WO2011032359A1 - Peptide mc-jj6 issu de momordica charantia et son procédé de synthèse sur phase solide assistée par micro-ondes - Google Patents

Peptide mc-jj6 issu de momordica charantia et son procédé de synthèse sur phase solide assistée par micro-ondes Download PDF

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WO2011032359A1
WO2011032359A1 PCT/CN2010/001421 CN2010001421W WO2011032359A1 WO 2011032359 A1 WO2011032359 A1 WO 2011032359A1 CN 2010001421 W CN2010001421 W CN 2010001421W WO 2011032359 A1 WO2011032359 A1 WO 2011032359A1
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ala
lys
gly
met
pro
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PCT/CN2010/001421
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Chinese (zh)
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黄文龙
金晶
钱海
张惠斌
陈巍
王敬杰
杜阔
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中国药科大学
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Priority claimed from CN2009101833191A external-priority patent/CN101691394B/zh
Priority claimed from CN200910183321A external-priority patent/CN101691400A/zh
Priority claimed from CN200910183322A external-priority patent/CN101691395A/zh
Priority claimed from CN200910183318A external-priority patent/CN101691399A/zh
Application filed by 中国药科大学 filed Critical 中国药科大学
Publication of WO2011032359A1 publication Critical patent/WO2011032359A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
    • 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
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a microwave-assisted solid phase synthesis method of MC-JJ6 polypeptide and its application and MC-JJ6 polypeptide. Background technique
  • Diabetes is an important disease that threatens human health today. About 5% to 7% of people worldwide suffer from diabetes. Therefore, diabetes is considered to be the third leading cause of human disability and death after cardiovascular and cerebrovascular diseases and malignant tumors.
  • Type I diabetes is an autoimmune disease. It refers to insulin deficiency caused by autoimmunity and other causes. It is prone to ketosis and must be treated with insulin. It is more common in adolescents. This type of diabetes is rare, accounting for only about 5%. Type II diabetes accounts for about 95% of the total number of patients with diabetes.
  • the cause of type 2 diabetes is a disorder of islet cell metabolism, insufficient insulin secretion, or insulin resistance, which is a metabolic disease caused by insulin resistance mainly accompanied by insufficient insulin secretion, or insulin resistance caused by relatively insufficient insulin secretion. Insufficient insulin secretion, or insulin resistance, can cause excessive glucose in the blood. When the blood sugar level exceeds the renal threshold, glucose is transferred to the urine to produce glucoseuria.
  • Momordica charantia is a vegetable that has a long history of safe eating.
  • many fruits with hypoglycemic activity have been found in the fruit of bitter gourd.
  • Some bitter melon ingredients have been applied for as patents for the treatment of diabetes drugs.
  • the patents or products introduced from the scientific literature and based on the existing patents or products are mainly based on the understanding of a known component in many hypoglycemic active ingredients of bitter gourd, and do not realize that the components with hypoglycemic activity in bitter gourd are known.
  • some active ingredients with blood sugar lowering in bitter gourd are unknown. Therefore, the limitations of existing literature, patents or products are also obvious.
  • U.S. Patent No. 6,391,854 B1 and Chinese Patent CN 98805001 provide a water-soluble extract of bitter gourd Known as MC6, MC6 contains three peptides, and its preparation method is characterized in that the centrifugally clarified bitter gourd juice passes through 30kDal, lOkDal and 3kDal ultrafiltration membranes in sequence.
  • the MC6 molecular weight is less than lOkDal and can be retained by the lOkDal ultrafiltration membrane for the 3kDal membrane.
  • the active MC6 is characterized by migration in a single band on SDS-PAGE gel electrophoresis with a molecular weight of less than 10 kDal and contains three peptides. Also provided are peptide components of MC6, referred to as MC6. 1, and derivatives and mimetics of MC6.1. Active MC6, MC6. 1, MC6. 2, MC6. 3 has hypoglycemic effect, especially diabetes, wherein these active drugs are preferably administered orally. In this patent, the bitter melon used is also active after hypoglycemia orally.
  • MC6 does not increase insulin activity in plasma of model animals.
  • the invention of an international patent application provides a highly effective hypoglycemic polypeptide, k, which is extracted from bitter melon.
  • the polypeptide-k preparation method introduced by the patent applicant is: treating the bitter gourd seed (or fruit) with a non-polar solvent, removing unnecessary oil, flavonoids and saponin, and then extracting the residue with 80% aqueous acetone. The protein is finally selectively crystallized to isolate the polypeptide-k.
  • Applicants describe that peptide-k is insoluble in water, and its mechanism of hypoglycemia is to activate inactive insulin in the blood.
  • both MC6 and peptide-k are proteins with hypoglycemic activity in bitter gourd.
  • MC6 is soluble in water, while polypeptide-k is insoluble in water. According to the preparation method of MC6, the polypeptide-k in bitter gourd is lost. According to the preparation method of polypeptide-k, MC6 in bitter gourd was also lost.
  • a bitter melon hypoglycemic capsule and a preparation process thereof, the invention uses fresh bitter gourd as raw material, is cleaned and frozen, low temperature pulping, pressure filtration, and the filter residue is dried through 100 mesh.
  • Sieve made of bitter gourd slag powder; filtrate clarified and filtered, acid (pH 3. 0)
  • the ethanol was stirred under a condition of 15 Torr for 3 hours, and the precipitate was allowed to stand for 12 hours. After filtration, the precipitate was dried at a low temperature and passed through a 100 mesh sieve to prepare a bitter melon protein; the filtrate was concentrated under vacuum in 60 Torr, dissolved in 2 times water, and doubled.
  • the invention discloses a method for preparing natural momordicin hypoglycemic agent, including the following Steps: a. Cut the mature bitter gourd into pieces and dry it; b. Soak the dried bitter gourd slices in water and extract the infusion; c. Concentrate the bitter gourd extract into a concentrate; d. Performing bio-enzymatic hydrolysis to obtain a highly active degradation liquid; e. subjecting the degradation liquid to constant temperature drying to spray granulation; f. loading the granule into a capsule into a medicine; the method has simple process equipment, meets large-scale production requirements, and has high yield. Good therapeutic effect, no toxic side effects.
  • the invention is a traditional Chinese medicine for treating diabetes, the main components of the traditional Chinese medicine are animal pancreas, bitter melon, pumpkin, scutellaria, yam, production method It is the animal's pancreas smashed and dried. It is mixed with the extract of bitter gourd, pumpkin and astragalus extracted by water extraction or alcohol precipitation method and then added to yam powder.
  • the drug has the benefits of qi, spleen and moistening, nourishing yin and reducing fire. It also enhances the body's immune function, promotes insulin secretion, and lowers blood sugar.
  • the precipitate was washed three times with acetone and dried under vacuum to obtain a crude plant insulin.
  • the gel affinity chromatography was carried out by dissolving 0.1 L Tris. cl solution (pH 7.5), and the eluted peak solution was collected and concentrated by acetone or ethanol to obtain a plant-based insulin product.
  • the method can extract and prepare high-purity plant insulin components, and can be used for the treatment of diabetes.
  • a bitter melon lozenge and a preparation method thereof the invention is a bitter melon lozenge and a preparation method thereof.
  • the bitter melon lozenge mainly comprises bitter gourd powder, dextrin, citric acid and mannitol.
  • fresh bitter gourd is firstly juiced, filtered, freeze-dried to obtain bitter gourd lyophilized powder, and then mixed with other ingredients and pressed. Into pieces.
  • the preparation method of the invention is scientific and reasonable, and fully retains various nutrients in the bitter gourd, and the prepared tablet contains no activity of bitter gourd at normal temperature, and has the functions of clearing away heat and detoxifying, widening thirst, stimulating thirst, lowering blood sugar, and improving immunity.
  • the ability to suppress cancer cells and other functions is a convenient necessity for daily life, especially in disaster relief, military operations, tourism, and expeditions, to quickly replenish physical strength and improve immunity.
  • the Sovereignty Item (A61K35/78) requires: a bitter melon lozenge that is characterized by bitter melon powder, dextrin, mannitol, spices, pigments, and magnesium stearate.
  • bitter gourd saponin is a tetracyclic three mushroom compound, which has many different components and is hardly soluble in water. It can be extracted from dried bitter gourd fruit by ethanol (Chang Fenggang: Chemical constituents of bitter gourd, Chinese herbal medicine, 1995) , 26 (10) 507-510) Momordica saponin does not affect the plasma insulin content in the animal model, mainly to promote the synthesis of muscle glycogen and hepatic glycogen (Wang Xianyuan, Jin Hong, Xu Zhiqin, etc.: the hypoglycemic effect of bitter melon saponin and Mechanisms, Amino Acids and Biological Resources, 2001, 23 (3): 42-45), and thus have significant hypoglycemic effects (Sun Shuqing, Overview of Pharmacology and Clinical Research of Momordica charantia L., Chinese Medicinal Materials, 1997, (8): 428-429 ), animal experiments have shown that the blood sugar reduction is even better than "Yi Jiang Tang
  • the water-soluble polypeptide MC6 the water-insoluble polypeptide-k and the bitter melon saponin belong to each other due to differences in their chemical properties. Moreover, the bitter melon hypoglycemic active ingredient is not only known saponins and proteins.
  • hypoglycemic functional actives of bitter gourd are known, and there is also an unknown hypoglycemic function of bitter gourd, which is actually the result of synergistic action of various components having hypoglycemic activity contained therein.
  • the known active ingredients are concerned, it is difficult to simultaneously extract them with a solvent because of their different chemical properties.
  • the hypoglycemic active of bitter gourd a component which is not heat-resistant is contained, and the extraction process should not be heated.
  • the fresh bitter gourd pulp tissue cells are fully disrupted by refining, and all the contents of the cells, including the hypoglycemic active ingredients dissolved in water and poorly soluble in water, are released into the pulp.
  • the crude fiber in the pulp (from the cell wall of the pulp) is filtered off and then freeze-dried.
  • This lyophilized product of bitter gourd juice undoubtedly contains a relatively comprehensive hypoglycemic active ingredient.
  • the content of hypoglycemic active substances in bitter gourd juice is actually very low, and the direct freeze-drying is expensive.
  • the sugar substances present in bitter gourd juice are easily deliquescent, so that the freeze-dried products cannot be divided under normal conditions at all. Packing, storage and use. Although a large amount of drying aid or moisture-proofing agent can be added to solve the problem of deliquescence of the freeze-dried product of bitter gourd juice, it is impossible to obtain a high content of bitter melon hypoglycemic product.
  • the preparation of the active ingredients of bitter gourd disclosed in the literature and patents is basically the extraction of one or several specific chemical components by physical and chemical methods, or a certain solvent of bitter gourd under heating conditions (
  • the total extracts, such as water, ethanol or mixtures thereof, do not mention how to use the hypoglycemic active ingredients of the chemically unknown in bitter gourd, or the thermal damage of certain thermosensitive hypoglycemic actives in bitter gourd.
  • the polypeptide can be obtained by genetic engineering or chemical synthesis.
  • the method of genetic engineering is superior to chemical methods in obtaining long peptides (amino acid residue lengths greater than 50) or proteins, but the chemical synthesis methods of peptides, especially after the appearance of solid phase synthesis strategies, are prepared to have amino acid residues less than 40 in length.
  • Polypeptides or small peptides have incomparable flexibility, diversity and efficiency in genetic engineering methods.
  • 1963 Merrifield founded and developed a method for solid phase synthesis of peptides. There are two strategies for solid phase peptide synthesis: Boc/Bzl orthogonal protection solid phase synthesis strategy and Fraoc/tBu orthogonal protection solid phase synthesis strategy.
  • Merrifield's solid phase synthesis method is Boc/Bzl orthogonal protection solid phase. Synthesis strategy.
  • Boc/Bzl orthogonal protection solid phase synthesis strategy there are some disadvantages in the Boc/Bzl orthogonal protection solid phase synthesis strategy, such as many side reactions, harsh conditions, and loss of the polypeptide chain from the solid phase during the extension of the peptide chain.
  • the subsequent Fmoc/tBu orthogonal protection solid phase synthesis strategy has a milder reaction conditions.
  • microwave technology has been applied to the solid phase synthesis of peptides in recent years, the synthesis technology of peptides has made a leap. The microwave promotes the chemical reaction because it rapidly rotates the polar molecules in the microwave field, so that some reaction rates are 10 to 1000 times faster than conventional heating methods, and the yield is greatly improved.
  • a first object of the present invention is to modify a polypeptide obtained from a homologous plant bitter gourd having hypoglycemic activity to obtain a MC-JJ6 polypeptide having a higher hypoglycemic activity.
  • the present invention provides a bitter melon MC-JJ6 polypeptide having hypoglycemic activity, wherein the bitter melon MC-JJ6 polypeptide comprises the amino acid sequence shown in SEQ. ID NO: 1.
  • Xaa for Lys, Gly, Ala, Val, Leu, lie, Phe, Pro, Met, Cys, D- Lys, D-Ala, D-Val, D-Leu, D-lie D-Phe, D-Pro, D-Met or D-Cys,
  • Xaa2 is Lys, Gly, Ala, Val, Leu, lie, Phe, Pro, Met, Cys, Thr, D-Lys, D-Ala, D-Val, D-Leu, D-lie D-Phe, D-Pro , D-Met or D-Cys,
  • Xaa3 is Lys, Gly, Ala, Val, Leu, lie, Phe, Pro, Met, Cys, Asn, D-Lys, D-Ala, D-Val, D-Leu, D-Ile D-Phe, D-Pro , D-Met or! ) -Cys, and
  • Xaa4 is Ala or Gly.
  • the present invention also provides a bitter melon MC-JJ6222 polypeptide having hypoglycemic activity, wherein the bitter melon MC-JJ6222 polypeptide comprises the amino acid sequence shown in SEQ. ID NO: 115,
  • ⁇ 33 1 is Asn, Ala, Gly or D-Ala
  • Xaa 2 is Ala, Gly, His or D-Ala
  • Xaa 3 is 413, 01, 1115 or 0-eight 13; 3 is Ala, Gly, Tyr or D-Ala;
  • Xaa 5 is Ala, Gly, Tyr or D-Ala
  • Xaa 6 is Ala, Gly, Lys or D-Ala
  • Xaa 7 is Ala, Gly, Lys or D-Ala.
  • the inventors of the present application have recognized that the use of microwaves to promote solid phase synthesis of polypeptides can overcome difficult peptide sequences and synthesize polypeptides that are not available in conventional solid phase methods, and the synthesis time is greatly shortened, and the purity and yield of the crude polypeptide are significantly improved.
  • the purification of the product is greatly facilitated, as long as a preparative HPLC can be used to obtain a product with higher purity; in addition, it is also very convenient to use a D-type amino acid and an unnatural amino acid to modify the polypeptide to find a more active, biological Polypeptides with longer half-lives are beyond the reach of genetic engineering techniques.
  • a second object of the present invention is to provide a solid phase preparation method for the above polypeptide, which can be efficiently and rapidly synthesized using a microwave-assisted Fmoc/tBu orthogonal protection solid phase synthesis strategy.
  • the method using microwave to promote Fmoc/tBu orthogonal protection solid phase synthesis strategy, firstly synthesizing the first Fmoc protected amino acid resin on the solid phase carrier, and removing the Finoc after the ninhydrin method is negative.
  • the protecting group obtains the resin carrying the first amino acid residue; then proceeds to the next coupling cycle, repeats the coupling and deprotection steps with different protected amino acids according to the corresponding peptide sequence, and sequentially prolongs the desired amino acid sequence, and synthesizes
  • the resin carrying the corresponding polypeptide is obtained, and finally the polypeptide is cleaved from the resin with a lysing agent to obtain a crude polypeptide.
  • the crude product was purified by preparative high performance liquid chromatography and freeze-dried to obtain a pure polypeptide.
  • the synthesis of the resin carrying the first Fmoc protected amino acid is obtained by coupling the Fmoc-protected amino acid to the solid phase carrier after activation by an activator under microwave irradiation, and adding a hydroxy-benzotriazole to the reaction.
  • H0BT hydroxy-benzotriazole
  • the solid phase carrier used in the synthesis of the resin carrying the first Fmoc protected amino acid is Rink resin, Wang resin or 2- Chlorotrityl chloride resin.
  • the activator is dicyclohexylcarbodiimide (DCC), hydrazine, ⁇ '-diisopropylcarbodiimide (DIC), N,N"-carbonyldiimidazole (CDI), 1-ethyl- (3 -Dimethylaminopropyl)carbodiimide hydrochloride (EDC.
  • HC1 2-(7-azobenzotriazole) _N, N, N', N '-tetramethylurea hexafluoro Phosphate (HATU), benzotriazole N, N', N'-tetramethylurea hexafluorophosphate (HBTU) or 1-hydroxy-benzotriazole (H0BT) derivative used as N- Hydroxysuccinimide (H0SU), 1-hydroxy-7-azobenzotriazole (H0AT) or 3-hydroxy-1,2,3-benzotriazine-4(3H)-one (H00BT)
  • the organic base is triethylamine (TEA), N-methylmorpholine (NMM) or diisopropylethylamine (DIEA); the microwave-promoted fr is: microwave frequency 2450MHz, reaction temperature: 20 ⁇ 100°C, The reaction time is 5 to 15 min.
  • the removal of the Fmoc protecting group is carried out by a microwave-assisted reaction using a solution of 1-hydroxy-benzotriazole (H0BT) containing hexahydropyridine containing 0.1 mol. L- ', using dimethyl ketone
  • H0BT 1-hydroxy-benzotriazole
  • DMS0 dimethyl sulfoxide
  • NMP N-methylpyrrolidone
  • the microwave promotion conditions are: microwave frequency 2450 MHz, reaction temperature: 20 to 100 ° C, and reaction time is l 10 10 min.
  • the proposed MC-JJ6 polypeptide or MC-JJ6222 polypeptide can improve stability and prolong the action time on the basis of retaining hypoglycemic activity.
  • Microwave-assisted solid phase synthesis of the polypeptide greatly increases the coupling reaction rate.
  • Conventional solid phase synthesis methods fully couple an amino acid to the resin, often ranging from 2 hours to 20 hours or even longer.
  • the microwave promotion takes only about 10 minutes on average; the conventional solid phase synthesis method takes 10 minutes to 1 hour to remove the Fmoc protecting group, while the microwave promotion takes only about 5 minutes on average, which greatly improves the peptide synthesis.
  • the efficiency of the synthesis cycle is shortened.
  • the purity of the crude product synthesized by microwave-assisted solid phase synthesis MC-JJ6 peptide is more than 60%, and the purity of the crude product synthesized by microwave-assisted solid phase synthesis MC-JJ6221 is more than 80%, which is much better than the conventional solid phase synthesis method. Subsequent purification work requires only one preparative liquid phase purification and lyophilization to obtain the target pure product.
  • the microwave-assisted solid phase method for the synthesis of MC-JJ6 peptides has a low cost. Due to the high coupling efficiency, the average amino acid required to be protected needs only a 2-fold excess, which is much lower than the conventional solid-phase synthesis method by 4 to 5 times.
  • the MC-JJ6 peptide method is easy to automate and large-scale, which makes it more suitable for industrial production.
  • the above-mentioned polypeptide is prepared by the microwave-assisted solid phase synthesis method provided by the invention, and has high yield, short synthesis cycle, easy purification of crude product, low production cost and easy industrial automation production.
  • the prepared MC-JJ6 series polypeptide is more stable and has a longer action time than the natural MC-JJ6 polypeptide, and is suitable as an active ingredient for treating diabetes drugs.
  • Et 3 N triethylamine; NMM: N-methylmorpholine; DIEA: ⁇ , ⁇ '-diisopropylethylamine; DMF: dimethylformamide; DM SO: dimethyl sulfoxide; DCM: Methyl chloride; Finoc: N-9 methoxycarbonyl; DIC: ⁇ , ⁇ '-diisopropylcarbodiimide; CDI: ⁇ , ⁇ '-carbonyldiimidazole; DMAP: 4-dimethylaminopyridine; H0SU : ⁇ -hydroxysuccinimide; EDC.
  • HC1 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU: 2-(7-azobenzotriazole) ⁇ ''-tetramethylurea hexafluorophosphate ; HBTU: benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate; HCTU : 6-chlorobenzotriazine Oxazole-1,1,3,3-tetramethylurea hexafluorophosphate; H0AT: 1-hydroxy-7-azobenzotriazole; H0BT: 1-hydroxy-benzotriazole; PyBOP: Benzyltrifluoro-l-yl-oxytripyrrolephosphonium fluorophosphate; HPLC: high performance liquid chromatography; ESI-MS: electrospray ionization mass spectrometry; Gly: glycine; Ser: serine; Ala: electros
  • Fmoc-Ala rink (0.04 ol) HBTU (0.04 ol) H0BT (0.04 ol) and DIPEA (0,08 mmol) were dissolved in 10 mL of NMP, and this solution was added to the above resin in a microwave reactor. The reaction was carried out for 7 min, the microwave power was 25 W, the reaction temperature was controlled at 50 Torr, and air cooling was performed using an air compressor. After the completion of the reaction, the reaction solution was filtered off, and 7 mL of the varnish resin of DCM and hydrazine was used for three times.
  • the coupling efficiency of the resin is legally determined by the ninhydrin method or bromophenol blue, and the color reaction is negative to enter the next coupling cycle.
  • the ninhydrin method a small amount of resin particles were washed with ethanol, and placed in a transparent vial, 5% ninhydrin ethanol, KCN pyridine solution (2 ml of 0.001 M KCN diluted in 98 ml of pyridine), and 2 drops of 80% phenol ethanol solution. Heat at 100 Torr for 5 minutes, if the resin is blue, it is positive.
  • Bromophenol blue method Take a small amount of resin particles and wash with diacetyl acetamide, add 3 drops of 1% bromophenol blue dimethylacetamide solution into a transparent vial, shake at room temperature for 3 minutes, if the resin is blue That is positive.
  • the crude product of Ala 3 -MOJJ6-(1 11)-NH2 obtained above was dissolved in a small amount of water, and the crude product was purified by the reversed phase HPLC.
  • Purification medium was prepared by C18 reverse phase preparative column (340 mm ⁇ 28 mm, 5 Mm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); mobile phase Gradient: Mobile phase B 13% 15%, 30 min; flow rate 6 mL/min; detection wavelength 214 nm.
  • the collected solution was lyophilized to a pure product to finally obtain a pure product of 29.7 mg.
  • mice Male Kunming mice (body weight 18 ⁇ 22 g) of 10 weeks old were randomly divided into groups of 6 rats. The tail vein was injected with alloxan to be administered at a dose of 60 mg/kg. After 72 hours, the mice were fasted for 5 to 6 hours to measure blood glucose. The mice with blood glucose values ranging from 13 to 23 were randomly assigned to each group. only. The modeled mice were intraperitoneally injected with MC-JJ6 polypeptide for 10 days. The blood glucose level was measured by a blood glucose meter at 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes.
  • hypoglycemic activity of the C-JJ6 polypeptide was stronger than that of metformin.
  • Fmoc- Ser- OH (0. 04 mmol), HBTU (0. 04 ol H0BT (0. 04 mmol) and DIPEA (0. 08 mmol) were dissolved in 10 mL of NMP, and this solution was added to the above resin.
  • the reaction was carried out in a microwave reactor for 7 min, the microwave power was 25 W, the reaction temperature was controlled at 50 V, and air cooling was performed using an air compressor. After the reaction, the reaction solution was filtered off, and 7 mL of each of DCM and NMP was used to wash the resin. Times.
  • the coupling efficiency of the resin is legally determined by the ninhydrin method or bromophenol blue, and the color reaction is negative to enter the next coupling cycle.
  • Ninhydrin method take a small amount of resin particles and wash with ethanol, put into a transparent vial, add 5% ninhydrin ethanol, KCN pyridine solution (2ml 0. 001M KCN diluted in 98ml pyridine), 80% phenol ethanol solution 2 drops , heated at 100 ° C for 5 minutes, if the resin is blue, it is positive.
  • Bromophenol blue method Take a small amount of resin particles and wash with diacetyl acetamide, add 3 drops of 1% bromophenol blue dimethylacetamide solution into a transparent vial, shake at room temperature for 3 minutes, if the resin is blue That is positive.
  • the resin obtained by the above-mentioned Al ai -MC-JJ6222-(Guang 21)-NH 2 was placed in a reaction flask, and each of the cracking agent Reagent K (TFA/phenylsulfide/water/phenol/EDT 82.5) was added. : 5 :5 : 5 :2. 5, V/V) lOmL, first shake at 0 °C for 30 min, then react at room temperature for 3 h. After completion of the reaction, the mixture was suction filtered, washed with a small amount of TFA and DCM, and the filtrate was combined.
  • the cracking agent Reagent K TFA/phenylsulfide/water/phenol/EDT 82.5
  • the crude product of Alal-MC-JJ6222-(1 ⁇ 21)-NH 2 obtained above was dissolved in a small amount of water, and the crude product was purified by the reversed phase HPLC.
  • the C18 reverse phase preparative column (340 X 28 5 Mm) was used for the purification; mobile phase A: 0.1% TFA/water (V/V), mobile phase B: 0.1% TFA/acetonitrile (V/V); Mobile phase gradient: Mobile phase B 20% ⁇ 65% 40 min; flow rate 6 mL/min; detection wavelength 214 nm. 7 ⁇
  • the collected solution was lyophilized to a pure product, and finally obtained a pure product 29. 7 ⁇ .
  • Example 186 Lys-Thr-Gly-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Gly-His-Pro-Ala-Tyr-Ser-Ile-Lys-Lys-Ser (SEQ. ID NO: 188); The theoretical relative molecular mass is 2117.573.
  • ESI-MSm/z found [M+3H] 3 ' 724.9, [M+4H] 4t 543.9 [M+5H] 5t 435.3; calu[M+3H] 3 ' 724.8, [M+4H] 4t 543.8, [ M+5H] 5+ 435.3.
  • mice Male Kunming mice (body weight 18 ⁇ 22 g) of 10 weeks old were randomly divided into groups of 6 rats. The tail vein was injected with alloxan to be administered at a dose of 60 mg/kg. After 72 hours, the mice were fasted for 5 to 6 hours to measure blood glucose. The mice with blood glucose values ranging from 13 to 23 were randomly assigned to each group. only. The modeled mice were intraperitoneally injected with MC-JJ6222 polypeptide for 10 consecutive days. The blood glucose level was measured with a blood glucose meter at 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne les peptides MC-JJ6 et MC-JJ6222 issus de Momordica charantia ayant une activité hypoglycémique et leur procédé de synthèse sur phase solide assistée par micro-ondes. Les peptides MC-JJ6 et MC-JJ6222 comprennent les séquences d'acides aminés de SEQ ID N°: 1 et SEQ ID N°: 115, respectivement.
PCT/CN2010/001421 2009-09-17 2010-09-17 Peptide mc-jj6 issu de momordica charantia et son procédé de synthèse sur phase solide assistée par micro-ondes WO2011032359A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN2009101833191A CN101691394B (zh) 2009-09-17 2009-09-17 微波促进固相合成苦瓜mc-jj6多肽类似物及其应用
CN200910183321A CN101691400A (zh) 2009-09-17 2009-09-17 微波促进固相合成苦瓜mc-jj6222多肽类似物及其应用
CN200910183318.7 2009-09-17
CN200910183322A CN101691395A (zh) 2009-09-17 2009-09-17 微波促进固相合成苦瓜mc-jj62多肽类似物及其应用
CN200910183319.1 2009-09-17
CN200910183321.9 2009-09-17
CN200910183322.3 2009-09-17
CN200910183318A CN101691399A (zh) 2009-09-17 2009-09-17 微波促进固相合成苦瓜mc-jj6221多肽类似物及其应用

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WO2011032359A1 true WO2011032359A1 (fr) 2011-03-24

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CN1255044A (zh) * 1997-04-01 2000-05-31 卡里克斯治疗公司 苦瓜中的口服活性成分,其活性肽,及它们治疗糖尿病的用途
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CN101691400A (zh) * 2009-09-17 2010-04-07 中国药科大学 微波促进固相合成苦瓜mc-jj6222多肽类似物及其应用
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