CN113754752B - Polypeptide and preparation method and application thereof - Google Patents

Polypeptide and preparation method and application thereof Download PDF

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CN113754752B
CN113754752B CN202111026493.2A CN202111026493A CN113754752B CN 113754752 B CN113754752 B CN 113754752B CN 202111026493 A CN202111026493 A CN 202111026493A CN 113754752 B CN113754752 B CN 113754752B
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polypeptide
ser
glu
gly
application
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CN113754752A (en
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曾能
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Fitch Biomedical Zhongshan Research Institute Co ltd
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Fitch Biomedical Zhongshan Research Institute Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The application discloses a polypeptide, a preparation method and application thereof. The amino acid sequence of the polypeptide is His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (P EG2-PEG 2-gamma-Glu-CO (CH) 2 ) 18 CO 2 H) The polypeptide prepared by the scheme of the application has better and excellent long-acting blood glucose control effect than that of the Soxhlet Ma Lutai, has more stable blood concentration, high enzymolysis stability, high biological activity and no adverse reaction, and has important significance in preparing medicines for treating obesity and diabetes caused by hyperglycemia.

Description

Polypeptide and preparation method and application thereof
Technical Field
The application belongs to the field of biological pharmacy, and particularly relates to a polypeptide, a preparation method and application thereof.
Background
GLP-1 (glucagon-like peptide-1) is an intestinal sex hormone in human body, after eating, the hormone can promote insulin secretion (insulin produced by the 'incretin effect' accounts for more than 50% of the total amount of insulin after eating), plays a role in glucose concentration-dependent blood glucose reduction, is very intelligent, only acts when blood glucose is high, and therefore has no risk of hypoglycemia of injected insulin. More attractive is that GLP-1 has the effect of protecting islet beta cells: promote the transcription of insulin gene, the synthesis and secretion of insulin, stimulate the proliferation and differentiation of beta cells of pancreatic islet, inhibit the apoptosis of beta cells of pancreatic islet, and increase the quantity of beta cells of pancreatic islet. GLP-1 can also act on islet alpha cells to strongly inhibit the release of glucagon, and act on islet delta cells to promote the secretion of somatostatin, which can also be used as a paracrine hormone to participate in the inhibition of the secretion of glucagon.
Disadvantages of GLP-1: GLP-1 produced by a human body is easily degraded by dipeptidyl peptidase IV (DPP-4) in the human body, the plasma half-life period is less than 2 minutes, and continuous intravenous drip or continuous subcutaneous injection is required to produce the curative effect, so that the clinical application of the GLP-1 is greatly limited.
Cord Ma Lutai is a potent GLP-1 receptor agonist. The cable Ma Lutai has more than 30 amino acids and is structurally characterized by replacing Aib with amino acid of DPP-4 hydrolase catalytic site 8 (2-aminoisobutyric acid) at amino acid Lys 26 The stearyl diacid is grafted by a spacer to combine albumin and delay kidney clearance. Although cord Ma Lutai has improved long-term efficacy, the patient's pursuit of long-term efficacy is not satisfactory.
Disclosure of Invention
The present application aims to solve at least one of the technical problems in the prior art described above. Therefore, the application provides a polypeptide which can control sugar for a long time.
The application also provides a preparation method of the polypeptide.
The application also provides application of the polypeptide.
According to one aspect of the present application, there is provided a polypeptide having an amino acid sequence His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (PEG) 2 -PEG 2 -γ-Glu-CO(CH 2 ) 18 CO 2 H) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly, abbreviated as: hsegtftsdvssylegqaa-K (PEG) 2 -PEG 2 -γGlu-CO(CH 2 ) 18 COOH) -EFIAWLVRGRG; the molecular weight is 4171.73.
According to a second aspect of the present application, there is provided a method for producing the polypeptide, comprising the steps of: taking solid-phase synthetic resin as an initial raw material, and adopting Fmoc/t-Bu solid-phase synthetic strategy to sequentially connect amino acids to obtain a polypeptide main chain with an amino acid protecting group; grafting side chains on a polypeptide main chain, and carrying out deprotection groups and peptide cutting; the side chain is PEG2-PEG 2-gamma-Glu-CO (CH) 2 ) 18 COOH; the amino acid connection sequence of the polypeptide main chain is His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
In some embodiments of the application, the grafted side chain is located at the 20 th amino acid lysine of the main chain.
In some embodiments of the application, the Resin is a Wang Resin.
In some embodiments of the application, sequentially ligating amino acids using Fmoc/t-Bu solid phase synthesis strategy further comprises assembling Fmoc-Gly-Wang Resin vector.
In some embodiments of the application, when assembling Fmoc-Gly-Wang Resin carrier, the Fmoc-Gly-OH to Resin addition ratio is (4-8): 1.
in some embodiments of the application, the obtaining of the polypeptide backbone further comprises the step of coupling the amino acid with 1-Hydroxybenzotriazole (HOBT), N-Diisopropylcarbodiimide (DIC) as coupling agent, removing the Fmoc protecting group with 10-30% Piperidine (Piperidine)/N, N-Dimethylformamide (DMF) solution in N, N-Dimethylformamide (DMF) as solvent.
In some embodiments of the application, the Fmoc group removal is by washing the resin with 10-30% piperidine/DMF (v/v) solution to remove protecting groups.
In some embodiments of the application, further comprising the step of detecting the coupled amino acid and removing the Fmoc group using ninhydrin.
In some embodiments of the application, the amino acid to DIC and HOBt addition ratio is 1 (1-2): 1-3.
In some embodiments of the application, the amino acid protecting group is one or more of pbf, boc, fmoc, tBu, otBu and Trt.
In some embodiments of the application, the cleavage solution is used to perform simultaneous deprotection and peptide cleavage steps.
In some embodiments of the application, the composition of the lysate comprises TFA (trifluoroacetic acid), phenols (Phenol), thioanisole (anisole) and EDT (1, 2-ethanedithiol), in a mass ratio of (80-90): (3-6): (3-6): (2-5).
In a third aspect, the application provides an application of the polypeptide in preparing a medicament for treating or preventing diabetes.
In some embodiments of the application, the use of the polypeptide in the preparation of a glucagon-like peptide-1 receptor agonist
In some embodiments of the application, the polypeptide is used in the preparation of a glucagon receptor (GCGR) agonist.
In some embodiments of the application, the use of the polypeptide in the manufacture of a medicament for the treatment or prevention of hyperglycemia-induced obesity.
A medicament for treating diabetes, the medicament comprising the polypeptide.
In some embodiments of the application, the preparation of the medicament for treating diabetes further comprises a pharmaceutically acceptable carrier.
In some embodiments of the application, the pharmaceutically acceptable carrier is a pharmaceutical carrier conventional in the pharmaceutical arts.
In some embodiments of the application, the pharmaceutically acceptable carrier comprises at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, sweeteners, and flavoring agents.
In some embodiments of the application, the excipient comprises water.
In some embodiments of the application, the filler comprises at least one of starch and sucrose.
In some embodiments of the application, the binder comprises at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.
In some embodiments of the application, the humectant comprises glycerin.
In some embodiments of the application, the disintegrant comprises at least one of agar, calcium carbonate, and sodium bicarbonate.
In some embodiments of the application, the absorption enhancer comprises a quaternary ammonium compound.
In some embodiments of the application, the surfactant comprises cetyl alcohol.
In some embodiments of the application, the adsorption carrier comprises at least one of kaolin clay and soap clay.
In some embodiments of the application, the lubricant comprises at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.
In some embodiments of the application, the mass fraction of the polypeptide in the medicament is 0.01% -99%.
According to some preferred embodiments of the application, the mass fraction of the polypeptide in the medicament is 0.05% -95%.
According to some preferred embodiments of the application, the mass fraction of the polypeptide in the medicament is 0.05% -20%.
In some embodiments of the application, the diabetic drug is administered at the following standard: the polypeptide compound is 0.01 mg/day to 10 mg/day.
In some embodiments of the application, the pharmaceutical dosage form is various dosage forms conventional in the art, preferably solid, semi-solid or liquid form, and may be an aqueous solution, non-aqueous solution or suspension, more preferably a tablet, capsule, soft capsule, granule, pill, oral liquid, dry suspension, drop pill, dry extract, injection or infusion, transdermal agent, transdermal microneedle.
In some embodiments of the application, the mode of administration of the drug may be conventional in the art, including but not limited to injection or oral administration. The injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
The term "administered dose" as used herein is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. May depend on the particular disease being treated, as well as other factors including age, weight, health, severity of symptoms, route of administration, frequency of treatment, and whether additional medications are concomitantly used during the treatment.
According to an embodiment of the application, at least the following advantages are achieved:
1. obesity can induce endoplasmic reticulum stress, liver lipid accumulation and insulin resistance, obesity-induced endoplasmic reticulum stress damages the insulin signaling pathway by activating JNK and serine phosphorylation levels of its downstream insulin receptor, endoplasmic reticulum stress has been considered as a new important mechanism for insulin resistance to occur in obesity. The polypeptide compound provided by the application can weaken insulin signal path and reduce AKT phosphorylation level and IRS-1 tyrosine phosphorylation level by replacing Aib amino isobutyric acid with D-serine of a second sequence. Inhibiting the expression of cytochrome P450A reduces hepatic endoplasmic reticulum stress and insulin resistance. Endoplasmic reticulum stress plays an important role in insulin resistance, and can be used as a potential therapeutic target for diabetes. The novel design ensures that the peptide chain has stronger activity, strong targeting effect and more obvious weight-reducing and sugar-controlling effects.
2. The amino acid sequence of the present application is a "sidearm" short peptide chain linked to Lys at position 20 in the amino acid sequence by amino acid substitution at a specific site, namely: compared with the positive medicine cable Ma Lutai without the modification of the side arm, the polypeptide medicine designed by the application greatly prolongs the half-life of the action, realizes the super-long-acting of the polypeptide medicine, and has the overall medicine action effect greatly superior to that of the positive medicine cable Ma Lutai. In addition, the polypeptide prepared by the application combines albumin in blood by using lipophilic substituent groups to protect the albumin from enzymatic degradation, thereby improving half-life. The polypeptides prepared by the present application 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 prepared polypeptide has 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).
3. The polypeptide prepared by the scheme of the application has better long-acting blood glucose control effect than the bisoprost Ma Lutai, has more stable blood concentration, and has the effects of high enzymolysis stability, high biological activity and basically no adverse reaction, thereby having important significance in preparing obesity drugs and diabetes drugs caused by hyperglycemia.
Drawings
The application is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a plot of the change in blood glucose in mice after administration of glucose in the form of eudragit Ma Lutai and cable Ma Lutai for 24 hours in the test example of the present application;
FIG. 2 is a plot of the change in blood glucose in mice after administration of glucose in the test example of the present application for gastric lavage of mice for Ma Lutai and cable Ma Lutai hours;
FIG. 3 is a graph comparing the rate of change of blood glucose per minute after administration of Uygur autonomous Ma Lutai and Soxhlet Ma Lutai in a test example of the present application, wherein P < 0.00001;
fig. 4 is a graph of AUC results of the effects of eudragit Ma Lutai and cable Ma Lutai on blood glucose in the test cases of the present application, wherein P < 0.005 and P < 0.00001.
Detailed Description
The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.
Example 1 preparation of Polypeptides
The present example prepared a polypeptide which was synthesized manually according to Fmoc/t-Bu strategy, scale of synthesis: 0.5mmol, the following polypeptides were synthesized: boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (PEG 2-PEG 2-gamma-Glu-CO (CH) 2 ) 18 COOH)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-WangThe Resin comprises the following concrete processes:
1. main peptide chain assembly
(1) 1.47g Wang Resin (loading 0.47mmol/g, western An Lan Xiao) was weighed, added to the reaction column, swollen for 30min in 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 deprotection and coupling effects are monitored by ninhydrin hydrate during Fmoc removal and coupling. 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, fmoc-Ser (tBu-OH), boc-His (Trt) -OH above amino acid feed (relative to 5eq on synthesis scale) gave Boc-His-D-Ser (t-Bu) -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. The reaction was completed and washed 6 times with N, N-Dimethylformamide (DMF) and 6 times with Dichloromethane (DCM).
(3) Removal of allyloxycarbonyl (Alloc) and introduction of lipophilic substituents
The resin was washed twice with N, N-Dimethylformamide (DMF)/Dichloromethane (DCM) =1:1 (volume ratio) solution, a solution of tetraphenylphosphine palladium (Pd (PPh 3) 4) in DCM and morpholine was added, the reaction mixture was shaken at room temperature for 2 hours, and then filtered to give Boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-gin-Ala-Lys-Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-wangsin). Thereafter, the resin was washed thoroughly three times with N, N-Dimethylformamide (DMF), dichloromethane (DCM), N, N-Dimethylformamide (DMF) in this order.
FmocNH-PEG2-OH (CAS#: 872679-70-4,Quanta BioDesign) was added to 5 times the scale of synthesis, 1-Hydroxybenzotriazole (HOBT) and N, N-Diisopropylcarbodiimide (DIC) were used as coupling agents, in amounts of 1.2 and 1.3 times the amount of amino acid added, respectively, and after shaking for 2 hours, the mixture was filtered. Obtaining Boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln-Ala-Ala-Lys (Fmoc-PEG 2) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-WangResin. Thereafter, the resin was washed thoroughly three times with N, N-Dimethylformamide (DMF), dichloromethane (DCM), N, N-Dimethylformamide (DMF) in this order.
Fmoc groups were removed (twice 5 min+7min) in a 20% Piperidine (Pieridine)/N, N-Dimethylformamide (DMF) solution, fmocNH-PEG2-OH (CAS#: 872679-70-4,Quanta BioDesign) was added to correspond to a 5-fold synthesis scale, 1-Hydroxybenzotriazole (HOBT), N, N-Diisopropylcarbodiimide (DIC) was used as a coupling agent in an amount 1.2 times and 1.3 times the amount of the amino acid added, and after shaking for 2 hours, boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (OtBu) -Val-Ser (t-Bu) -Tyr (t-Bu) -Leu-Gly-Ala-Lys (Fm-2-Glu) -Glu (OtBu) -Gly-Glu (t-Bu) -Phe-Thr (t-Bu) -Val-Ser (t-Bu) -Tyr (t-Bu) -Leu) -Val (t-Bu-L (Arg-L-Ala-Ala-Arg (PbL-Arg). Thereafter, the resin was washed thoroughly three times with N, N-Dimethylformamide (DMF), dichloromethane (DCM), N, N-Dimethylformamide (DMF) in this order.
Fmoc groups were removed (twice 5min+7 min) in a 20% Piperidine (Pieridine)/N, N-Dimethylformamide (DMF) solution, fmoc-Glu-OtBu was added to correspond to a synthesis scale of 5 times, 1-Hydroxybenzotriazole (HOBT), N, N-Diisopropylcarbodiimide (DIC) was used as a coupling agent in an amount of 1.2 times and 1.3 times the amount of the amino acid added, and after shaking for 2 hours, boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln-Ala-Lys (Fmoc-PEG 2-gamma-PEG) -Gly-Bu-Glu (t-Bu) -Glu (Pbf) -Arg (Pbf-Arg-Pbf) -Arg (Pbf). Thereafter, the resin was washed thoroughly three times with N, N-Dimethylformamide (DMF), dichloromethane (DCM), N, N-Dimethylformamide (DMF) in this order.
Fmoc groups are removed from 20% Piperidine (Pieridine)/N, N-Dimethylformamide (DMF) solution (5 min+7min twice), mono-tert-butyl eicosanoate is added to be equivalent to 5 times of synthesis scale, 1-Hydroxybenzotriazole (HOBT), N, N-Diisopropylcarbodiimide (DIC) are used as coupling agents, the dosage is 1.2 times and 1.3 times of the dosage of amino acid respectively, after shaking for 2 hours, boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln-Ala-Lys (PEG 2-PEG 2-gamma-CO (CH) 2 ) 18 COOH) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-Wang Resin. After this time the resin was washed three times with N, N-Dimethylformamide (DMF), dichloromethane (DCM) and Dichloromethane (DCM) in sequence, and dried in vacuo.
2. Removal of full protection of polypeptides
To dry Boc-His (Boc) -D-Ser (t-Bu) -Glu (OtBu) -Gly-Thr (t-Bu) -Phe-Thr (t-Bu) -Ser (t-Bu) -Asp (OtBu) -Val-Ser (t-Bu) -Ser (t-Bu) -Tyr (t-Bu) -Leu-Glu (OtBu) -Gly-Gln-Ala-Ala-Lys (PEG 2-PEG 2-gamma-Glu-CO (CH) 2 ) 18 COOH) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-Wang Resin, adding lysate TFA/Phenoll/thioanisole/EDT/H 2 O (82.5:5:5:2.5:5, volume ratio) 10 times the weight volume was reacted at room temperature for 2.5 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 glacial diethyl ether with stirring. Standing for more than 0.5 hr, standing for complete precipitation, centrifuging, washing with diethyl ether for 3 times, and vacuum drying to obtain crude compound H-His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (PEG 2-PEG 2-gamma-Glu-CO (CH) 2 ) 18 COOH)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
3. Purification of polypeptide compounds
Dissolving the crude product obtained in the previous step in Acetonitrile (ACN)/H 2 O=1:2 (volume ratio) of solution, preparative HPLC purification was performed on a 10 μm reverse phase C18 preparative column (20 mm x 250 mm). With 30% acetonitrile (containing 0.05% trifluoroacetic acid)/H 2 O (0.05% trifluoroacetic acid) as an initial component, eluting the column at a gradient (ratio of acetonitrile increase at 1.33%/min) and a flow rate of 15mL/min for 30min, collecting the peptide-containing fraction, freeze-drying to obtain a pure polypeptide with an HPLC purity of greater than 95%, designated as excellent Ma Lutai (yomaglutide) (if the purity does not meet the requirement, HPLC purification can be repeated once). The purity of the product separated by liquid chromatography-mass spectrometry analysis is more than 95 percent.
Test examples
1. Determination of agonistic Activity of Excellent Ma Lutai on GLP-1R and GCGR
This test example tested the agonistic activity of Excellent Ma Lutai and Soxhlet Ma Lutai prepared in the examples on GLP-1R and GCGR.
Experimental materials: cord Ma Lutai is purchased from Zhejiang surge peptide biology Inc. (CAS No.: 910463-68-2). In GLP-1R-Luciferase-293T cells and GCGR-Luciferase-293T cell models, optimal Ma Lutai was subjected to primary screening for agonistic activities of GLP-1R and GCGR.
The experimental method comprises the following steps: digestion cells were plated in 96-well plates (medium containing 10% FBS, GLP-1R-Luciferase-293T:20000 cells/well, GCGR-Luciferase-293T: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 euonymus Ma Lutai and cable Ma Lutai were prepared 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.
Experimental results: EC of cable Ma Lutai GLP-1 50 EC of 265.2pM and GCGR 50 8600875pM; EC of the excellent Ma Lutai polypeptide compound GLP-1 of the application 50 EC of 315.2pM and GCGR 50 26008755pM. The results show that the excellent Ma Lutai polypeptide compound prepared by the scheme of the application has obvious agonistic activity on GLP-1R and GCGR as well as cable Ma Lutai, and has a certain specificity on the activation of GLP-1R receptor.
2. Effect of eudragit Ma Lutai and cable Ma Lutai on oral glucose tolerance (OGTT)
The experimental method comprises the following steps: male C57BL/6J mice (university of Nanj model animal research center) at 8 weeks of age were randomly grouped according to similar blood glucose (blood sample assessment obtained from the tail tip), 8 per group. Animals were dosed after a fast (6 hours) and the instant application with the polypeptide compound of eudragit Ma Lutai, positive control doxofyll Ma Lutai, were dosed subcutaneously at 100ug/kg, with PBS in the control group. 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. Mice were fasted for 8h prior to gavage and were given 2.5g/kg of gavage glucose and ddH for gavage glucose at 24h and 48h, respectively 2 O is prepared into a stock solution with the concentration of 0.5g/mL, and the stock solution is stored at normal temperature. Mice were fasted for 8h prior to intragastric administration, and glucose was infused at doses of 2.5g/kg at 24h and 48h, respectively, 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, respectively. Blood glucose changes were detected and blood glucose was tracked up to 48 hours. Using software graphpadrism processing the data produced a blood glucose change line graph (as shown in fig. 1 and 2) and calculated the area under the curve to give an AUC graph (as shown in fig. 3 and 4).
Experimental results: the results are shown in fig. 1-4, and fig. 1 is a graph showing the change line of the blood sugar of the mice after the mice are subjected to gastric lavage glucose after being administrated with excellent Ma Lutai and cable Ma Lutai hours; FIG. 2 is a plot of changes in glucose in mice after administration of glucose in the stomach and after administration of glucose in the stomach for Ma Lutai and Ma Lutai hours; FIG. 3 is a graph comparing the rate of change of blood glucose per minute after administration of Ubbelow Ma Lutai and Soxhlet Ma Lutai to a control group; FIG. 4 is a graph of AUC results of effects of UO Ma Lutai and Song Ma Lutai on blood glucose; as can be seen from the figure, both the cord Ma Lutai and the optimal Ma Lutai have significantly reduced AUC (P < 0.05) in the first OGTT curve period (0-120 min) compared to vehicle (PBS) 24h after administration, and increased AUC to a different extent starting in the subsequent OGTT curve period 48 h. AUC, cord Ma Lutai, at the period of the OGTT curve of 48h has lost pharmacodynamic activity.
In conclusion, the activity of the You Ma Lutai and the activity of the cable Ma Lutai prepared by the scheme of the application are equivalent, but the effect of the You Ma Lutai on long-acting glucose reduction are obviously better than that of the cable Ma Lutai. Compared with a carrier (PBS), the excellent Ma Lutai polypeptide compound can improve the glucose tolerance effect of mice, and has more excellent and obvious glucose tolerance effect, and meanwhile, the result also shows that the excellent Ma Lutai has more excellent and obvious long-acting glucose reducing effect.
The embodiments of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.

Claims (11)

1. A polypeptide, characterized in that the amino acid sequence of the polypeptide is His- (D-Ser) -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (PEG 2-PEG 2-gamma-Glu-CO (CH) 2 ) 18 CO 2 H)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly。
2. A method of producing the polypeptide of claim 1, comprising the steps of: taking solid-phase synthetic resin as an initial raw material, and adopting Fmoc/t-Bu solid-phase synthetic strategy to sequentially connect with protective amino acid or polypeptide to obtain a polypeptide main chain with an amino acid protective group; and (3) grafting side chains on the polypeptide main chain, and carrying out deprotection and peptide cutting.
3. Use of the polypeptide of claim 1 for the preparation of glucagon-like peptide-1 receptor agonists
4. Use of the polypeptide of claim 1 for the preparation of a glucagon receptor agonist.
5. Use of the polypeptide of claim 1 for the manufacture of a medicament for the treatment or prevention of obesity caused by hyperglycemia.
6. Use of the polypeptide of claim 1 for the preparation of a medicament for the treatment or prevention of diabetes.
7. A medicament for treating diabetes comprising the polypeptide of claim 1.
8. The medicament for treating diabetes of claim 7, wherein the raw materials for preparing the medicament further comprise a pharmaceutically acceptable carrier.
9. The drug for treating diabetes according to claim 7, wherein the mass fraction of the polypeptide in the drug is 0.01% -99%.
10. The medicament for treating diabetes according to claim 9, wherein the mass fraction is 0.05% -95%.
11. The medicament for treating diabetes according to claim 10, wherein the mass fraction is 0.05% -20%.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104302772A (en) * 2012-05-18 2015-01-21 爱德迪安（北京）生物技术有限公司 Protein and protein conjugate for diabetes treatment, and applications thereof
CN107129538A (en) * 2010-04-27 2017-09-05 西兰制药公司 Peptide conjugate of the receptor stimulating agents of GLP 1 and gastrin and application thereof
CN110922470A (en) * 2019-12-26 2020-03-27 杭州肽佳生物科技有限公司 Preparation method of somaglutide
CN112552391A (en) * 2019-09-25 2021-03-26 北京志道生物科技有限公司 Recombinant interleukin-15 analogue

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107129538A (en) * 2010-04-27 2017-09-05 西兰制药公司 Peptide conjugate of the receptor stimulating agents of GLP 1 and gastrin and application thereof
CN104302772A (en) * 2012-05-18 2015-01-21 爱德迪安（北京）生物技术有限公司 Protein and protein conjugate for diabetes treatment, and applications thereof
CN112552391A (en) * 2019-09-25 2021-03-26 北京志道生物科技有限公司 Recombinant interleukin-15 analogue
CN110922470A (en) * 2019-12-26 2020-03-27 杭州肽佳生物科技有限公司 Preparation method of somaglutide

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