 and the name of long-acting GLP-1 polypeptide analogue, a preparation method and application thereof, which are submitted by 2021, 07 and 19 days.
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a long-acting GLP-1 polypeptide analogue for treating or preventing diabetes or obesity, and a preparation method and application thereof.
Background
Diabetes is a chronic disease in which the body is at a high blood sugar level for a long time to cause the body to have a disturbed sugar metabolism, and is mainly characterized in that: chronic hyperglycemia, and concomitantly with insulin secretion deficiency or insulin dysfunction, further causes chronic damage to various organs by affecting fat, carbohydrate and protein metabolism, leading to progressive organ dysfunction and even organ failure.
Worldwide diabetic patients have increased three times over the past three decades. Worldwide, approximately 9% of adults suffer from type 2 diabetes (T2 DM). The advent of T2DM and its complications greatly exacerbates the worldwide risk of disability and death. For example, global disease risk study 2013 identified diabetes (all forms) as the ninth leading cause of reduced life expectancy. Diabetes has become another important chronic non-infectious disease that seriously jeopardizes human health after cardiovascular and cerebrovascular diseases and tumors.
Obesity and diabetes are metabolic diseases, and obesity is closely related to diabetes occurrence. Diabetes occurs mainly due to decline of islet beta cell function and insulin resistance, obesity being a key factor for insulin resistance. Because the weight of the obese patient exceeds standard and the fat content is high, insulin resistance is easy to generate, and the insulin resistance can not exert the corresponding hypoglycemic effect on the insulin in the body. Insulin is the only hypoglycemic hormone in the body, and the body must increase the insulin secretion capacity of islet beta cells in order to control blood sugar, and diabetes mellitus occurs when the insulin secretion is increased and the blood sugar is still not normal, so obesity is the root cause of insulin resistance and is also an important cause of diabetes mellitus.
In the 60 s of the 20 th century, mcIntyre and Elrick et al observed an interesting phenomenon, with oral glucose having a significantly higher promoting effect on insulin secretion than intravenous injection, this effect being termed the "incretin effect", followed by the discovery of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin release peptide (GIP) in small intestinal mucosal extracts. GLP-1 is a hormone that induces insulin secretion and has beneficial effects on a variety of important organs including pancreas, heart, liver, and the like. The GLP-1 receptor (GLP-1R) agonist drugs have the advantages of effectively controlling blood sugar, obviously reducing the occurrence rate of hypoglycemic events, and also having the benefits of obviously reducing weight and reducing the risk of cardiovascular events. However, GLP-1 drugs are unstable due to their own specific polypeptide structure, can be degraded by gastric acid after oral administration, can be administered basically only by subcutaneous injection, and have a short half-life.
With intensive studies on diabetes and its treatment, GLP-1 receptor agonists such as liraglutide and cable Ma Lutai have been approved for the market in recent years. Among them, the affinity of the increased carbon chain of cord Ma Lutai to albumin is greatly enhanced, and the clearance of kidney to albumin is greatly slowed down. Two modifications can prolong the half-life period of the rat to about 8 hours, and only one subcutaneous injection is needed in clinic.
Nevertheless, how to solve the problems of short half-life and insufficient metabolic stability of polypeptides, particularly complex polypeptides, in a breakthrough manner remains a major scientific and core problem in the art; the development of the ultra-long-acting polypeptide drug molecule modification technology is key, and is also a bottleneck to be broken through internationally in the field of research.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a novel class of longer acting GLP-1 polypeptide analogues.
It is a further object of the present invention to provide a method for preparing such long acting GLP-1 polypeptide analogues.
It is a further object of the present invention to provide a composition comprising a long acting GLP-1 polypeptide analogue as described above.
It is a further object of the present invention to provide the use of a GLP-1 polypeptide analogue as described above.
A long acting GLP-1 polypeptide analogue according to an embodiment of the invention has the amino acid sequence as follows:
His-D-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-L
ys{(Gly) x -(Ser-Gly) y -γGlu-CO(CH 2 ) n CO 2 H}-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gl y
wherein:
xaa9 is Asp or Glu;
xaa21 is Glu or Gln or Asp;
x is an integer from 1 to 4; y is an integer from 1 to 4; n is an integer from 12 to 20, i.e., n is 12, 13, 14, 15, 16, 17, 18, 19 or 20.
Wherein,,
His-D-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly as the main peptide chain;
(Gly) x -(Ser-Gly) y -γGlu-CO(CH 2 ) n CO 2 h is a side arm structure;
the "sidearm" structure includes a "sidearm" short peptide chain- (Gly) x- (Ser-Gly) y- γGlu-and a fatty acid substituent-CO (CH) attached thereto 2 ) n CO 2 H。
The side chain amino group of the 20 th Lys in the amino acid sequence of the main peptide chain is connected with a side arm short peptide chain structure in an amide bond forming way with the carboxyl of the glycine residue at the other end;
further, the amino group of the "side arm" short peptide chain end "Z" amino acid residue is attached to the fatty acid substituent by amide bond formation with the carboxyl group.
The carboxyl end of the amino acid sequence of the main peptide chain is not modified, or is modified by amino to form-CONH 2 A group.
Preferably Xaa9 is Asp and Xaa21 is Glu or Gln.
Preferably Xaa9 is Glu and Xaa21 is Asp.
Preferably, x is 1 or 2, y is 1, 2 or 3; n is any integer from 14 to 18.
A long acting GLP-1 polypeptide analogue according to an embodiment of the invention, wherein x is 2, y is 3 and n is 18 in its amino acid sequence, has the following sequence: ,
His-D-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{(Gly) 2 -(Ser-Gly) 3 -γGlu-CO(CH 2 ) 18 CO 2 H}-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
preferably, the GLP-1 polypeptide analog is any one of the following compounds:
compound 6 (SEQ ID No. 6):
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2 ) 18 CO 2 H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
shorthand: hsegtftsdvssylegqaak (GGSGSGSG-gamma-E-CO (CH) 2 ) 18 CO 2 H)EFIAWLVRGRG；
Compound 7 (SEQ ID No. 7):
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2 ) 18 CO 2 H)-Gln-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
shorthand: hsegtftsdvssylegqaak (GGSGSGSG-gamma-E-CO (CH) 2 ) 18 CO 2 H)QFIAWLVRGRG；
Compound 8 (SEQ ID No. 8):
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Glu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2 ) 18 CO 2 H)-Asp-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
shorthand: hsegtftsevssylegqaak (GGSGSGSG-gamma-E-CO (CH) 2 ) 18 CO 2 H)DFIAWLVRGRG；
The preparation method of the long-acting GLP-1 polypeptide analogue according to the specific embodiment of the invention comprises the following steps:
step 1: synthesizing main peptide resin corresponding to the main peptide chain of the long-acting GLP-1 polypeptide analogue according to Fmoc/t-Bu strategy, wherein the main peptide chain is
His-D-Ser--Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
Step 2: based on the main peptide resin, coupling a side arm structure corresponding to the long-acting GLP-1 polypeptide analogue according to an Fmoc/t-Bu strategy to obtain a polypeptide resin corresponding to the long-acting GLP-1 polypeptide analogue; wherein the side arm structure is (Gly) x -(Ser-Gly) y -γGlu-CO(CH 2 ) n CO 2 H；
Step 3: adding a cracking solution into the polypeptide resin, performing a cracking reaction, removing the full protection of the polypeptide, and extracting a crude compound;
purifying the crude compound to obtain the long-acting GLP-1 polypeptide analogue.
According to a method for preparing a GLP-1 polypeptide analogue of an embodiment of the invention,
in the step 2, 1-hydroxybenzotriazole and N, N-diisopropylcarbodiimide are used as coupling agents, N-dimethylformamide is used as a solvent, and Fmoc groups are removed by 20% piperidine/N, N-dimethylformamide solution;
in step 3, the lysate is prepared from TFA, DODT, m-cresol, H 2 The volume ratio of O is 92.5:2.5:2.5: 2.5; the crude compound extraction mode comprises filtration, precipitation and/or methyl tertiary butyl ether extraction.
In the step 4, the purity of the obtained long-acting GLP-1 polypeptide analogue is more than 96%.
It is a further object of the present invention to provide a composition comprising a long acting GLP-1 polypeptide analog, further comprising a pharmaceutically acceptable carrier or adjuvant. For example, carriers capable of reducing degradation and loss of drugs, reducing side effects, such as carriers for micelles, microemulsions, gels, and the like; adjuvants refer to materials added to make the drug into a suitable dosage form, such as buffers, lyophilization excipients, and the like, to enable the pharmaceutical composition containing the polypeptide analog of the invention to be formulated as a solution or lyophilized powder for parenteral administration, which may be reconstituted by adding an appropriate solvent or other pharmaceutically acceptable carrier prior to use, liquid formulations typically being buffers, isotonic solutions, and aqueous solutions. The buffer solution can be phosphate buffer solution, the isotonic solution can be 0.9% sodium chloride solution, and the aqueous solution is directly dissolved by purified water.
It will be appreciated by those skilled in the art that the pharmaceutical compositions prepared by adding pharmaceutically acceptable carriers and/or excipients to the long-acting GLP-1 polypeptide analogs as active ingredients are suitable for various modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, and the like. Depending on the mode of administration employed, the pharmaceutical compositions of the polypeptide analogs of the invention may be formulated in a variety of suitable dosage forms comprising at least one effective dose of the polypeptide analog of the invention and at least one pharmaceutically acceptable carrier. Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions for injection.
The amount of the pharmaceutical composition of the present invention may vary widely and may be determined by one skilled in the art based on objective factors such as the kind of disease, the severity of the disease, the weight of the patient, the dosage form, the route of administration, etc.
It is a further object of the present invention to provide the use of the long acting GLP-1 polypeptide analogues and compositions described above.
The invention obtains a series of GLP-1 polypeptide analogues and develops and researches the pharmacodynamic effect of the series of medicaments. GLP-1 receptor agonistic activity, blood glucose and lipid reducing, weight reducing, diabetic nephropathy and other activities of the synthesized serial polypeptide analogue medicaments are evaluated, and the pharmacokinetics of the polypeptide analogue medicaments are initially studied. The results show that the long-acting effect of the compound is far better than that of the cable Ma Lutai (the half life of the compound is up to or more than 2 times that of the cable Ma Lutai) by comparison with the cable Ma Lutai, and the compound is also better than that of the cable Ma Lutai in the aspects of treating and improving diabetic nephropathy.
The long-acting GLP-1 polypeptide analogue has longer half-life period, insulinotropic activity and no adverse reaction, can be used for treating diabetes and obesity, and can be potentially used as a new-generation medicament for treating diabetes and obesity.
The application of the long-acting GLP-1 polypeptide analogue specifically comprises the following steps:
in preparing medicines for preventing or treating diabetes and obesity.
The invention has the beneficial effects that:
the solution of the half-life and stability of the polypeptide is the key of the design of the polypeptide medicine and whether the polypeptide can be prepared or not, and is a major scientific and core problem of the research in the field. The development of the ultra-long-acting polypeptide drug molecule modification technology is key, and is also a bottleneck to be broken through internationally in the field of research. The invention develops site-specific side arm structure modification technology through bioinformatics, structure biology, computer aided design, structure-activity relationship research and the like, breaks through the ultra-long-acting polypeptide and protein drug molecule modification technology, greatly prolongs the half-life of the synthesized polypeptide analogue, and realizes the ultra-long-acting of the polypeptide drug.
The parent peptide in the long acting GLP-1 polypeptide analogues of the invention is a homologous polypeptide, wherein the homologous polypeptide refers to a polypeptide which originally has the amino acid sequence of glucagon-like peptide (GLP-1) and cable Ma Lutai (Semaglutide), but one or more amino acid residues are replaced by different amino acid residues, and the structural sequence is added with a 'side arm' short peptide amino acid residue sequence, the amino acid residues are conserved with each other, and the obtained polypeptide can be used for implementing the invention.
Specifically, we have a "sidearm" short peptide chain linked to a specific site (Lys at position 20 in the amino acid sequence) by amino acid substitution at the specific site, namely: the site-specific side arm modification technology successfully breaks through the ultra-long-acting polypeptide drug molecule modification technology. Compared with the positive drug, namely the somalunin without side arm modification, the polypeptide drug designed by the invention greatly prolongs the action half-life period, realizes the super-long-acting of the polypeptide drug, and has the overall drug action effect which is greatly superior to that of the positive drug, namely the somalunin.
In addition, the GLP-1 polypeptide analogues of the invention use lipophilic substituents to bind albumin in the blood and protect them from enzymatic degradation, thereby increasing half-life. The GLP-1 polypeptide analogues of the invention have improved potency and/or selectivity towards the glucagon-like peptide 1 receptor (GLP-1R) through the helical structure of the intramolecular bridge stabilizing molecule.
The GLP-1 polypeptide analogue has high synthesis yield, good stability, easy mass production and low cost.
By comparison with the cable Ma Lutai, the long-acting effect and stability of the drug are far better than those of the cable Ma Lutai (the half life of the drug is up to 2 times or more than that of the cable Ma Lutai).
The GLP-1 polypeptide analogues of the invention are also superior to cable Ma Lutai in treating and improving diabetes and nephropathy when the mass numbers are the same.
At the same time, the GLP-1 polypeptide analogues of the invention have better weight-reducing pharmacodynamic effects, and the GLP-1 polypeptide analogues can be used for preventing weight increase or promoting weight loss by causing food intake to be reduced and/or energy consumption to be increased, so that the GLP-1 polypeptide analogues of the invention can be also used for directly or indirectly treating other diseases caused by overweight or characterized by the overweight, such as treating and/or preventing obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder diseases and sleep apnea caused by obesity, and the effects of the invention in the diseases can be due to the effects of the GLP-1 polypeptide analogues on the weight directly or indirectly or on other aspects of the body besides the weight.
Compared with the cable Ma Lutai, the compounds 2, 5, 6 and 7 synthesized by the embodiment of the invention have more excellent and obvious glucose tolerance effect improvement, longer drug effect and half-life period, can obviously reduce the liver weight and epididymal fat content of mice, and are also superior to the cable Ma Lutai in improving the oral glucose tolerance (OGTT) and insulin resistance (ITT) of diabetic mice.
The abbreviations used in the present invention have the following specific meanings:
DCM is dichloromethane, DMF is N, N-dimethylformamide, HOBt is 1-hydroxybenzotriazole, fmoc is fluorenylmethoxycarbonyl, resin is resin, FBS is fetal bovine serum, GLP-1R is glucagon-like peptide 1 receptor, GLP-1 is glucagon-like peptide, his is histidine, ser is serine, D-Ser is D-serine, gln is glutamine, gly is glycine, glu is glutamic acid, ala is alanine, thr is threonine, lys is lysine, arg is arginine, tyr is tyrosine, asp is aspartic acid, trp is tryptophan, phe is phenylalanine, IIe is isoleucine, leu is leucine, cys is cysteine, pro is proline, val is valine, met is methionine, asn is asparagine. Aib is 2-aminoisobutyric acid and Iva is isovaline.
Drawings
FIG. 1 is a graph of 24h and 48h blood glucose monitoring after dosing based on experimental animals in example 3; wherein:
FIG. 1A is a graph showing the change in blood glucose concentration within 120 minutes of gastric lavage glucose 24 hours after administration;
FIG. 1B is the area under the curve (AUC) calculated in FIG. 1A;
FIG. 1C is a graph showing the change in blood glucose concentration within 120 minutes of gastric lavage glucose 48 hours after administration;
FIG. 1D is the area under the curve (AUC) calculated in FIG. 1C;
FIG. 2 is a graph of blood glucose monitoring based on 72h and 96h post-dosing of experimental animals in example 3; wherein:
FIG. 2A is a graph showing the change in blood glucose concentration within 120 minutes of gastric lavage glucose after 72 hours of administration;
FIG. 2B is the area under the curve (AUC) calculated in FIG. 2A;
FIG. 2C is a graph showing the change in blood glucose concentration within 120 minutes of gastric lavage glucose 96 hours after administration;
FIG. 2D is the area under the curve (AUC) calculated in FIG. 2C;
FIG. 3 is a graph of body monitoring of 8 week old male db/db diabetic mice of example 4 after administration, wherein:
FIG. 3A is a graph of the body weight monitoring of the mice in example 4;
FIG. 3B is a graph of the blood glucose monitoring of the mice in example 4;
FIG. 3C is a graph of the food intake monitoring of the mice in example 4;
FIG. 4 is a serological index graph of the draw assay 6 weeks after administration of treatment to 8 week old db/db diabetic mice, wherein:
FIGS. 4A and 4B are mouse serum ALT and AST results, p <0.05, respectively;
FIGS. 4C and 4D are mouse serum TG and T-CHO results, respectively, p <0.05;
FIGS. 4E, 4F and 4G are graphs of results of mouse serum HDL-C, LDL-C and GHb, p <0.05, respectively;
FIG. 5 is a graph of physiological index at week 6 of mouse administration in example 4, wherein,
FIG. 5A is a graph of liver weight (liver weight) of mice;
FIG. 5B is a graph of liver index (liver/body weight) of mice;
FIG. 5C is a body weight (body weight) diagram of a mouse;
FIG. 5D is a graph of the Body Mass Index (BMI) of the mice;
FIG. 5E is a graph of epididymal fat weight (EAM weight) of mice;
FIG. 5F is a graph of epididymal fat index (EAM/body weight) of mice;
FIG. 6 is a graph of Oral Glucose Tolerance Test (OGTT) and insulin resistance test (ITT) experiments in mice; wherein, fig. 6A is a graph of OGTT results at week 4 of mice dosing in example 4;
FIG. 6B is a bar graph of the AUC corresponding to FIG. 6A;
FIG. 6C is a graph of ITT results measured at week 5 of mouse dosing in example 4;
FIG. 6D is a histogram of the corresponding AUC administration of FIG. 6C at week 5; p <0.05;
fig. 7 is a graph of blood concentration collected over various time periods after a single dose of an 8 week old SD rat, wherein:
FIGS. 7A1 and 7A2 are graphs of blood concentration of SD rat tail vein and subcutaneous injection cord Ma Lutai, respectively;
FIGS. 7B1 and 7B2 are graphs of blood concentration of SD rat tail vein and subcutaneous injection of Compound 2, respectively;
FIGS. 7C1, 7C2 are plasma concentration curves for SD rat tail vein and subcutaneous injection of compound 5, respectively;
FIGS. 7D1 and 7D2 are graphs of blood concentration of SD rat tail vein and subcutaneous injection of compound 6, respectively;
FIGS. 7E1 and 7E2 are graphs of blood concentration of SD rat tail vein and subcutaneous injection of compound 7, respectively;
FIG. 8 shows the formula and structure of fatty acid substituents.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Materials:
all amino acids were purchased from Shanghai Jier Biochemical company, soxhlet Ma Lutai from Zhejiang surge peptide Biol.Co., ltd., CAS No.:910463-68-2; all other reagents were analytically pure, purchased from Sigma, unless otherwise specified. Adopts a Protein Technologies PRELUDE channel polypeptide synthesizer Phenomenex Luna C 18 A column (46 mm x250 mm) was prepared for purification of the polypeptide, and a high performance liquid chromatograph was available from Thermo company, and mass spectrometry was performed using a Thermo liquid mass spectrometer.
Materials and methods:
Boc-His (Trt) -OH, fmoc-Aib-OH was purchased from ShanghaiJil, eicosanedioic acid mono-tert-butyl ester self-made. The remaining amino acids were purchased from Chengdu Zheng Yuan, and the condensing agent was purchased from Suzhou-Haihan. All other reagents were analytically pure and solvents were purchased from Shanghai Taitan, inc., unless otherwise specified. The centrifuge was purchased from Lu Xiangyi. 5.0m phase C 18 A column (46 mm X250 mm) was prepared for purification of the polypeptide. The high performance liquid chromatograph is the product of the Siemens company. Mass spectrometry was performed using a Waters mass spectrometer.
EXAMPLE 1 Synthesis of Compound 5
Amino acid sequence of compound 5:
His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Glym-γ-Glu-CO(CH 2 ) 18 CO 2 H)-Glu-Phe-Ile-Ala-Trp
-Leu-Val-Arg-Gly-Arg-Gly
the abbreviation is: H-Aib-EGT FTSDV SSYLE GQAAK (GGSGSGSG-gamma-E-CO (CH) 2 ) 18 CO 2 H) EFIAW LVRGR G (acetate salt),
the method comprises the following steps:
step 1, synthesizing a main peptide resin
Manual synthesis according to Fmoc/t-Bu strategy, scale of synthesis: 0.5mmol, the following main peptide resin was synthesized:
Boc-His-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-Wang Resin。
(1): 1.47 g of Wang Resin (loading 0.47mmol/g, siemens Lan Xiao) was weighed into a reaction column, swollen for 30min with N, N-Dimethylformamide (DMF), and Fmoc-Gly-OH was weighed: 1.233g (6 eq), HOBT:0.672g (7.2 eq), DMAP:0.06g (0.72 eq) for use. DMF was removed and the resin was washed thoroughly with N, N-Dimethylformamide (DMF) 2 times and the above weighed material was added to the reaction column. Appropriate amount of DMF was added, and the mixture was stirred well with nitrogen, and 0.83mL (7.8 eq) of N, N-Diisopropylcarbodiimide (DIC) was added. The reaction is carried out for 2 hours, and the reaction is finished. The reaction solution was removed, washed 3 times with DMF and blocked by the addition of acetic anhydride/pyridine (7:6, v/v) for 4h. The blocking solution was removed and washed 6 times with DMF to give Fmoc-Gly-Wang Resin.
(2): fmoc-Gly-Wang Resin is used as a carrier, 1-Hydroxybenzotriazole (HOBT), N, N-Diisopropylcarbodiimide (DIC) is used as a coupling agent, N, N-Dimethylformamide (DMF) is used as a solvent, 20% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) solution is used for removing Fmoc groups (twice 5min+7 min), and the coupling effect is monitored by ninhydrin hydrate in the coupling process. Performing manual charging, and sequentially performing condensation reaction from the C end to the N end to connect Fmoc-Arg (pbf) -OH, fmoc-Gly-OH, fmoc-Arg (pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys (Alloc) -OH, fmoc-Ala-OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Ser (tBu-OH), boc-His (Trt) -OH above amino acid feed (5 eq relative to the synthesis scale) gave Boc-His-Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (Alloc) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-Wang Resin.
There are several points to be described:
1) Fmoc-Gly-Wang Resin is synthesized, and the substitution degree of the Wang Resin is low, so that the feeding amount of Fmoc-Gly-OH is large, otherwise, the substitution degree is low, and materials are wasted. Blocking with acetic anhydride/pyridine prevents the appearance of defective peptides.
2) In each subsequent condensation reaction, fmoc-protected amino acid, 1-Hydroxybenzotriazole (HOBT), N, N-Diisopropylcarbodiimide (DIC) was fed 5 times, and the reaction time was 2 hours.
(3): removal of allyloxycarbonyl (Alloc)
Dichloromethane (DCM) was added to the resin, morpholine 0.5mL (12 eq) was added, 0.173g of tetrakis triphenylphosphine palladium (0.3 eq) was weighed into the reaction column and reacted for 1h. At the end of the reaction, the reaction solution was withdrawn, washed 3 times with N, N-Dimethylformamide (DMF) and 6 times with DCM. Obtaining main peptide resin: boc-His-Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys-Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-Wang Resin.
Step 2, coupling a side arm structure:
Fmoc-Gly-OH was coupled according to Fmoc/t-Bu strategy: fmoc-Gly-OH, HOBT and proper amount of N, N-Dimethylformamide (DMF) are added into the main peptide resin product, nitrogen is uniformly stirred, DIC is added, nitrogen is stirred for 2 hours for reaction, ninhydrin is used for detecting the coupling effect, and the reaction is colorless and transparent. The reaction solution was removed, washed 3 times with N, N-Dimethylformamide (DMF), fmoc group was removed with 20% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) (5 min+7min twice), after Fmoc removal, washed 6 times with DMF, sampled ninhydrin detection was performed, and developed.
The above operations were repeated to couple Fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu-OtBu, and eicosanedioic acid mono-tert-butyl ester in sequence. Boc-His-Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (Gly-Gly-Ser (tBu) Gly-Ser (tBu) -Gly-Ser (tBu) -Gly-Glu-OtBu-eicosanedioic acid mono-tert-butyl) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-Wang Resin. Washing with N, N-Dimethylformamide (DMF) 3 times, washing with Dichloromethane (DCM) 3 times, shrinking with Methanol (Methanol) 2 times, and vacuum drying to obtain dry polypeptide resin.
Step 3, removing the full protection of the polypeptide
Lysate: TFA, DODT, m-cresol, H 2 The volume ratio of O is 92.5:2.5:2.5:2.5, and freezing in a refrigerator for 2 hours.
To the dried polypeptide Resin Boc-His-Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (Gly-Gly-Ser (tBu) -Gly-Ser (tBu) -Gly-Ser (tBu) -Gly-Glu-OtBu-eicosanoid-mono-tert-butyl) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-Wang Resin was added a 10mL of lysate, the lysate was warmed to room temperature, and the reaction was allowed to proceed for 3 hours. Filtering, washing the filter cake with a small amount of lysate for 3 times, and combining the filtrates. The filtrate was slowly poured into ice methyl tert-butyl ether with stirring. Standing for more than 2 hours until the precipitation is complete. Removing supernatant, centrifuging the precipitate, washing 3 times with methyl tertiary butyl ether, centrifuging, and drying the solid with nitrogen. Obtaining crude compound His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (Gly-Gly-Ser-Gly-Ser-Gly-Ser-Ser-Ser-Gly-Glu-eicosanedioic acid) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly.
Step 4. Refining and purifying the crude compound
The crude compound obtained in step 2 was dissolved in Acetonitrile (ACN): h 2 O=1: 2 (volume ratio) of C in reversed phase by 5.0m 18 Is subjected to preparative HPLC purification on a packed 46mm x250mm column. With 45% acetonitrile/H 2 O (containing 1% trifluoroacetic acid) was used as an initial step, the column was eluted at a gradient (ratio of acetonitrile increase at 0.33%/min) and a flow rate of 10mL/min for 60 minutes, and fractions containing the polypeptide were collected to obtain a sample having an HPLC purity of more than 90%. HPLC purification was repeated once, starting with 31% acetonitrile/20 mM sodium dihydrogen phosphate aqueous solution adjusted to pH 6.5 with 1M sodium hydroxide solution at a flow rate of 10mL/min at a gradient of 0.33%/min, eluting the column for 60min, collecting the fractions containing the polypeptide, and freeze-drying to give a purity of greater than 96.86% with a total yield of 15%.
Step 5. Product confirmation
The isolated product polypeptide was identified by liquid chromatography-mass spectrometry and found to have an m/z value of the ionic peak of the protonated molecule of: 4397.16 as the target compound 5, the theoretical molecular weight of compound 5 was 4397.87.
Synthesis of Compound 2
Since compound 2 differs from compound 5 by the sequence of the "side arm" short peptide chain, the synthetic steps of compound 2 and compound 5 differ by the coupling of the "side arm" structure of step 2:
in this step, fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Ser (tBu) -OH, fmoc-Gly-OH, fmoc-Glu-OtBu, and eicosanedioic acid mono-tert-butyl ester need to be coupled in sequence. To obtain Boc-His-Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (Gly-Gly-Ser (tBu) Gly-Ser (tBu) -Gly-Glu-OtBu-eicosanedioic acid mono-tert-butyl) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-Arg (pbf) -Gly-Wang Resin.
The purification of compound 2 and the confirmation of the product were carried out in the same manner as in compound 5, to obtain a purity of more than 97.66% and a total yield of 12%. The isolated product was identified by liquid chromatography-mass spectrometry and found to have an m/z value of the ionic peak of the protonated molecule of: 4253.07 is the target compound 2, and the theoretical molecular weight of compound 2 is 4253.74.
Based on the above synthesis steps, purification and product confirmation methods, according to the difference of amino acids in main peptide chains and side arm short peptide chains of the compounds 1, 3, 4, 6, 7 and 8, the coupling sequence of amino acids in the compound synthesis steps 1 and 2 is adjusted, the corresponding target product is finally synthesized, the separated product is identified by liquid chromatography-mass spectrometry, the m/z value of the ion peak of the protonated molecule (the actual measurement value in table 1) is confirmed, the actual measurement value is compared with the theoretical value of molecular weight, the synthesized and purified product is confirmed to be the target product, and the molecular weight theoretical value, the actual measurement value, sequence and molecular formula of the liquid chromatography-mass identification of the compounds 1 to 8 are respectively shown in table 1.
Table 1 amino acid sequence table and LC-MS identification results of long-acting GLP-1 polypeptide analogues
Example 2 evaluation of agonistic Activity of Compounds 1-8 and Soxhlet Ma Lutai on GLP-1R (glucagon-like peptide 1 receptor)
In the GLP-1R-Luciferase-HEK293 cell model (cell line constructed in the present laboratory), the agonistic activity of GLP-1R was measured on compounds 1-8 and cable Ma Lutai.
First, the digested cells were plated in 96-well plates (medium containing 10% FBS, GLP-1R-Luciferase-HEK293:20000 cells/well, 100. Mu.L); after 36h, medium in 96-well plates was discarded and 90 μl of serum-free medium was added; after 6h, serial concentrations (0.01, 0.1, 1, 10, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000 pM) of polypeptide drug (8 polypeptide analogs and cord Ma Lutai) were formulated with serum-free medium, 10 μl (i.e. diluted 10 times) was added per well, and cells were incubated for 5h; adding 100 mu L of cell lysate into each hole, carrying out ice lysis for 10min, then vibrating uniformly, adding 2 mu L of lysate into a 384 enzyme-labeled white plate, firstly adding 10 mu L of firefly luciferase reaction solution, reading, and then adding 10 mu L of Renilla luciferase reaction solution, and reading; data processing, namely dividing the reading of firefly luciferase by the reading of Renilla luciferase, and subtracting the values of the blank groups to obtain the excitation multiples under different concentrations.
TABLE 2 Compounds 1-8 and Cable Ma Lutai agonize the EC of GLP-1R 50
EC 50
Rope Ma Lutai 222.2pM
Compound 1 699.1pM
Compound 2 64.73pM
Compound 3 1003.7pM
Compound 4 1349.9pM
Compound 5 146.3pM
Compound 6 203.5pM
Compound 7 302.4pM
Compound 8 1599.2pM
As can be seen from the experimental results, each of the compounds 1 to 8 of the present invention activates GLP-1R, and the compounds 2, 5, 6 and 7 have better agonistic activity to GLP-1R (Table 2), wherein the half maximum effector concentration of the compounds 2, 5 (hereinafter abbreviated as EC 50 Refers to a concentration that causes 50% of the maximum effect) is lower than that of cable Ma Lutai, indicating that both agonistic activities to GLP-1R are superior to that of cable Ma Lutai, whereas compounds 6, 7 have agonistic activity to GLP-1R comparable to that of cable Ma Lutai.
Example 3 effects of Compounds 1-8 and Soxhlet Ma Lutai on oral glucose tolerance (OGTT)
Male C57BL/6J mice (university of Zhongshan laboratory animal center) of about 8 weeks old were raised for one week to adapt to the environment, and randomly grouped according to similar blood glucose (blood sample evaluation obtained from the tail tip), 8 animals per group.
Compounds 1-8 (corresponding to numbers 1-8, respectively) and cable Ma Lutai (corresponding to semaglutinide, respectively) of the present invention were administered subcutaneously at a dose of 120ug/kg, and the control group was given the same dose of PBS. ddH for lavage of glucose 2 O is prepared into a stock solution with the concentration of 0.5g/mL, and the stock solution is stored at normal temperature. Will be before stomach irrigationMice were fasted for 8h, and were perfused with 2.5g/kg of gastric glucose at 24h, 48h, 72h and 96h, respectively, after dosing, and blood glucose was measured at t=0 min, t=15 min, t=30 min, t=60 min, t=90 min and t=120 min. The data were processed using the software GraphPadPrism to create a blood glucose change line graph and calculate the area under the curve to obtain an AUC graph, and the results are shown in fig. 1 and 2.
According to the AUC (area under concentration-time curve) counted by the OGTT curve, the bioavailability is high if the AUC is large, and otherwise, the bioavailability is low. In fig. 1 and 2: represents p <0.05; * *: represents p <0.01; * **: represents p <0.001.
As can be seen from fig. 1A, B, cable Ma Lutai and compounds 1-8 significantly reduced AUC compared to vehicle (PBS) 24h after administration, indicating that cable Ma Lutai and compounds 1-8 had significant glucose tolerance.
As can be seen from fig. 1, C, D and fig. 2, the effect of cable Ma Lutai on AUC was not significantly different from that of vehicle (PBS) after 48h, 72h, 96h administration, indicating that cable Ma Lutai had failed to improve glucose tolerance. After 48h, 72h, 96h dosing, compounds 2, 5, 6 and 7 still significantly reduced AUC compared to PBS group, thus improving glucose tolerance, and thus compounds 2, 5, 6 and 7 were all better than cord Ma Lutai in improving glucose tolerance in mice.
Taken together, compounds 1-8 of the present invention all exhibited a different degree of improvement in glucose tolerance in mice than in vehicle (PBS), with compounds 2, 5, 6 and 7 exhibiting more excellent and significant improvement in glucose tolerance over 4 OGTT curve periods (24, 48, 72, 96 h), while the results also demonstrate that compounds 1-8, especially compounds 2, 5, 6 and 7, exhibited more excellent and significant long-acting hypoglycemic effects over 4 OGTT curve periods (24, 48, 72, 96 h) relative to cord Ma Lutai.
Example 4 treatment of diabetes by Compounds 2, 5, 6, 7 and Soxhlet Ma Lutai on diabetic mice
48 male Lepr db/db mice (db/db) of 8 weeks of age and 8 normal mice (WT) of littermates were purchased from Nanjing university-Nanjing biomedical research institute.
The db/db mice diabetic model (purchased from Nanjing university-Nanjing biomedical research institute, about 8 weeks, and assayed for blood glucose and body weight to ensure that subsequent experiments were performed smoothly) was obtained, and the db/db mice were randomly divided into 6 groups (compound 2, 5, 6, 7 and cord Ma Lutai, physiological saline group) according to blood glucose, 8 in each group, with no difference in basal body weight and blood glucose. Each group of mice was subcutaneously injected every other day with compound 2, 5, 6, 7 (120 μg/kg), cord Ma Lutai (120 μg/kg), and physiological saline (WT and db/db groups), respectively. Blood glucose and body weight of mice were measured after each administration of mice, fasted for 6 hours every other day; the intake and feed intake were measured every 7 days. OGTT was measured at week 4, insulin resistance (ITT) at week 5, and various serological and physiological indices were measured at week 6. The monitoring results of blood sugar, body weight, water intake and food intake of mice are shown in figure 3, the serum indexes obtained by the method are shown in figure 4, and the physiological indexes are shown in figure 5.
A model of type 2 diabetes characterized by: obesity, insulin resistance, hyperglycosemia, dyslipidemia, and liver fat vacuolation-like degeneration, etc. Oral glucose tolerance experiments (OGTT) and insulin resistance tests (ITT) were performed at the time of 4 weeks and 5 weeks of treatment of diabetic mice with compounds 2, 5, 6, 7 and cable Ma Lutai, respectively, and the results are shown in fig. 6.
FIG. 3 is a graph of body monitoring of male db/db diabetic mice of middle 8 weeks of age after administration, in which physiological saline, compound 2 (120. Mu.g/kg), compound 5 (120. Mu.g/kg), compound 6 (120. Mu.g/kg), compound 7 (120. Mu.g/kg) and cable Ma Lutai (120. Mu.g/kg) were injected subcutaneously, respectively, and administration was continued every other day for 6 weeks; mice body weight and blood glucose (3A and 3B) measured every 3 days fasted for 6h, mice food intake (3C) measured every 7 days, wherein: represents p <0.05; * *: represents p <0.01; * **: represents p <0.001.
The results in fig. 3 (A, C) show that compounds 2, 5, 6, and 7 all significantly reduced the feeding capacity of the mice compared to db/db group, thereby reducing the weight of the mice, and the weight reduction effect was superior to that of cable Ma Lutai.
The experimental results in fig. 3B show that compounds 2, 5, 6, 7 and the positive control drug, rope Ma Lutai, each significantly reduced fasting blood glucose in db/db mice compared to db/db group, indicating that these drugs all had significant hypoglycemic effects, blood glucose had fallen to normal levels by week 1, and thereafter blood glucose was relatively stable and superior to rope Ma Lutai.
Fig. 4 (A, B) shows that compounds 2, 5, 6 and 7 also correspond to cable Ma Lutai in terms of liver protection function.
Glycosylated hemoglobin (GHb) can be used as a control index reflecting that a diabetic patient gets blood sugar for a long period of time (4-10 weeks), blood sugar is poorly controlled for a long period of time, and thus glycosylated hemoglobin is increased, so that glycosylated hemoglobin measurement contributes to control, and has an important role in the study of peripheral blood vessels and cardiovascular complications of diabetes, and experimental results in fig. 4G show that compounds 2, 5, 6 and 7 have a remarkable effect of reducing glycosylated hemoglobin and are superior to cord Ma Lutai.
The results in fig. 5E show that compounds 2, 5, 6 and 7 all significantly reduced liver weight and epididymal fat content in mice and were superior to cord Ma Lutai.
The results in fig. 6 show that compounds 2, 5, 6 and 7 are superior to the positive control drug cord Ma Lutai in improving the oral glucose tolerance (OGTT) and insulin resistance (ITT) of diabetic mice.
EXAMPLE 5 pharmacokinetic study of Compounds 2, 5, 6 and 7 and Soxhlet Ma Lutai in SD rats
SD rats (experimental animal center of Zhongshan university) of about 8 weeks of age were raised for one week to adapt to the environment, and the male and female animals were randomly grouped according to body weight, and the number of the SD rats was 6 (male and female animals) in each group, and the number of the SD rats was 5.
In this example, compounds 2, 5, 6 and 7 and cable Ma Lutai were administered subcutaneously and caudally in a dose of 50ug/kg, a volume of 2mL/kg, a concentration of 60ug/mL, and a vehicle as normal saline.
The single tail vein dosing group rats were blood-sampled via the jugular vein before (0 min) and 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 24h, 36h, 48h, 72h, 4d, 5d, 6d, 7d, 8d, 9d, 10d after dosing, with a dose of about 0.2mL per time point. Rats in the single subcutaneous injection group were given 10min, 20min, 30min, 1h, 2h, 4h, 6h, 8h, 24h before (0 min) and after administrationBlood samples were collected via jugular vein at 36h, 48h, 72h, 4d, 5d, 6d, 7d, 8d, 9d, 10d, at about 0.2mL per time point. Placed in a container containing anticoagulant K 2 In an EP tube of EDTA and centrifuged (3500 g,10 min) at 2-8℃within 1 hour after blood collection, the plasma separated after centrifugation was placed in a labeled EP tube and the drug concentrations of compounds 2, 5, 6, 7 and cable Ma Lutai in the plasma were determined by LC-MS/MS analysis. The data processing uses a non-compartmental model in WinNonlin 6.4 software to calculate pharmacokinetic parameters.
As can be seen from the experimental results in FIG. 7, the absorption in rats was slow after a single subcutaneous injection of Compounds 2, 5, 6 and 7 (50. Mu.g/kg) for peak time T max All are about 24h, half-life period t 1/2 Average 14.99h, 15.4h, 16.6h and 15.42h, respectively, compounds 2, 5, 6 and 7 were all Yu Suoma lutide (7.87 h) high. Half-life t after a single intravenous injection of Compounds 2, 5, 6 and 7 (50. Mu.g/kg) 1/2 Average 14.49h, 15.2h, 16.2h and 15.1h respectively, are all better than cord Ma Lutai (average 8 h).
The foregoing is merely illustrative embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think about variations or substitutions within the technical scope of the present invention, and the invention should be covered. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims (10)

1. A long acting GLP-1 polypeptide analogue, characterized in that the amino acid sequence of said long acting GLP-1 polypeptide analogue is as follows:
His-D-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{(Gly) x -(Ser-Gly) y -γGlu-CO(CH 2 ) n CO 2 H}-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gl y
wherein:
xaa9 is Asp or Glu;
xaa21 is Glu or Gln or Asp;
x is an integer from 1 to 4; y is an integer from 1 to 4; n is an integer of 12-20.
2. The long acting GLP-1 polypeptide analogue of claim 1 wherein Xaa9 is Asp and Xaa21 is Glu or gin.
3. The long acting GLP-1 polypeptide analogue of claim 1 wherein Xaa9 is Glu and Xaa21 is Asp.
4. The long acting GLP-1 polypeptide analogue of claim 1, wherein x is 1 or 2 and y is 1, 2 or 3; n is any integer from 14 to 18.
5. The long acting GLP-1 polypeptide analogue of any one of claims 1 to 4 wherein x is 2, y is 3 and n is 18.
6. The long acting GLP-1 polypeptide analogue of claim 1, wherein the long acting GLP-1 polypeptide analogue is any one of the following compounds:
compound 6:
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2 ) 18 CO 2 H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
compound 7:
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2 ) 18 CO 2 H)-Gln-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
compound 8:
His-(D-Ser)-Glu-Gly-Thr-Phe-Thr-Ser-Glu-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2 ) 18 CO 2 H)-Asp-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
7. the method for preparing the long-acting GLP-1 polypeptide analogue according to any one of claims 1-6, which is characterized by comprising the following steps:
step 1: synthesizing main peptide resin corresponding to a main peptide chain of the long-acting GLP-1 polypeptide analogue according to an Fmoc/t-Bu strategy, wherein the main peptide chain is as follows:
His-D-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Xaa9-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Xaa21-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly；
step 2: based on the main peptide resin, coupling a side arm structure corresponding to the long-acting GLP-1 polypeptide analogue according to an Fmoc/t-Bu strategy to obtain a polypeptide resin corresponding to the long-acting GLP-1 polypeptide analogue; wherein the side arm structure is (Gly) x -(Ser-Gly) y -γGlu-CO(CH 2 ) n CO 2 H；
Step 3: adding a cracking solution into the polypeptide resin, performing a cracking reaction, removing the full protection of the polypeptide, and extracting a crude compound;
step 4: purifying the crude compound to obtain the long-acting GLP-1 polypeptide analogue.
8. The method for producing a long-acting GLP-1 polypeptide analog according to claim 7, wherein,
in the step 2, 1-hydroxybenzotriazole and N, N-diisopropylcarbodiimide are used as coupling agents, N-dimethylformamide is used as a solvent, and Fmoc groups are removed by 20% piperidine/N, N-dimethylformamide solution;
in step 3, the lysate is prepared from TFA, DODT, m-cresol, H 2 The volume ratio of O is 92.5:2.5:2.5: 2.5; the crude compound extraction mode comprises filtration, precipitation and/or methyl tertiary butyl ether extraction.
9. A composition comprising the long acting GLP-1 polypeptide analogue of any one of claims 1 to 6 and a pharmaceutically acceptable carrier or adjuvant.
10. Use of a long acting GLP-1 polypeptide analogue of any one of claims 1 to 6 for the manufacture of a medicament for the prevention or treatment of diabetes;
or, the long-acting GLP-1 polypeptide analogue is applied to the preparation of a medicament for preventing or treating obesity.
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