WO2017101144A1 - Adipose tissue targeting polypeptide, preparation method therefor and application thereof - Google Patents

Adipose tissue targeting polypeptide, preparation method therefor and application thereof Download PDF

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Publication number
WO2017101144A1
WO2017101144A1 PCT/CN2015/098741 CN2015098741W WO2017101144A1 WO 2017101144 A1 WO2017101144 A1 WO 2017101144A1 CN 2015098741 W CN2015098741 W CN 2015098741W WO 2017101144 A1 WO2017101144 A1 WO 2017101144A1
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Prior art keywords
adipose tissue
targeting polypeptide
fmoc
amino acid
seq
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PCT/CN2015/098741
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French (fr)
Chinese (zh)
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张晓�
厉保秋
路杨
杨东晖
何南海
沈柯
周俊
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杭州阿诺生物医药科技股份有限公司
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Publication of WO2017101144A1 publication Critical patent/WO2017101144A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • 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

Definitions

  • the invention relates to the field of protein polypeptides, in particular to a kind of adipose tissue targeting polypeptides and a preparation method and application thereof.
  • obesity As a common chronic endocrine and metabolic disease, obesity has a rising incidence year by year and has become a global public health problem. When the body eats more calories than it consumes, it is stored in the body in the form of fat. More than 20% of normal body weight is obesity. Obesity can occur at any age, but after 40 years of age, the incidence of women is high. Obesity is divided into two types: simple obesity, patients with obesity as the main performance, without obvious changes in neurological and endocrine system functions, but with metabolic regulation disorders, such obesity is the most common. Second, secondary obesity, often secondary encephalitis, meningitis, brain damage, pituitary disease, adrenal hyperfunction, hypothyroidism, excessive insulin secretion.
  • Ideal weight-loss drugs should reduce energy intake, increase energy expenditure, and improve obesity-related cardiovascular risk factors. So far, obesity drugs can be mainly divided into three categories according to the mechanism of action: drugs that suppress appetite, drugs that affect nutrient absorption, and drugs that increase energy consumption.
  • the appetite-suppressing drugs have the following types.
  • the drugs acting on norepinephrine directly act on the central nervous system, stimulate the release of norepinephrine (NE) or block its reuptake, leading to depletion of nerve endings NE, thereby suppressing appetite.
  • NE norepinephrine
  • Such drugs are, for example, amfepramone.
  • Serotonin (5-HT) receptor agonists achieve weight loss by promoting the release of 5-HT and/or inhibiting its reuptake.
  • Sibutramine is a representative of neurotransmitter reuptake inhibitors, which reduce the intake of food and reduce body weight through reuptake of NE and 5-HT; unlike fenfluramine and dexfenfluramine, West Butrexide does not induce the release of 5-HT and is therefore considered to be unrelated to the development of cardiovascular disease.
  • the problem of the prior art solved by the present invention is that the currently used weight-loss drugs for clinical use actually help to reduce weight and reduce body weight rebound, but lack long-term safety and efficacy evaluation, and long-term use may lead to nervous, cardiovascular and other systems. disease.
  • the present inventors have discovered a novel drug intervention target by studying the mechanism of obesity occurrence, thereby finding the adipose tissue targeting polypeptide of the invention.
  • the present invention provides an adipose tissue targeting polypeptide comprising a cell targeting polypeptide peptide, an apoptotic polypeptide peptide, and a cell-targeting polypeptide peptide and apoptosis A bridge of a polypeptide peptide.
  • the cell-targeting polypeptide peptide is selected from one of the following sequences: SEQ1, SEQ2, SEQ3, SEQ4, SEQ5, SEQ6, SEQ7, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ. , SEQ 17, SEQ 18, SEQ 19, SEQ 20, SEQ 21, SEQ 22, SEQ 23, SEQ 24, SEQ 25, SEQ 26, SEQ 27, SEQ 28, SEQ 29, SEQ 30;
  • amino acids in the peptides of the cell-targeting polypeptides described herein are independently glycine and a D- or L-form amino acid, preferably a glycine and an L-form amino acid.
  • the amino acid in the peptide segment of the cell targeting polypeptide is a natural or unnatural amino acid, and the natural amino acid is preferably a natural amino acid modified by a protecting group; the protecting group is preferably a tert-butoxycarbonyl group. , O-t-butyl, trityl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, One or more of allyl, (N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene-1)ethyl).
  • the apoptotic polypeptide peptide segment is selected from the group consisting of an apoptotic factor or one of the following peptide chain fragments: SEQ31, SEQ32;
  • amino acid in the peptide chain fragment is independently a D-form or an L-form amino acid; preferably all of the amino acids are D-form amino acids or L-form amino acids; further preferably all of the amino acids are D-form amino acids.
  • the apoptotic polypeptide peptide segment is selected from the group consisting of an apoptotic factor or a peptide chain fragment SEQ33;
  • amino acid in the peptide chain fragment is independently a glycine and a D-form or an L-form amino acid; preferably a glycine and a D-form amino acid.
  • the amino acid in the peptide chain fragment is a natural or unnatural amino acid;
  • the natural amino acid is preferably a natural amino acid modified by a protecting group; and the protecting group is preferably a tert-butoxycarbonyl group or an oxygen-free one.
  • the apoptotic factor is selected from the group consisting of interleukin-1, interleukin-2, interleukin-5, interleukin-10, interleukin-11, and leukocyte mediator. -12, interleukin-18, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , tumor necrosis factor- ⁇ , macrophage colony-stimulating factor.
  • the bridge is from 2 to 10 natural or unnatural amino acid fragments.
  • the bridge is a fragment of 2 to 6 amino acids, more preferably 2 amino acid fragments.
  • the bridge chain comprises glycine or alanine.
  • the adipose tissue targeting polypeptide is a chain or a circular adipose tissue targeting polypeptide; and the cyclic adipose tissue targeting polypeptide is preferably intramolecularly looped.
  • the ring formation is S-S, CO-NH or CO-S bond.
  • the adipose tissue targeting polypeptide is modified with polyethylene glycol, and the adipose tissue targeting polypeptide binds to polyethylene glycol at any position of the C-terminus, the N-terminus or the side chain.
  • the abranche of the adipose tissue targeting polypeptide is bound to polyethylene glycol.
  • the polyethylene glycol is selected from the group consisting of PEG10, PEG100, PEG200, PEG300, PEG400, PEG500, PEG600, PEG700, PEG800, PEG900, PEG1000, PEG1100, PEG1200, PEG1300, PEG1400, PEG1500, PEG1600, PEG1700, PEG1800, PEG2000 One or more of PEG3000, PEG4000, PEG10000, PEG20000, and PEG40000.
  • the polyethylene glycol is selected from one or more of PEG10, PEG100, PEG200, PEG300, PEG400, PEG500.
  • the polyethylene glycol is selected from one or more of PEG 10, PEG 100, and PEG 200.
  • the invention also provides a preparation method of an adipose tissue targeting polypeptide, comprising the following steps:
  • the coupling agent comprises a condensing agent and a reaction solvent
  • the condensing agent is selected from one of the following combinations: (1) N, N'-diisopropylcarbodiimide and 1-hydroxyl Benzotriazole,
  • the reaction solvent is one or more selected from the group consisting of N,N'-dimethylformamide, dichloromethane, N-methylpyrrolidone, and dimethyl sulfoxide.
  • the preparation method further comprises the step of cyclizing the chain adipose tissue targeting polypeptide obtained in the step (2) to obtain a cyclic adipose tissue targeting polypeptide; the cyclization process comprises liquid phase cyclization and Solid phase cyclization.
  • the liquid phase cyclization comprises the following steps:
  • the solid phase cyclization is to add a chain adipose tissue targeting polypeptide to an oxidizing agent to obtain a cyclic adipose tissue targeting polypeptide.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the adipose tissue-targeting polypeptide of any of the above forms in free form or in a pharmaceutically acceptable salt form as an active ingredient, and one or more A pharmaceutically acceptable carrier material and/or diluent.
  • the invention also provides the use of the adipose tissue targeting polypeptide of any of the above or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment and/or prevention of weight loss or other diseases associated with weight loss.
  • the present invention also provides the use of the adipose tissue-targeting polypeptide of any of the above or the pharmaceutical composition described above for the treatment and/or prevention of weight loss or other diseases related to weight loss.
  • the beneficial effects obtained by the present invention are as follows:
  • the present invention obtains a novel targeted polypeptide drug.
  • the structure of the compound is to link a specific cell-targeting polypeptide and an apoptotic polypeptide through a bridge chain, which can inhibit the expression of the target in the fat vascular endothelial cells and cause the ablation of the visceral adipose tissue, which can promote the normalization of metabolism and Rapid reversal of obesity in animal experiments and disruption of receptor-mediated intracellular mitochondrial membranes, including D-form amino acids, provides resistance to peptidase enzymatic hydrolysis in plasma.
  • Such compounds can specifically cause apoptosis of fat cells, thereby achieving the effect of reducing fat and reducing weight.
  • the targeting mechanism contained in the molecule itself it can avoid the common adverse reactions of the existing slimming drugs, and has more direct effects and fewer side effects than the existing slimming drugs.
  • an adipose tissue targeting polypeptide comprising a cell targeting polypeptide peptide, an apoptotic polypeptide peptide, and a cell-targeting polypeptide peptide and cell A bridge of an apoptotic polypeptide peptide.
  • the invention mainly relates to a kind of synthetic adipose tissue targeting polypeptide, which is characterized in that a cell targeting polypeptide and an apoptotic polypeptide are linked by a bridge chain. Different cell targeting polypeptides, bridges and apoptotic polypeptides were screened.
  • the adipose tissue-targeting polypeptide drug has remarkable curative effect and has low physiological toxicity.
  • the adipose tissue targeting polypeptide provided by the invention has a clinical dosage ranging from 3 to 15 mg/kg per day.
  • the inventors increased the stability of the adipose tissue-targeting polypeptide by chemical synthesis, such as: targeting the synthesized adipose tissue to form an intramolecular disulfide ring to increase stability, and obtaining the target by changing the amino acid sequence.
  • the polypeptide is less susceptible to hydrolysis and the polypeptide improves renal clearance.
  • Adipose tissue targeting polypeptides consist of contiguous amino acids that can selectively act on general human organs, tissues or cells, and in particular on adipose tissue or cells.
  • the adipose tissue targeting polypeptide has a recognition function on adipose tissue cells and can selectively act on adipose tissue cells. Selective localization allows the adipose tissue targeting polypeptide to be enriched twice or more on the target tissue or cell.
  • the synthetic adipose tissue-targeting polypeptide has properties that reduce physiological toxicity, wherein physiological toxicity includes renal toxicity.
  • the cell-targeting polypeptide of the present invention can specifically act on a general human organ, tissue or cell, and particularly on an adipose tissue or a cell, and specifically localize the synthesized adipose tissue-targeting polypeptide to adipose tissue or fat.
  • the apoptotic polypeptide can specifically act on the cells at the adipose tissue to cause apoptosis and obtain the purpose of slimming and slimming.
  • the adipose tissue targeting polypeptide of the present invention may be modified by using polyethylene glycol (PEG), and the polyethylene glycol may be in a single or multiple groups with the C-terminus, N-terminus or side chain of the peptide chain. Binding at any position, the modified polypeptide compound can prolong its half-life in vivo.
  • PEG polyethylene glycol
  • the targeted adipose tissue polypeptide peptide chain of the present invention can be intramolecularly looped, thereby prolonging its half-life in vivo, and the ring-forming manner can be a disulfide bond or an amide bond.
  • the disulfide bond uses an oxidizing agent to cause intramolecular ring formation of a cysteine side chain thiol group, and the oxidizing agent is preferably hydrogen peroxide, iodine or air.
  • the amide bond is prepared by condensing a carboxyl group and an amino group of a side chain of an amino acid with a coupling agent.
  • the invention also provides a method for preparing an adipose tissue targeting polypeptide, but is not limited to the following methods:
  • the route of the present invention is described by taking CRGGRAKDC-GG-D (KLAKLAKKLAKLAK) as an example.
  • Solid phase synthesis method using a resin or a 2-chlorotrityl chloride resin (2-CTC resin) as a starting resin, and sequentially coupling the N-terminal Fmoc according to the adipose tissue-targeting polypeptide main chain peptide sequence Protected and side chain protected amino acids; finally peptide resin is cleaved, cyclized, purified, and lyophilized to give the target compound.
  • 2-CTC resin 2-chlorotrityl chloride resin
  • the present invention provides a method for synthesizing two adipose tissue targeting polypeptides, the steps of which are as follows:
  • the resin solid carrier in the step (1) may be a 2-CTC resin
  • the activator system is selected from DIEA, TMP or NMM
  • the Fmoc-D-Lys (Boc)-resin is 0.10 to 0.50 mmol/
  • the Fmoc-D-Lys(Boc)-CTC resin having a degree of g substitution can also be obtained directly by purchase.
  • the resin solid carrier in the step (1) may also be a king resin, the activator system consisting of DIC, HOBt and DMAP, and the Fmoc-D-Lys (Boc)-resin is 0.10 to 0.50 mmol/g.
  • the degree of substitution of Fmoc-D-Lys(Boc)-king resin can also be obtained directly by purchase.
  • the coupling agent system comprises a condensing agent selected from the group consisting of DIC/HOBt, PyBOP/HOBt/DIEA, HATU/DIEA, PyBOP/HOBt/NMM or HATU/NMM; and the reaction solvent is selected from the group consisting of DMF , DCM, NMP, DMSO or any combination therebetween; preferably a combination of DCM and DMF.
  • liquid phase cyclization method comprises the steps of: (1) dissolving the chain peptide in a suitable solvent, and (2) adjusting the solution to be alkaline and adding an oxidizing agent for cyclization, (3) The solution was adjusted to an acidic quenching reaction to obtain a cyclic peptide crude peptide.
  • the second method differs from the first method in that the method (3) in the second method adopts solid phase cyclization, cleavage, purification, and lyophilization to obtain an adipose tissue targeting polypeptide.
  • the method for solid phase cyclization in the second method is: (1) adding a oxidizing agent to the peptide resin to obtain a cyclic polypeptide peptide resin, and (2) washing the obtained cyclic polypeptide peptide resin.
  • the adipose tissue targeting polypeptide of the present invention can be used for treating related diseases caused by obesity or obesity, in particular, a drug for treating a disease caused by obesity or obesity which is more effective in producing a more effective side effect of adipose tissue targeting polypeptide. It will be useful.
  • polypeptide sequences mentioned in the present invention are as follows:
  • polypeptide sequence SEQ1 up to the Homo-Arg appearing in SEQ33 is homoarginine.
  • the cell-targeting polypeptide peptide is SEQ1: Cys Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter as CRGGRAKDC; and the apoptotic polypeptide peptide is SEQ31 : Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKKLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is glycine or alanine.
  • the cell-targeting polypeptide peptide is SEQ2: Cys Homo-Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter: C Homo-RGGRAKDC; apoptotic polypeptide peptide SEQ31: Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKKLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is glycine or alanine.
  • the cell-targeting polypeptide peptide is SEQ30: Lys Gly Gly Gly Homo-Arg Ala Arg Glu, which is represented by a single letter KGGG Homo-RARE;
  • the apoptotic polypeptide peptide is SEQ31: Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys Leu Ala Lys, which is represented by a single letter : KLAKLAKKLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge chain is glycine or alanine.
  • the cell-targeting polypeptide peptide is SEQ1: Cys Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter: CRGGRAKDC;
  • the apoptotic polypeptide peptide is SEQ32: Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKRLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is glycine or alanine.
  • the cell-targeting polypeptide peptide is SEQ2: Cys Homo-Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter: C Homo-RGGRAKDC; apoptotic polypeptide peptide SEQ32: Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKRLAKLAK; the bridge is glycine or alanine.
  • the cell-targeting polypeptide peptide is SEQ30: Lys Gly Gly Gly Homo-Arg Ala Arg Glu, which is represented by a single letter: KGGG Homo-RARE;
  • the apoptotic polypeptide peptide is SEQ32: Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKRLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is glycine or alanine.
  • the cell-targeting polypeptide peptide is SEQ1: Cys Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter: CRGGRAKDC;
  • the apoptotic polypeptide peptide is SEQ31: Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys Leu Ala Lys, which is represented by a single letter KLAKLAKKLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid;
  • the bridge is a polyethylene glycol modified glycine or alanine.
  • the amino acid in the adipose tissue-targeting polypeptide of the present invention may be a D-form or an L-form amino acid when it is involved in the D configuration or the L configuration, and is used in the following examples.
  • the amino acid defaults to an L-form amino acid when it does not indicate a configuration, and is specifically indicated as a D-form amino acid or a D in the lower left corner when a D-form amino acid is involved.
  • HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • Trt trityl
  • TMP 2,4,6-trimethylpyridine.
  • Fmoc-D-Lys (Boc)-King resin purchased from Jill Biochemical (Shanghai) Co., Ltd.;
  • DMF N, N'-dimethylformamide, purchased from Zhejiang Jiangshan Chemical Co., Ltd.;
  • DCM dichloromethane
  • TFA trifluoroacetic acid
  • HOBt (1-hydroxybenzotriazole), purchased from Shanghai Yuhong Chemical Technology Co., Ltd.;
  • Tetrakis(triphenylphosphine)palladium (Pd(PPh 3 ) 4 ), purchased from Angie Chemical;
  • a stranded SEQ34:CRGGRAKDC-GG-KLAKLAKKLAKLAK adipose tissue targeting polypeptide was first prepared, wherein the amino acids at positions 12 to 25 were all D-type amino acids.
  • the side chain of the Cys at position 1 and the side chain of Cys at position 9 are then subjected to a ring of disulfide bonds by a cyclization process.
  • the preparation process is as follows:
  • Liquid phase cyclization After dissolving the crude peptide in 250 ml of water, the pH was adjusted to 8 with aqueous ammonia, 50 ⁇ l of hydrogen peroxide was added, and after reacting for 2 hours, the pH was adjusted to 6 with acetic acid.
  • a SEQ35:CHomo-RGGRAKDC-GG-KLAKLAKKLAKLAK adipose tissue-targeting polypeptide having a chain form is first prepared, wherein the amino acids 12 to 25 are D-form amino acids; and then the first one is obtained by cyclization.
  • the side chain of the Cys and the side chain of the Cys of the 9th position form a disulfide bond ring, and the preparation process is as follows:
  • Liquid phase cyclization After dissolving the crude peptide in 250 ml of water, adjust the pH to 8 with ammonia water, and then add 50 ⁇ l of hydrogen peroxide, after reacting for 2 h, the pH was adjusted to 6 with acetic acid.
  • a chained SEQ36:KGGG Homo-RARE-GG-KLAKLAKKLAKLAK adipose tissue targeting polypeptide was prepared, wherein the 11th to the 24th amino acids were D-form amino acids;
  • the side chain of Lys at position 1 and the side chain of Glu at position 8 form an amide bond ring, and the preparation process is as follows:
  • the lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA, the filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for precipitation for 1 hour, centrifuged, and dried with diethyl ether. It was washed 6 times and dried under vacuum to give 236.5 mg of crude peptide.
  • Example 4 prepared a chained SEQ37: CRGGRAKDC-GG-KLAKLAKRLAKLAK adipose tissue targeting polypeptide, wherein the 12th to 25th amino acids are D-type amino acids; then, by cyclization, the first position of Cys The side chain and the side chain of Cys at position 9 form a disulfide bond ring, and the preparation process is as follows:
  • Liquid phase cyclization After dissolving the crude peptide in 250 ml of water, the pH was adjusted to 8 with aqueous ammonia, 50 ⁇ l of hydrogen peroxide was added, and after reacting for 2 hours, the pH was adjusted to 6 with acetic acid.
  • Embodiment 5 is a diagrammatic representation of Embodiment 5:
  • a chained SEQ38:C Homo-RGGRAKDC-GG-KLAKLAKRLAKLAK adipose tissue targeting polypeptide is prepared, wherein the amino acids 12 to 25 are D-form amino acids;
  • the side chain of Cys at position 1 and the side chain of Cys at position 9 form a disulfide bond ring, and the preparation process is as follows:
  • the reaction was carried out for 1 hour, and the criterion was applied to the subsequent amino acid coupling to determine the end point of the reaction by the ninhydrin method, thereby preparing Fmoc-D-Ala-D-Lys(Boc)-king resin.
  • Liquid phase cyclization After dissolving the crude peptide in 250 ml of water, the pH was adjusted to 8 with aqueous ammonia, 50 ⁇ l of hydrogen peroxide was added, and after reacting for 2 hours, the pH was adjusted to 6 with acetic acid.
  • Example 6 prepared a chained SEQ39:KGGG Homo-RARE-GG-KLAKLAKRLAKLAK adipose tissue targeting polypeptide, wherein the 11th to 24th amino acids are D-type amino acids; then, by cyclization, the first of them The side chain of the Lys and the side chain of the Glu of the 8th position form an amide bond ring, and the preparation process is as follows:
  • SEQ40 CRGGRAKDC-G-(PEG)10-CH 2 CH 2 -G-KLAKLAKKLAKLAK adipose tissue targeting polypeptide was prepared, wherein the 12th to 25th amino acids were D-form amino acids; Cyclization, wherein the side chain of the Cys at position 1 and the side chain of Cys at position 9 form a disulfide bond ring, and the preparation process is as follows:
  • Liquid phase cyclization After dissolving the crude peptide in 250 ml of water, the pH was adjusted to 8 with aqueous ammonia, 50 ⁇ l of hydrogen peroxide was added, and after reacting for 2 hours, the pH was adjusted to 6 with acetic acid.
  • the target product (19.7 mg, 6.37%) was obtained by two-time purification on a C18 column, salt-transfer, and lyophilization.
  • the product was analyzed by MALDI-TOF, and the m/z value of the protonated molecular ion peak was found. Is 3093.5676 (theoretical amount is 3093).
  • C57BL/6 mice were selected to build an obesity model by feeding a high-fat diet to observe the therapeutic effect of adipose tissue-targeted peptides on obese mice, and to screen out the best therapeutic effect of adipose tissue-targeting peptides and doses.
  • Relevant experimental research provides a reference basis.
  • C57BL/6 mice were selected as experimental animals because the rodent has rich background information and is the most commonly used rodent experimental system at home and abroad, and is conducive to comparison with similar test results.
  • C57BL/6 mice were purchased from Shandong Hongli Medical Animal Experimental Research Co., Ltd. After the experimental animals are delivered to the company, they are checked by the quarantine personnel, and the quarantine personnel are responsible for the adaptive feeding for 3 days. The daily behaviors, irritation, muscle tension, breathing, feces, exogenous system, skin and mucous membranes of the animals Observe with eyes and so on.
  • the quarantine period is marked by the tail line method.
  • the animal number is marked by picric acid smearing, and the experiment number, animal group and dosage are indicated on the cage.
  • Manure treatment method replace with the litter, 2 times / week;
  • Cleaning and disinfection methods Wipe the cage and the ground with a cloth to remove the venom. During the experiment, clean the ground and the workbench after each experiment, and wipe off the venom with a cloth (disinfectant: 1% nevi, 1% 84 disinfectant, 0.2% peracetic acid, once a week).
  • Storage conditions stored in dedicated feed, low temperature, dry and sanitary
  • Feed testing Each batch of feed has a quality certificate from the manufacturer.
  • Feeding method free feeding
  • Water supply method Free access to the drinking fountain set by the drinking water bottle
  • test article is the adipose tissue-targeting polypeptide prepared in the first embodiment to the fifth embodiment, and the solvent is sterile water for injection.
  • the configuration method is: using an electronic balance to weigh different numbers of adipose tissue targets in a clean bench To the polypeptide, it is placed in a sterile container and dissolved in sterile water for injection to a final concentration of 1 g/L.
  • Example 5 The targeting polypeptide stock solution was diluted to different doses with PBS.
  • the corresponding experimental label is marked with the experiment number, the test sample number, the preparation concentration, the formulator, the reviewer, and the preparation time.
  • mice pre-test feeding
  • mice were fed a high fat diet TD97366 (25.4% fat, 21.79% protein, 38.41% carbon compound, purchased from Harlan Teklad) prior to the anti-obesity test, giving an average body weight of 50 g before the test.
  • TD97366 25.4% fat, 21.79% protein, 38.41% carbon compound, purchased from Harlan Teklad
  • mice were randomly divided into six groups of 10 mice each. The specific doses and groupings are shown in Table 1.
  • the route of administration is a subcutaneous injection of the neck and the side, and the route of administration is consistent with the route of administration of the clinical drug.
  • the administration time is between 9:30 and 10:30 every morning.
  • mice The best weight loss targeting polypeptide (the adipose tissue targeting polypeptide prepared in Example 5) was selected, and the mice were randomly divided into different experimental groups according to different administration routes and different administration doses. Group 10 mice, the specific dose and grouping are shown in Table 2.
  • the administration route part IP represents intraperitoneal injection
  • IM represents intramuscular injection
  • the administration time is between 9:30 and 10:30 every morning.
  • mice The body weight of the mice was measured before the administration and at different experimental points after the administration. Experimental mice were dosed once a day while weight measurements were taken every three days. At the same time, the data were entered and statistically analyzed using EXCEL2013 and SPSS13.0 software; the mouse body weight was averaged ( ) ⁇ standard deviation (SD or S).
  • adipose tissue-targeting polypeptides of Examples 1 to 5 were administered to mice, and the body weight changes of the mice were measured at different times, and the experimental results are shown in Table 3.
  • mice were administered the adipose tissue-targeting polypeptide prepared in the different examples, and the weights of the mice of Example 1 to Example 5 were significantly decreased.
  • the body weight decreased by 1.62 g
  • the weight of other mice generally decreased by 3 to 5 g, indicating that the targeted polypeptides of Examples 1 to 5 have obvious therapeutic effects and can reduce the mice.
  • the weight of the body relieves the symptoms of obesity in mice.
  • the change in body weight of mice gradually decreased with time, showing a significant time dependence.
  • the target polypeptide of Example 5 was selected and then diluted with sterile water to different concentrations, using different routes of administration, including intraperitoneal (IP) and intramuscular (IM), and then determined at different times.
  • IP intraperitoneal
  • IM intramuscular
  • mice regardless of intraperitoneal injection or intramuscular injection of adipose tissue-targeting polypeptide, the body weight of the mice showed significant weight loss with increasing concentration; and the weight loss values showed significant time-dependent . Especially at doses of 10 mg/kg and 15 mg/kg, the mice showed a very significant weight loss effect.
  • mice were given the adipose tissue-targeting polypeptides prepared in the first to the fifth examples, and the body weight of the mice showed a significant decrease, indicating the targets of Examples 1 to 5. It has a significant therapeutic effect on the polypeptide, can reduce the body weight of the mouse, and alleviate the symptoms of obesity in the mouse. Moreover, the change in body weight of mice gradually decreased with time, showing a significant time dependence. The sample screening results indicated that the targeted polypeptide of Example 5 exhibited the best anti-obesity therapeutic effect.
  • the dose screening results showed that the target polypeptide of Example 5 had obvious weight loss effect at the doses of 10 mg/kg and 15 mg/kg, and could be used for relieving obesity in mice, reducing body weight of mice, and having a dose effect.

Abstract

Provided are an adipose tissue targeting polypeptide and a preparation method therefor. The polypeptide comprises a cell targeting peptide fragment, an apoptotic peptide fragment, and a bridging chain linking the two peptide fragments, and can be used for fat reduction and weight loss.

Description

脂肪组织靶向多肽及其制备方法和应用Adipose tissue targeting polypeptide and preparation method and application thereof 技术领域Technical field
本发明涉及蛋白多肽领域，具体涉及一类脂肪组织靶向多肽及其制备方法和应用。The invention relates to the field of protein polypeptides, in particular to a kind of adipose tissue targeting polypeptides and a preparation method and application thereof.
背景技术Background technique
肥胖症作为一种常见的慢性内分泌代谢疾病，发病率呈逐年上升趋势，已成为全球性的公共健康难题。当人体进食的热量多于消耗量时便以脂肪形式存于体内，超过正常体重的20％即为肥胖。肥胖可发生于任何年龄，但以40岁以后者为多，女性发生率偏高。肥胖分为两大类型：一是单纯性肥胖，病人以肥胖为主要表现，不伴明显的神经和内分泌系统功能的改变，却伴有代谢调节障碍，这类肥胖最为多见。二是继发性肥胖，常继发脑炎、脑膜炎、脑部损害、垂体疾病、肾上腺皮质机能亢进、甲状腺机能低下、胰岛素分泌过多等。As a common chronic endocrine and metabolic disease, obesity has a rising incidence year by year and has become a global public health problem. When the body eats more calories than it consumes, it is stored in the body in the form of fat. More than 20% of normal body weight is obesity. Obesity can occur at any age, but after 40 years of age, the incidence of women is high. Obesity is divided into two types: simple obesity, patients with obesity as the main performance, without obvious changes in neurological and endocrine system functions, but with metabolic regulation disorders, such obesity is the most common. Second, secondary obesity, often secondary encephalitis, meningitis, brain damage, pituitary disease, adrenal hyperfunction, hypothyroidism, excessive insulin secretion.
多年来，肥胖症的行为和药物干预一直未被广泛接受。直至20世纪60年代开始了肥胖症行为治疗，使通过改变不良的饮食和运动习惯来减轻体重成为可能。研究表明，现有药物治疗可使体重减轻并能维持3～6个月。虽然研究病例有限，但却提出了一个与以往截然不同的概念，即肥胖症应与其他慢性疾病一样，通过长期的药物治疗来改善。尽管芬氟拉明等药物由于严重的心血管不良反应致使其最终撤出市场，但大量事实证明肥胖症有必要视作一种慢性疾病进行长期的药物治疗。Obesity behavior and drug intervention have not been widely accepted for many years. It was not until the 1960s that obesity behavioral therapy began, making it possible to lose weight by changing bad diet and exercise habits. Studies have shown that existing medications can reduce weight and last for 3 to 6 months. Although the research case is limited, it has proposed a completely different concept from the past that obesity should be improved by long-term drug treatment like other chronic diseases. Although drugs such as fenfluramine have eventually withdrawn from the market due to severe cardiovascular adverse effects, a large number of facts have proven that obesity is necessary as a chronic disease for long-term medical treatment.
理想的减肥药物应能减少能量摄取，增加能量消耗，并改善肥胖相关的心血管疾病危险因子。迄今为止，肥胖药物按作用机制主要可分为三类：抑制食欲的药物，影响营养吸收的药物，增加能量消耗的药物。抑制食欲的药物有以下几类，作用于去甲肾上腺素的药物直接作用于中枢神经系统，刺激去甲肾上腺素(NE)的释放或阻断其再摄取，导致神经末梢NE耗竭，进而抑制食欲，减少摄食，降低体重。此类药物如安非拉酮。5-羟色胺(5-HT)受体激动剂通过促进5-HT的释放和(或)抑制其再摄取而达到减肥的目的。短期研究显示，此类药物有部分减轻体重的作用，疗效与作用与去甲肾上腺素类似，如：芬氟 拉明、右芬氟拉明、氟西汀和舍曲林等。西布曲明为神经递质再摄取抑制剂类药物的代表，该药物通过NE和5-HT的再摄取，减少摄食，降低体重；不同于芬氟拉明和右芬氟拉明的是，西布曲明不诱导5-HT的释放，因而被认为与心血管疾病的发生无关。目前，FDA批准的减少营养物吸收的药物只有奥利司他，该药物可特异性地抑制胃肠道胰脂肪酶，抑制膳食中脂肪(甘油三酯)水解为可吸收的游离脂肪酸和甘油单脂，减少饮食中脂肪的吸收达30％，从而减轻体重，该药只通过外周组织发挥作用，全身吸收量甚微(﹤1％)。该药是第一个被认可的非食欲抑制性抗肥胖药物，其药理作用呈剂量依赖性，推荐剂量为一日3次，每次120mg。严格来说，目前临床上尚无增加能量消耗类的减肥药物，不过西布曲明除抑制食欲和增加饱腹感外，也有研究发现它能提高基础代谢率及促进产热效应。Ideal weight-loss drugs should reduce energy intake, increase energy expenditure, and improve obesity-related cardiovascular risk factors. So far, obesity drugs can be mainly divided into three categories according to the mechanism of action: drugs that suppress appetite, drugs that affect nutrient absorption, and drugs that increase energy consumption. The appetite-suppressing drugs have the following types. The drugs acting on norepinephrine directly act on the central nervous system, stimulate the release of norepinephrine (NE) or block its reuptake, leading to depletion of nerve endings NE, thereby suppressing appetite. Reduce food intake and reduce weight. Such drugs are, for example, amfepramone. Serotonin (5-HT) receptor agonists achieve weight loss by promoting the release of 5-HT and/or inhibiting its reuptake. Short-term studies have shown that these drugs have partial weight-reducing effects, and their effects and effects are similar to norepinephrine, such as: fenflurine Lamin, dexfenfluramine, fluoxetine and sertraline. Sibutramine is a representative of neurotransmitter reuptake inhibitors, which reduce the intake of food and reduce body weight through reuptake of NE and 5-HT; unlike fenfluramine and dexfenfluramine, West Butrexide does not induce the release of 5-HT and is therefore considered to be unrelated to the development of cardiovascular disease. Currently, the FDA-approved drug that reduces nutrient absorption is only orlistat, which specifically inhibits gastrointestinal pancreatic lipase and inhibits the hydrolysis of fat (triglyceride) in the diet to absorbable free fatty acids and glycerol. Lipid, which reduces the absorption of fat in the diet by 30%, thereby reducing body weight. The drug only works through peripheral tissues, and the systemic absorption is very small (<1%). This drug is the first approved non-or appetite suppressant anti-obesity drug, its pharmacological effect is dose-dependent, the recommended dose is 3 times a day, 120mg each time. Strictly speaking, there is currently no weight-loss drug for increasing energy consumption in the clinic. However, in addition to suppressing appetite and increasing satiety, sibutramine has also been found to increase basal metabolic rate and promote thermogenic effects.
发明内容Summary of the invention
本发明所解决的现有技术的问题在于：目前临床应用的减肥药物，确实有助于减轻体重和减少体重反弹，但缺乏长期的安全性及疗效评估，长期服用易导致神经、心血管等系统疾病。本发明人通过研究肥胖症发生的机制，发现了全新的药物干预靶点，从而找到了发明所述的脂肪组织靶向多肽。The problem of the prior art solved by the present invention is that the currently used weight-loss drugs for clinical use actually help to reduce weight and reduce body weight rebound, but lack long-term safety and efficacy evaluation, and long-term use may lead to nervous, cardiovascular and other systems. disease. The present inventors have discovered a novel drug intervention target by studying the mechanism of obesity occurrence, thereby finding the adipose tissue targeting polypeptide of the invention.
具体而言，本发明提供了一种脂肪组织靶向多肽，所述的脂肪组织靶向多肽包括细胞靶向多肽肽段、细胞凋亡多肽肽段以及连接细胞靶向多肽肽段和细胞凋亡多肽肽段的桥链。In particular, the present invention provides an adipose tissue targeting polypeptide comprising a cell targeting polypeptide peptide, an apoptotic polypeptide peptide, and a cell-targeting polypeptide peptide and apoptosis A bridge of a polypeptide peptide.
优选的，其中细胞靶向多肽肽段选自以下序列中的一种：SEQ1，SEQ2，SEQ3，SEQ4，SEQ5，SEQ6，SEQ7，SEQ8，SEQ9，SEQ10，SEQ11，SEQ12，SEQ13，SEQ14，SEQ15，SEQ16，SEQ17，SEQ18，SEQ19，SEQ20，SEQ21，SEQ22，SEQ23，SEQ24，SEQ25，SEQ26，SEQ27，SEQ28，SEQ29，SEQ30；Preferably, wherein the cell-targeting polypeptide peptide is selected from one of the following sequences: SEQ1, SEQ2, SEQ3, SEQ4, SEQ5, SEQ6, SEQ7, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ. , SEQ 17, SEQ 18, SEQ 19, SEQ 20, SEQ 21, SEQ 22, SEQ 23, SEQ 24, SEQ 25, SEQ 26, SEQ 27, SEQ 28, SEQ 29, SEQ 30;
其中所述的细胞靶向多肽肽段中的氨基酸独立地为甘氨酸和D型或者L型氨基酸，优选为甘氨酸和L型氨基酸。The amino acids in the peptides of the cell-targeting polypeptides described herein are independently glycine and a D- or L-form amino acid, preferably a glycine and an L-form amino acid.
优选的，所述的细胞靶向多肽肽段中的氨基酸为天然或非天然氨基酸，所述的天然氨基酸优选为经保护基团修饰的天然氨基酸；所述的保护基团优选为叔丁氧羰基、氧叔丁基、三苯甲基、2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基、 烯丙基、(N-1-(4,4-二甲基-2,6-二氧环亚己基-1)乙基)中的一种或几种。Preferably, the amino acid in the peptide segment of the cell targeting polypeptide is a natural or unnatural amino acid, and the natural amino acid is preferably a natural amino acid modified by a protecting group; the protecting group is preferably a tert-butoxycarbonyl group. , O-t-butyl, trityl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, One or more of allyl, (N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene-1)ethyl).
优选的，所述的细胞凋亡多肽肽段选自细胞凋亡因子或下列所述的肽链片段中的一种：SEQ31，SEQ32；Preferably, the apoptotic polypeptide peptide segment is selected from the group consisting of an apoptotic factor or one of the following peptide chain fragments: SEQ31, SEQ32;
其中所述肽链片段中的氨基酸独立地为D型或者L型氨基酸；优选为氨基酸全部为D型氨基酸或者L型氨基酸；进一步优选为氨基酸全部为D型氨基酸。Wherein the amino acid in the peptide chain fragment is independently a D-form or an L-form amino acid; preferably all of the amino acids are D-form amino acids or L-form amino acids; further preferably all of the amino acids are D-form amino acids.
优选的，所述的细胞凋亡多肽肽段选自细胞凋亡因子或肽链片段SEQ33；Preferably, the apoptotic polypeptide peptide segment is selected from the group consisting of an apoptotic factor or a peptide chain fragment SEQ33;
其中所述肽链片段中的氨基酸独立地为甘氨酸和D型或者L型氨基酸；优选为甘氨酸和D型氨基酸。Wherein the amino acid in the peptide chain fragment is independently a glycine and a D-form or an L-form amino acid; preferably a glycine and a D-form amino acid.
优选的，所述的肽链片段中的氨基酸为天然或非天然氨基酸；所述的天然氨基酸优选为经保护基团修饰的天然氨基酸；所述的保护基团优选为叔丁氧羰基、氧叔丁基、三苯甲基、2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基、烯丙基、(N-1-(4,4-二甲基-2,6-二氧环亚己基-1)乙基)中的一种或几种。Preferably, the amino acid in the peptide chain fragment is a natural or unnatural amino acid; the natural amino acid is preferably a natural amino acid modified by a protecting group; and the protecting group is preferably a tert-butoxycarbonyl group or an oxygen-free one. Butyl, trityl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, allyl, (N-1-(4,4-dimethyl-) One or more of 2,6-dioxocyclohexylene-1)ethyl).
优选的，所述的细胞凋亡因子选自以下中的一种：白细胞介素-1、白细胞介素-2、白细胞介素-5、白细胞介素-10、白细胞介素-11、白细胞介素-12、白细胞介素-18、干扰素-γ，干扰素-α、干扰素-β、肿瘤坏死因子-α、巨噬细胞集落刺激因子。Preferably, the apoptotic factor is selected from the group consisting of interleukin-1, interleukin-2, interleukin-5, interleukin-10, interleukin-11, and leukocyte mediator. -12, interleukin-18, interferon-γ, interferon-α, interferon-β, tumor necrosis factor-α, macrophage colony-stimulating factor.
优选的，所述的桥链为2～10个的天然或非天然氨基酸片段。Preferably, the bridge is from 2 to 10 natural or unnatural amino acid fragments.
优选的，所述的桥链为2～6个氨基酸组成的片段，更优选为2个氨基酸片段。Preferably, the bridge is a fragment of 2 to 6 amino acids, more preferably 2 amino acid fragments.
更优选的，所述的桥链为包含甘氨酸或丙氨酸。More preferably, the bridge chain comprises glycine or alanine.
优选的，所述的脂肪组织靶向多肽为链状或者是环状脂肪组织靶向多肽；所述的环状脂肪组织靶向多肽优选为分子内成环的方式。Preferably, the adipose tissue targeting polypeptide is a chain or a circular adipose tissue targeting polypeptide; and the cyclic adipose tissue targeting polypeptide is preferably intramolecularly looped.
优选的，所述的成环方式为化学键，选自C-N、C-O、C＝N、C-C、C＝C、C-S、S-S、CO-NH、CO-S键中的一种或几种以上。Preferably, the ring-forming method is a chemical bond selected from one or more of C-N, C-O, C=N, C-C, C=C, C-S, S-S, CO-NH, and CO-S bonds.
更优选的，所述的成环方式为S-S、CO-NH或CO-S键。More preferably, the ring formation is S-S, CO-NH or CO-S bond.
优选的，所述的脂肪组织靶向多肽采用聚乙二醇修饰，所述的脂肪组织靶向多肽的C端、N端或侧链的任一位置与聚乙二醇进行结合。Preferably, the adipose tissue targeting polypeptide is modified with polyethylene glycol, and the adipose tissue targeting polypeptide binds to polyethylene glycol at any position of the C-terminus, the N-terminus or the side chain.
更优选的，所述的脂肪组织靶向多肽的桥链与聚乙二醇进行结合。 More preferably, the abranche of the adipose tissue targeting polypeptide is bound to polyethylene glycol.
优选的，所述聚乙二醇选自PEG10，PEG100，PEG200，PEG300，PEG400，PEG500，PEG600，PEG700，PEG800，PEG900，PEG1000，PEG1100，PEG1200，PEG1300，PEG1400，PEG1500，PEG1600，PEG1700，PEG1800，PEG2000，PEG3000，PEG4000，PEG10000，PEG20000，PEG40000中的一种或几种以上。Preferably, the polyethylene glycol is selected from the group consisting of PEG10, PEG100, PEG200, PEG300, PEG400, PEG500, PEG600, PEG700, PEG800, PEG900, PEG1000, PEG1100, PEG1200, PEG1300, PEG1400, PEG1500, PEG1600, PEG1700, PEG1800, PEG2000 One or more of PEG3000, PEG4000, PEG10000, PEG20000, and PEG40000.
优选的，所述聚乙二醇选自PEG10，PEG100，PEG200，PEG300，PEG400，PEG500中的一种或几种以上。Preferably, the polyethylene glycol is selected from one or more of PEG10, PEG100, PEG200, PEG300, PEG400, PEG500.
更优选的，所述聚乙二醇选自PEG10，PEG100，PEG200中的一种或几种以上。More preferably, the polyethylene glycol is selected from one or more of PEG 10, PEG 100, and PEG 200.
本发明同时提供了一种脂肪组织靶向多肽的制备方法，包括如下步骤：The invention also provides a preparation method of an adipose tissue targeting polypeptide, comprising the following steps:
(1)采用固相合成法，依照脂肪组织靶向多肽的连接次序在偶联剂的作用下，依次进行氨基酸的偶联，合成具有N端保护的链状脂肪组织靶向多肽；(1) using solid phase synthesis method, according to the order of attachment of adipose tissue targeting polypeptides, under the action of a coupling agent, sequentially coupling amino acids to synthesize a chain adipose tissue targeting polypeptide having N-terminal protection;
(2)裂解去除N端保护的基团，得到链状的脂肪组织靶向多肽。(2) Cleavage to remove the N-terminal protected group to obtain a chain adipose tissue targeting polypeptide.
优选的，所述偶联剂包括缩合剂和反应溶剂，所述缩合剂选自以下几种组合中的一种：(1)N，N′-二异丙基碳化二亚胺和1-羟基苯并三唑，Preferably, the coupling agent comprises a condensing agent and a reaction solvent, and the condensing agent is selected from one of the following combinations: (1) N, N'-diisopropylcarbodiimide and 1-hydroxyl Benzotriazole,
(2)六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、1-羟基苯并三唑和N，N′-二异丙基乙胺，(2) benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxybenzotriazole and N,N'-diisopropylethylamine,
(3)2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯和N，N′-二异丙基乙胺，(3) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N'-diisopropylethylamine,
(4)六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、1-羟基苯并三唑和N-甲基吗啡啉，(4) benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxybenzotriazole and N-methylmorpholine,
(5)2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯和N-甲基吗啡啉；(5) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N-methylmorpholine;
所述反应溶剂选自N，N′-二甲基甲酰胺、二氯甲烷、N-甲基吡咯烷酮、二甲基亚砜中的一种或几种以上。The reaction solvent is one or more selected from the group consisting of N,N'-dimethylformamide, dichloromethane, N-methylpyrrolidone, and dimethyl sulfoxide.
优选的，所述的制备方法还包括将步骤(2)得到的链状脂肪组织靶向多肽进行环化过程，得到环状脂肪组织靶向多肽；所述的环化过程包括液相环化和固相环化。Preferably, the preparation method further comprises the step of cyclizing the chain adipose tissue targeting polypeptide obtained in the step (2) to obtain a cyclic adipose tissue targeting polypeptide; the cyclization process comprises liquid phase cyclization and Solid phase cyclization.
优选的，所述的液相环化包括如下步骤： Preferably, the liquid phase cyclization comprises the following steps:
(1)链状脂肪组织靶向多肽在溶剂中进行溶解，(1) a chain adipose tissue targeting polypeptide is dissolved in a solvent,
(2)调节溶液成碱性后加入氧化剂进行环化，(2) adjusting the solution to be alkaline and then adding an oxidizing agent to cyclize.
(3)调节溶液成酸性后得到环状脂肪组织靶向多肽；(3) adjusting the solution to be acidic to obtain a cyclic adipose tissue targeting polypeptide;
所述的固相环化为将链状脂肪组织靶向多肽加入氧化剂得到环状脂肪组织靶向多肽。The solid phase cyclization is to add a chain adipose tissue targeting polypeptide to an oxidizing agent to obtain a cyclic adipose tissue targeting polypeptide.
本发明还提供了一种药物组合物，该药物组合物包含治疗有效量的游离形式或可药用盐形式的以上任一项所述的脂肪组织靶向多肽作为活性成分，以及一种或多种药用载体物质和/或稀释剂。The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the adipose tissue-targeting polypeptide of any of the above forms in free form or in a pharmaceutically acceptable salt form as an active ingredient, and one or more A pharmaceutically acceptable carrier material and/or diluent.
本发明还提供了以上任一项所述的脂肪组织靶向多肽或以上所述的药物组合物在制备治疗和/或预防减肥或其他与减肥相关疾病药物方面的用途。The invention also provides the use of the adipose tissue targeting polypeptide of any of the above or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment and/or prevention of weight loss or other diseases associated with weight loss.
同时，本发明也提供了以上任一项所述的脂肪组织靶向多肽或以上所述的药物组合物在治疗和/或预防减肥或其他与减肥相关疾病方面的用途。Meanwhile, the present invention also provides the use of the adipose tissue-targeting polypeptide of any of the above or the pharmaceutical composition described above for the treatment and/or prevention of weight loss or other diseases related to weight loss.
本发明所取得的有益效果为：本发明获得了一种全新的靶向多肽药物。该化合物结构为将特异性细胞靶向多肽和细胞凋亡多肽通过桥链链接，其能够抑制目标物在脂肪血管内皮细胞中的表达和引起内脏脂肪组织的消融，该物质能促使新陈代谢正常化和在动物实验中快速逆转肥胖，并能破坏受体介导的细胞内化线粒体膜，其包括的D型氨基酸是提供了在血浆中肽酶酶解的抗性。此类化合物可以特异性的使脂肪细胞凋亡，从而达到降脂减肥的疗效。并且由于其分子本身包含的靶向作用机理，可以避免发生现有减肥药物的常见不良反应，相比现有减肥药物具有更直接的疗效和更少的副作用。The beneficial effects obtained by the present invention are as follows: The present invention obtains a novel targeted polypeptide drug. The structure of the compound is to link a specific cell-targeting polypeptide and an apoptotic polypeptide through a bridge chain, which can inhibit the expression of the target in the fat vascular endothelial cells and cause the ablation of the visceral adipose tissue, which can promote the normalization of metabolism and Rapid reversal of obesity in animal experiments and disruption of receptor-mediated intracellular mitochondrial membranes, including D-form amino acids, provides resistance to peptidase enzymatic hydrolysis in plasma. Such compounds can specifically cause apoptosis of fat cells, thereby achieving the effect of reducing fat and reducing weight. And because of the targeting mechanism contained in the molecule itself, it can avoid the common adverse reactions of the existing slimming drugs, and has more direct effects and fewer side effects than the existing slimming drugs.
具体实施方式detailed description
如上所述，本发明的目的在于提供一种脂肪组织靶向多肽，所述的脂肪组织靶向多肽包括细胞靶向多肽肽段、细胞凋亡多肽肽段以及连接细胞靶向多肽肽段和细胞凋亡多肽肽段的桥链。As described above, it is an object of the present invention to provide an adipose tissue targeting polypeptide comprising a cell targeting polypeptide peptide, an apoptotic polypeptide peptide, and a cell-targeting polypeptide peptide and cell A bridge of an apoptotic polypeptide peptide.
本发明主要涉及一类合成的脂肪组织靶向多肽，其特点为细胞靶向多肽和细胞凋亡多肽通过桥链链接而成。并对不同的细胞靶向多肽、桥链和细胞凋亡多肽进行了筛选。该脂肪组织靶向多肽药物具有显著的疗效，并具有较低的生理毒性。本发明提供的脂肪组织靶向多肽，其临床用量范围为每天3-15mg/kg。 The invention mainly relates to a kind of synthetic adipose tissue targeting polypeptide, which is characterized in that a cell targeting polypeptide and an apoptotic polypeptide are linked by a bridge chain. Different cell targeting polypeptides, bridges and apoptotic polypeptides were screened. The adipose tissue-targeting polypeptide drug has remarkable curative effect and has low physiological toxicity. The adipose tissue targeting polypeptide provided by the invention has a clinical dosage ranging from 3 to 15 mg/kg per day.
同时发明人通过化学合成法增加了脂肪组织靶向多肽的稳定性，如：将合成好的脂肪组织靶向多肽形成分子内二硫环从而增加了稳定性，并通过改变氨基酸序列使得获得的靶向多肽更加不容易被水解，且此多肽改良了肾脏的清除率。At the same time, the inventors increased the stability of the adipose tissue-targeting polypeptide by chemical synthesis, such as: targeting the synthesized adipose tissue to form an intramolecular disulfide ring to increase stability, and obtaining the target by changing the amino acid sequence. The polypeptide is less susceptible to hydrolysis and the polypeptide improves renal clearance.
脂肪组织靶向多肽由连续的氨基酸组成，可以选择性的作用在一般的人体器官、组织或细胞上且特别是脂肪组织或细胞上。脂肪组织靶向多肽对脂肪组织细胞具有识别作用，可以选择性的作用到脂肪组织细胞上。选择性定位可以使脂肪组织靶向多肽在目标组织或细胞上得以富集两倍或以上。此合成的脂肪组织靶向多肽具有降低生理毒性的特性，其中生理毒性包括肾脏毒性。Adipose tissue targeting polypeptides consist of contiguous amino acids that can selectively act on general human organs, tissues or cells, and in particular on adipose tissue or cells. The adipose tissue targeting polypeptide has a recognition function on adipose tissue cells and can selectively act on adipose tissue cells. Selective localization allows the adipose tissue targeting polypeptide to be enriched twice or more on the target tissue or cell. The synthetic adipose tissue-targeting polypeptide has properties that reduce physiological toxicity, wherein physiological toxicity includes renal toxicity.
其中，本发明中的细胞靶向多肽可以特异性的作用在一般的人体器官、组织或细胞上且特别是脂肪组织或细胞上，将合成的脂肪组织靶向多肽特异性定位在脂肪组织或者脂肪细胞处；细胞凋亡多肽可以特异性的作用于脂肪组织处的细胞，使其凋亡，得到减肥瘦身的目的。Wherein, the cell-targeting polypeptide of the present invention can specifically act on a general human organ, tissue or cell, and particularly on an adipose tissue or a cell, and specifically localize the synthesized adipose tissue-targeting polypeptide to adipose tissue or fat. At the cell site; the apoptotic polypeptide can specifically act on the cells at the adipose tissue to cause apoptosis and obtain the purpose of slimming and slimming.
本发明所述的脂肪组织靶向多肽可以通过采用聚乙二醇(PEG)的方式进行修饰，聚乙二醇可以以单一或者多组的方式与肽链的C端、N端或侧链的任一位置进行结合，修饰后的多肽化合物可以延长其在生物体内的半衰期。The adipose tissue targeting polypeptide of the present invention may be modified by using polyethylene glycol (PEG), and the polyethylene glycol may be in a single or multiple groups with the C-terminus, N-terminus or side chain of the peptide chain. Binding at any position, the modified polypeptide compound can prolong its half-life in vivo.
同时本发明所述的靶向脂肪组织多肽肽链可进行分子内成环，从而延长其在体内的半衰期，成环方式可以为二硫键或者酰胺键。其中二硫键采用氧化剂使半胱氨酸侧链巯基进行分子内成环，氧化剂优选为双氧水、碘及空气等。酰胺键采用偶联剂缩合氨基酸侧链的羧基和氨基制备可得。At the same time, the targeted adipose tissue polypeptide peptide chain of the present invention can be intramolecularly looped, thereby prolonging its half-life in vivo, and the ring-forming manner can be a disulfide bond or an amide bond. The disulfide bond uses an oxidizing agent to cause intramolecular ring formation of a cysteine side chain thiol group, and the oxidizing agent is preferably hydrogen peroxide, iodine or air. The amide bond is prepared by condensing a carboxyl group and an amino group of a side chain of an amino acid with a coupling agent.
本发明还提供了脂肪组织靶向多肽的制备方法，但不局限于下列方法：The invention also provides a method for preparing an adipose tissue targeting polypeptide, but is not limited to the following methods:
本发明的路线以CRGGRAKDC-GG-D(KLAKLAKKLAKLAK)为例进行描述。采用固相合成法，以王树脂或者2-氯三苯甲基氯树脂(2-CTC树脂)为起始树脂，按照所述的脂肪组织靶向多肽主链肽序依次偶联具有N端Fmoc保护且侧链保护的氨基酸；最后肽树脂裂解，环化，纯化，冻干后得到目标化合物。The route of the present invention is described by taking CRGGRAKDC-GG-D (KLAKLAKKLAKLAK) as an example. Solid phase synthesis method, using a resin or a 2-chlorotrityl chloride resin (2-CTC resin) as a starting resin, and sequentially coupling the N-terminal Fmoc according to the adipose tissue-targeting polypeptide main chain peptide sequence Protected and side chain protected amino acids; finally peptide resin is cleaved, cyclized, purified, and lyophilized to give the target compound.
为此本发明提供两种脂肪组织靶向多肽的合成方法，其步骤如下：To this end, the present invention provides a method for synthesizing two adipose tissue targeting polypeptides, the steps of which are as follows:
方法一：method one:
(1)在活化剂系统的存在下，由树脂固相载体和Fmoc-D-Lys(Boc)-OH 偶联得到Fmoc-D-Lys(Boc)-树脂；(1) In the presence of an activator system, a resin solid support and Fmoc-D-Lys(Boc)-OH Coupling to obtain Fmoc-D-Lys(Boc)-resin;
(2)通过固相合成法，按照脂肪组织靶向多肽主链肽序依次偶联具有N端Fmoc保护且侧链保护的氨基酸；(2) sequentially coupling an amino acid having an N-terminal Fmoc protection and a side chain protection according to a solid phase synthesis method according to an adipose tissue targeting polypeptide main chain peptide sequence;
(3)裂解，液相环化，纯化，冻干，得到脂肪组织靶向多肽。(3) cleavage, liquid phase cyclization, purification, and lyophilization to obtain an adipose tissue targeting polypeptide.
其中，步骤(1)中所述树脂固体载体可以采用2-CTC树脂，所述活化剂系统选自DIEA、TMP或NMM，所述Fmoc-D-Lys(Boc)-树脂为0.10～0.50mmol/g取代度的Fmoc-D-Lys(Boc)-CTC树脂，也可以直接通过购买得到。Wherein, the resin solid carrier in the step (1) may be a 2-CTC resin, the activator system is selected from DIEA, TMP or NMM, and the Fmoc-D-Lys (Boc)-resin is 0.10 to 0.50 mmol/ The Fmoc-D-Lys(Boc)-CTC resin having a degree of g substitution can also be obtained directly by purchase.
其中，步骤(1)中所述树脂固体载体也可以采用王树脂，所述活化剂系统由DIC、HOBt和DMAP组成，所述Fmoc-D-Lys(Boc)-树脂为0.10～0.50mmol/g取代度的Fmoc-D-Lys(Boc)-王树脂，也可以直接通过购买得到。Wherein, the resin solid carrier in the step (1) may also be a king resin, the activator system consisting of DIC, HOBt and DMAP, and the Fmoc-D-Lys (Boc)-resin is 0.10 to 0.50 mmol/g. The degree of substitution of Fmoc-D-Lys(Boc)-king resin can also be obtained directly by purchase.
其中，步骤(2)所述的固相合成方法，Wherein the solid phase synthesis method described in the step (2),
1)采用由体积比为1:4的哌啶和DMF组成的去保护液脱除Fmoc-D-Lys(Boc)-树脂上的Fmoc保护基，得到H-D-Lys(Boc)-树脂；1) removing the Fmoc protecting group on the Fmoc-D-Lys(Boc)-resin using a deprotecting solution consisting of piperidine and DMF in a volume ratio of 1:4 to obtain an H-D-Lys(Boc)-resin;
2)在偶联剂系统的存在下，H-D-Lys(Boc)-树脂和Fmoc保护且侧链保护的D型丙氨酸偶联得到Fmoc-D-Ala-D-Lys(Boc)-树脂；2) H-D-Lys(Boc)-resin and Fmoc-protected and side-chain protected D-alanine coupling in the presence of a coupling agent system to obtain Fmoc-D-Ala-D-Lys(Boc)-resin;
3)重复步骤1)、2)，按照脂肪组织靶向多肽主链肽序依次进行氨基酸的偶联，偶联氨基酸顺序为：3) Repeat steps 1) and 2) to sequentially perform amino acid coupling according to the adipose tissue-targeting polypeptide main chain peptide sequence. The amino acid sequence is as follows:
Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Cys(Trt)-OH；Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D- Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH;
所述偶联剂系统包括缩合剂和反应溶剂，所述缩合剂选自DIC/HOBt、PyBOP/HOBt/DIEA、HATU/DIEA、PyBOP/HOBt/NMM或HATU/NMM；所述反应溶剂选自DMF、DCM、NMP、DMSO或它们之间的任意组合；优选为DCM和DMF的组合。 The coupling agent system comprises a condensing agent selected from the group consisting of DIC/HOBt, PyBOP/HOBt/DIEA, HATU/DIEA, PyBOP/HOBt/NMM or HATU/NMM; and the reaction solvent is selected from the group consisting of DMF , DCM, NMP, DMSO or any combination therebetween; preferably a combination of DCM and DMF.
其中，步骤(3)所述的液相环化方法，其步骤为：(1)链状肽在合适的溶剂中进行溶解，(2)调节溶液成碱性加入氧化剂进行环化，(3)调节溶液成酸性淬灭反应，得到环肽粗肽。Wherein the liquid phase cyclization method according to the step (3) comprises the steps of: (1) dissolving the chain peptide in a suitable solvent, and (2) adjusting the solution to be alkaline and adding an oxidizing agent for cyclization, (3) The solution was adjusted to an acidic quenching reaction to obtain a cyclic peptide crude peptide.
方法二：Method Two:
方法二与方法一的不同之处在于，方法二中步骤(3)采用的是固相环化、裂解、纯化、冻干的方式，得到脂肪组织靶向多肽。The second method differs from the first method in that the method (3) in the second method adopts solid phase cyclization, cleavage, purification, and lyophilization to obtain an adipose tissue targeting polypeptide.
其中，方法二中的固相环化的方法为：(1)在肽树脂中加入氧化剂反应得环状多肽肽树脂，(2)洗涤获得的环状多肽肽树脂。The method for solid phase cyclization in the second method is: (1) adding a oxidizing agent to the peptide resin to obtain a cyclic polypeptide peptide resin, and (2) washing the obtained cyclic polypeptide peptide resin.
本发明的脂肪组织靶向多肽可用于治疗肥胖症或肥胖引起的相关疾病，具体地说，预计脂肪组织靶向多肽对于制备疗效更显著副作用更低的治疗肥胖症或肥胖引起的相关疾病的药物将是有用的。The adipose tissue targeting polypeptide of the present invention can be used for treating related diseases caused by obesity or obesity, in particular, a drug for treating a disease caused by obesity or obesity which is more effective in producing a more effective side effect of adipose tissue targeting polypeptide. It will be useful.
其中本发明提到的多肽序列如下：The polypeptide sequences mentioned in the present invention are as follows:
SEQ1：Cys Arg Gly Gly Arg Ala Lys Asp Cys，SEQ1: Cys Arg Gly Gly Arg Ala Lys Asp Cys,
SEQ2：Cys Homo-Arg Gly Gly Arg Ala Lys Asp Cys，SEQ2: Cys Homo-Arg Gly Gly Arg Ala Lys Asp Cys,
SEQ3：Cys Lys Gly Gly Homo-Arg Ala Lys Asp Cys，SEQ3: Cys Lys Gly Gly Homo-Arg Ala Lys Asp Cys,
SEQ4：Cys Lys Gly Gly Gly Arg Ala Lys Asp Cys，SEQ4: Cys Lys Gly Gly Gly Arg Ala Lys Asp Cys,
SEQ5：Cys Asp Gly Gly Gly Arg Ala Lys Lys Cys，SEQ5: Cys Asp Gly Gly Gly Arg Ala Lys Lys Cys,
SEQ6：Cys Glu Gly Gly Gly Arg Ala Lys Lys Cys，SEQ6: Cys Glu Gly Gly Gly Arg Ala Lys Lys Cys,
SEQ7：Cys Arg Gly Gly Gly Arg Ala Lys Asp Cys，SEQ7: Cys Arg Gly Gly Gly Arg Ala Lys Asp Cys,
SEQ8：Cys Homo-Arg Gly Gly Gly Arg Ala Lys Asp Cys，SEQ8: Cys Homo-Arg Gly Gly Gly Arg Ala Lys Asp Cys,
SEQ9：Cys Arg Gly Gly Gly Homo-Arg Ala Lys Asp Cys，SEQ9: Cys Arg Gly Gly Gly Homo-Arg Ala Lys Asp Cys,
SEQ10：Cys Lys Gly Gly Gly Homo-Arg Ala Lys Asp Cys，SEQ10: Cys Lys Gly Gly Gly Homo-Arg Ala Lys Asp Cys,
SEQ11：Cys Lys Gly Gly Gly Homo-Arg Ala Lys Glu Cys，SEQ11: Cys Lys Gly Gly Gly Homo-Arg Ala Lys Glu Cys,
SEQ12：Cys Homo-Arg Gly Gly Gly Arg Ala Arg Asp Cys，SEQ12: Cys Homo-Arg Gly Gly Gly Arg Ala Arg Asp Cys,
SEQ13：Cys Arg Gly Gly Gly Homo-Arg Ala Arg Asp Cys，SEQ13: Cys Arg Gly Gly Gly Homo-Arg Ala Arg Asp Cys,
SEQ14：Cys Lys Gly Gly Gly Homo-Arg Ala Arg Asp Cys，SEQ14: Cys Lys Gly Gly Gly Homo-Arg Ala Arg Asp Cys,
SEQ15：Cys Lys Gly Gly Gly Homo-Arg Ala Arg Glu Cys，SEQ15: Cys Lys Gly Gly Gly Homo-Arg Ala Arg Glu Cys,
SEQ16：Arg Gly Gly Arg Ala Lys Asp，SEQ16: Arg Gly Gly Arg Ala Lys Asp,
SEQ17：Homo-Arg Gly Gly Arg Ala Lys Asp， SEQ17: Homo-Arg Gly Gly Arg Ala Lys Asp,
SEQ18：Lys Gly Gly Homo-Arg Ala Lys Asp，SEQ18: Lys Gly Gly Homo-Arg Ala Lys Asp,
SEQ19：Lys Gly Gly Gly Arg Ala Lys Asp，SEQ19: Lys Gly Gly Gly Arg Ala Lys Asp,
SEQ20：Asp Gly Gly Gly Arg Ala Lys Lys，SEQ20: Asp Gly Gly Gly Arg Ala Lys Lys,
SEQ21：Glu Gly Gly Gly Arg Ala Lys Lys，SEQ21: Glu Gly Gly Gly Arg Ala Lys Lys,
SEQ22：Arg Gly Gly Gly Arg Ala Lys Asp，SEQ22: Arg Gly Gly Gly Arg Ala Lys Asp,
SEQ23：Homo-Arg Gly Gly Gly Arg Ala Lys Asp，SEQ23: Homo-Arg Gly Gly Gly Arg Ala Lys Asp,
SEQ24：Arg Gly Gly Gly Homo-Arg Ala Lys Asp，SEQ24: Arg Gly Gly Gly Homo-Arg Ala Lys Asp,
SEQ25：Lys Gly Gly Gly Homo-Arg Ala Lys Asp，SEQ25: Lys Gly Gly Gly Homo-Arg Ala Lys Asp,
SEQ26：Lys Gly Gly Gly Homo-Arg Ala Lys Glu，SEQ26: Lys Gly Gly Gly Homo-Arg Ala Lys Glu,
SEQ27：Homo-Arg Gly Gly Gly Arg Ala Arg Asp，SEQ27: Homo-Arg Gly Gly Gly Arg Ala Arg Asp,
SEQ28：Arg Gly Gly Gly Homo-Arg Ala Arg Asp，SEQ28: Arg Gly Gly Gly Homo-Arg Ala Arg Asp,
SEQ29：Lys Gly Gly Gly Homo-Arg Ala Arg Asp，SEQ29: Lys Gly Gly Gly Homo-Arg Ala Arg Asp,
SEQ30：Lys Gly Gly Gly Homo-Arg Ala Arg Glu；SEQ30: Lys Gly Gly Gly Homo-Arg Ala Arg Glu;
SEQ31：Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys，SEQ31: Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys,
SEQ32：Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys，SEQ32: Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys,
SEQ33：Lys Leu Ala Lys Leu Ala Lys Gly Arg Ala Lys Lys Leu Ala Lys Leu Ala Lys。SEQ33: Lys Leu Ala Lys Leu Ala Lys Gly Arg Ala Lys Lys Leu Ala Lys Leu Ala Lys.
其中，多肽序列SEQ1一直到SEQ33中出现的Homo-Arg为高精氨酸。Among them, the polypeptide sequence SEQ1 up to the Homo-Arg appearing in SEQ33 is homoarginine.
具体而言，在本发明的一种优选实施方式中，细胞靶向多肽肽段为SEQ1：Cys Arg Gly Gly Arg Ala Lys Asp Cys，其采用单字母表示为CRGGRAKDC；细胞凋亡多肽肽段为SEQ31：Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys，其采用单字母表示为：KLAKLAKKLAKLAK，细胞凋亡多肽肽段中的氨基酸为D型氨基酸；桥链为甘氨酸或丙氨酸。Specifically, in a preferred embodiment of the present invention, the cell-targeting polypeptide peptide is SEQ1: Cys Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter as CRGGRAKDC; and the apoptotic polypeptide peptide is SEQ31 : Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKKLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is glycine or alanine.
在本发明的一种优选实施方式中，细胞靶向多肽肽段为SEQ2：Cys Homo-Arg Gly Gly Arg Ala Lys Asp Cys，其采用单字母表示为：C Homo-RGGRAKDC；细胞凋亡多肽肽段为SEQ31：Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys，其采用单字母表示为：KLAKLAKKLAKLAK，细胞凋亡多肽肽段中的氨基酸为D型氨基酸；桥链为甘氨酸或丙氨酸。In a preferred embodiment of the invention, the cell-targeting polypeptide peptide is SEQ2: Cys Homo-Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter: C Homo-RGGRAKDC; apoptotic polypeptide peptide SEQ31: Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKKLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is glycine or alanine.
在本发明的一种优选实施方式中，细胞靶向多肽肽段为SEQ30：Lys Gly  Gly Gly Homo-Arg Ala Arg Glu，其采用单字母表示为KGGG Homo-RARE；细胞凋亡多肽肽段为SEQ31：Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys，其采用单字母表示为：KLAKLAKKLAKLAK，细胞凋亡多肽肽段中的氨基酸为D型氨基酸；桥链为甘氨酸或丙氨酸。In a preferred embodiment of the invention, the cell-targeting polypeptide peptide is SEQ30: Lys Gly Gly Gly Homo-Arg Ala Arg Glu, which is represented by a single letter KGGG Homo-RARE; the apoptotic polypeptide peptide is SEQ31: Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys, which is represented by a single letter : KLAKLAKKLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge chain is glycine or alanine.
在本发明的一种优选实施方式中，细胞靶向多肽肽段为SEQ1：Cys Arg Gly Gly Arg Ala Lys Asp Cys，其采用单字母表示为：CRGGRAKDC；细胞凋亡多肽肽段为SEQ32：Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys，其采用单字母表示为：KLAKLAKRLAKLAK，细胞凋亡多肽肽段中的氨基酸为D型氨基酸；桥链为甘氨酸或丙氨酸。In a preferred embodiment of the invention, the cell-targeting polypeptide peptide is SEQ1: Cys Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter: CRGGRAKDC; the apoptotic polypeptide peptide is SEQ32: Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKRLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is glycine or alanine.
在本发明的一种优选实施方式中，细胞靶向多肽肽段为SEQ2：Cys Homo-Arg Gly Gly Arg Ala Lys Asp Cys，其采用单字母表示为：C Homo-RGGRAKDC；细胞凋亡多肽肽段为SEQ32：Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys，其采用单字母表示为：KLAKLAKRLAKLAK；桥链为甘氨酸或丙氨酸。In a preferred embodiment of the invention, the cell-targeting polypeptide peptide is SEQ2: Cys Homo-Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter: C Homo-RGGRAKDC; apoptotic polypeptide peptide SEQ32: Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKRLAKLAK; the bridge is glycine or alanine.
在本发明的一种优选实施方式中，细胞靶向多肽肽段为SEQ30：Lys Gly Gly Gly Homo-Arg Ala Arg Glu，其采用单字母表示为：KGGG Homo-RARE；细胞凋亡多肽肽段为SEQ32：Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys，其采用单字母表示为：KLAKLAKRLAKLAK，细胞凋亡多肽肽段中的氨基酸为D型氨基酸；桥链为甘氨酸或丙氨酸。In a preferred embodiment of the present invention, the cell-targeting polypeptide peptide is SEQ30: Lys Gly Gly Gly Homo-Arg Ala Arg Glu, which is represented by a single letter: KGGG Homo-RARE; the apoptotic polypeptide peptide is SEQ32: Lys Leu Ala Lys Leu Ala Lys Arg Leu Ala Lys Leu Ala Lys, which is represented by a single letter: KLAKLAKRLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is glycine or alanine.
在本发明的一种优选实施方式中，细胞靶向多肽肽段为SEQ1：Cys Arg Gly Gly Arg Ala Lys Asp Cys，其采用单字母表示为：CRGGRAKDC；细胞凋亡多肽肽段为SEQ31：Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys，其采用单字母表示为KLAKLAKKLAKLAK，细胞凋亡多肽肽段中的氨基酸为D型氨基酸；桥链为聚乙二醇修饰的甘氨酸或丙氨酸。In a preferred embodiment of the invention, the cell-targeting polypeptide peptide is SEQ1: Cys Arg Gly Gly Arg Ala Lys Asp Cys, which is represented by a single letter: CRGGRAKDC; the apoptotic polypeptide peptide is SEQ31: Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys, which is represented by a single letter KLAKLAKKLAKLAK, the amino acid in the peptide segment of the apoptotic polypeptide is a D-type amino acid; the bridge is a polyethylene glycol modified glycine or alanine.
需要说明的是，本发明中脂肪组织靶向多肽中的氨基酸除甘氨酸以外，其他氨基酸若涉及到D构型或L构型时，均可以为D型或者L型氨基酸，以下实施例中用到的氨基酸在不表明构型时，默认为L型氨基酸，在涉及到D型氨基酸时，均会特别说明为D型氨基酸或者在左下角标注D来表示。It should be noted that, in addition to glycine, the amino acid in the adipose tissue-targeting polypeptide of the present invention may be a D-form or an L-form amino acid when it is involved in the D configuration or the L configuration, and is used in the following examples. The amino acid defaults to an L-form amino acid when it does not indicate a configuration, and is specifically indicated as a D-form amino acid or a D in the lower left corner when a D-form amino acid is involved.
下面通过具体的实施例对本发明作进一步的详细的说明。但是这些实施例 仅用于说明本发明，而不是对本发明范围的限制。The invention will now be further described in detail by way of specific examples. But these examples The invention is intended to be illustrative only and not to limit the scope of the invention.
本发明中一些常用的缩写具有以下含义；Some commonly used abbreviations in the present invention have the following meanings;
Fmoc：芴甲氧羰基Fmoc: fluorenylmethoxycarbonyl
Fmoc-AA：芴甲氧羰基保护的氨基酸Fmoc-AA: Aminocarbonyl protected amino acid
DIC：N，N′-二异丙基碳化二亚胺DIC: N, N'-diisopropylcarbodiimide
DCC：N，N′-二环己基碳二亚胺DCC: N, N'-dicyclohexylcarbodiimide
PyBOP：六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷PyBOP: benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate
HATU：2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯HATU: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
HOBt：1-羟基苯并三唑HOBt: 1-hydroxybenzotriazole
tBu：叔丁基tBu: tert-butyl
OtBu:氧叔丁基OtBu: Oxybutyl t-butyl
Trt：三苯甲基Trt: trityl
Boc：叔丁氧羰基Boc: tert-butoxycarbonyl
Dde：(N-1-(4,4-二甲基-2,6-二氧环亚己基-1)乙基)Dde: (N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene-1)ethyl)
Pbf：2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
Cys：半胱氨酸Cys: Cysteine
Pro：脯氨酸Pro: Proline
Leu：亮氨酸Leu: Leucine
Gly：甘氨酸Gly: glycine
Arg：精氨酸Arg: arginine
Ala：丙氨酸Ala: Alanine
Asp：天冬氨酸Asp: aspartic acid
Glu：谷氨酸Glu: glutamic acid
Lys：赖氨酸Lys: Lysine
DMF：N，N′-二甲基甲酰胺DMF: N, N'-dimethylformamide
MeOH：甲醇MeOH: methanol
DCM：二氯甲烷DCM: dichloromethane
NMP：N-甲基吡咯烷酮NMP: N-methylpyrrolidone
DMSO：二甲基亚砜 DMSO: dimethyl sulfoxide
TFA：三氟醋酸TFA: trifluoroacetic acid
Piperidine：六氢吡啶Piperidine: Hexahydropyridine
DMAP：4-二甲氨基吡啶DMAP: 4-dimethylaminopyridine
DIEA：N，N′-二异丙基乙胺DIEA: N,N'-diisopropylethylamine
TMP：2,4,6-三甲基吡啶。TMP: 2,4,6-trimethylpyridine.
其中，实施例中所用的试剂和仪器的厂商型号如下：Among them, the manufacturer models of the reagents and instruments used in the examples are as follows:
Fmoc-D-Lys(Boc)-王树脂，购自吉尔生化(上海)有限公司；Fmoc-D-Lys (Boc)-King resin, purchased from Jill Biochemical (Shanghai) Co., Ltd.;
保护氨基酸，购自吉尔生化(上海)有限公司；Protected amino acids, purchased from Jill Biochemical (Shanghai) Co., Ltd.;
DMF(N，N′-二甲基甲酰胺)，购自浙江江山化工股份有限公司；DMF (N, N'-dimethylformamide), purchased from Zhejiang Jiangshan Chemical Co., Ltd.;
哌啶，购自上海凌峰化学试剂有限公司；Piperidine, purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.;
DCM(二氯甲烷)，购自浙江衢化氟化学有限公司；DCM (dichloromethane), purchased from Zhejiang Suihua Fluorine Chemical Co., Ltd.;
DIC(N，N′-二异丙基碳化二亚胺)，购自淄博天堂山化工有限公司；DIC (N, N'-diisopropylcarbodiimide), purchased from Zibo Tianshan Chemical Co., Ltd.;
TFA(三氟醋酸)，购自浙江化工院科技有限公司；TFA (trifluoroacetic acid), purchased from Zhejiang Chemical Industry Technology Co., Ltd.;
苯甲硫醚，购自舒莱维化工科技(杭州)有限公司；Benzoyl sulfide, purchased from Shulaiwei Chemical Technology (Hangzhou) Co., Ltd.;
苯甲醚，购自上海凌峰化学试剂有限公司；Anisole, purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.;
HOBt(1-羟基苯并三唑)，购自上海瀚鸿化工科技有限公司；HOBt (1-hydroxybenzotriazole), purchased from Shanghai Yuhong Chemical Technology Co., Ltd.;
C18柱，C8柱，购自Daisogel；C18 column, C8 column, purchased from Daisogel;
四(三苯基膦)钯(Pd(PPh3)4)，购自安耐吉化学；Tetrakis(triphenylphosphine)palladium (Pd(PPh 3 ) 4 ), purchased from Angie Chemical;
电子天平，JY10001，上海精密科学仪器有限公司。Electronic balance, JY10001, Shanghai Precision Scientific Instrument Co., Ltd.
(一)脂肪组织靶向多肽的合成(1) Synthesis of adipose tissue targeting polypeptide
实施例一Embodiment 1
实施例一首先制备合成了链状的SEQ34：CRGGRAKDC-GG-KLAKLAKKLAKLAK脂肪组织靶向多肽，其中的第12位到第25位的氨基酸均为D型氨基酸。然后通过环化过程将其中第1位的Cys的侧链和第9位的Cys的侧链成一个二硫键的环。其制备过程如下：In the first embodiment, a stranded SEQ34:CRGGRAKDC-GG-KLAKLAKKLAKLAK adipose tissue targeting polypeptide was first prepared, wherein the amino acids at positions 12 to 25 were all D-type amino acids. The side chain of the Cys at position 1 and the side chain of Cys at position 9 are then subjected to a ring of disulfide bonds by a cyclization process. The preparation process is as follows:
1)称取0.1mmol取代度为0.45mmol/g的Fmoc-D-Lys(Boc)-王树脂，加入固相反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-D-Lys(Boc)-王树脂30分钟后，用DMF：哌啶体积比为4:1的混合溶液脱去Fmoc保护，得到 H-D-Lys(Boc)-王树脂，并用DMF洗涤6次。1) Weigh 0.1 mmol of Fmoc-D-Lys(Boc)-Wang resin with a degree of substitution of 0.45 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-D-Lys (Boc) with DMF. After 30 minutes of the king resin, the Fmoc protection was obtained by using a mixed solution of DMF: piperidine in a volume ratio of 4:1. H-D-Lys (Boc)-king resin and washing 6 times with DMF.
2)称取Fmoc-D-Ala-OH0.5mmol、HOBt0.5mmol加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入80μl DIC(0.5mmol)活化后，加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点；如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时；从而制备得到Fmoc-D-Ala-D-Lys(Boc)-王树脂。此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点。2) Weigh Fmoc-D-Ala-OH 0.5mmol and HOBt0.5mmol into a mixed solution of DCM and DMF in a volume ratio of 1:1. After adding 80μl DIC (0.5mmol) to the ice bath, activate the above resin. In the reaction column, after reacting at room temperature for 2 hours, the end point of the reaction is determined by the ninhydrin method; if the resin is colorless and transparent, the reaction is complete; when the resin develops color, the reaction is incomplete, and further reaction is required for 1 hour; Fmoc-D-Ala-D-Lys(Boc)-king resin was obtained. This criterion is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method.
3)重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，依次完成Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Cys(Trt)-OH的偶联。偶联完毕，将肽树脂用DMF洗涤3次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到0.751g粗肽树脂。3) repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D- Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc) -OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH , Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg ( Coupling of Pbf)-OH, Fmoc-Cys(Trt)-OH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH and dried to give 0.751 g of crude peptide resin.
4)裂解：为了去除树脂和肽链氨基酸侧链的保护基团，称取0.751g全保护的肽树脂，加入到25mL的三口圆底烧瓶中，按TFA：苯甲硫醚：苯甲醚＝95：3：2的体积比配置裂解液10mL，将裂解液加入上述树脂中，室温反应2小时，过滤，用少量TFA洗涤裂解后的树脂3次，合并滤液，浓缩，将浓缩后的液体加入到冰乙醚中沉淀1小时，离心，无水乙醚离心洗涤6次，真空干燥，得到粗肽257.2mg。4) Cleavage: In order to remove the protective groups of the resin and peptide chain amino acid side chains, 0.751 g of the fully protected peptide resin was weighed and added to a 25 mL three-neck round bottom flask according to TFA: thioanisole: anisole = The volume ratio of 95:3:2 was 10 mL of the lysate, the lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA, the filtrate was combined, concentrated, and the concentrated liquid was added. The mixture was precipitated into ice diethyl ether for 1 hour, centrifuged, washed with diethyl ether for 6 times, and dried in vacuo to give 257.2 g of crude peptide.
5)液相环化：将粗肽用250ml水溶解后，用氨水调节PH至8后，加入双氧水50μl，反应2h后，用醋酸调节PH至6。5) Liquid phase cyclization: After dissolving the crude peptide in 250 ml of water, the pH was adjusted to 8 with aqueous ammonia, 50 μl of hydrogen peroxide was added, and after reacting for 2 hours, the pH was adjusted to 6 with acetic acid.
6)纯化，冻干：通过C18柱2次纯化、转盐、冷冻干燥后得到目标产物(20.7mg，8.02％)，用MALDI-TOF分析产物，发现质子化的分子离子峰的m/z值为2582.5123(理论量为2582)。6) Purification, lyophilization: Purification by C18 column twice, salt transfer, lyophilization to obtain the target product (20.7 mg, 8.02%), and the product was analyzed by MALDI-TOF, and the m/z value of the protonated molecular ion peak was found. It is 2582.5123 (theoretical quantity is 2582).
实施例二 Embodiment 2
实施例二首先制备合成了链状的SEQ35：CHomo-RGGRAKDC-GG-KLAKLAKKLAKLAK脂肪组织靶向多肽，其中的第12位到第25位氨基酸均为D型氨基酸；然后通过环化，将其中第1位的Cys的侧链和第9位的Cys的侧链成一个二硫键的环，其制备过程如下：In the second embodiment, a SEQ35:CHomo-RGGRAKDC-GG-KLAKLAKKLAKLAK adipose tissue-targeting polypeptide having a chain form is first prepared, wherein the amino acids 12 to 25 are D-form amino acids; and then the first one is obtained by cyclization. The side chain of the Cys and the side chain of the Cys of the 9th position form a disulfide bond ring, and the preparation process is as follows:
1)称取0.1mmol取代度为0.45mmol/g的Fmoc-D-Lys(Boc)-王树脂，加入固相反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-D-Lys(Boc)-王树脂30分钟后，用DMF：哌啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，制备得到H-D-Lys(Boc)-王树脂。1) Weigh 0.1 mmol of Fmoc-D-Lys(Boc)-Wang resin with a degree of substitution of 0.45 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-D-Lys (Boc) with DMF. After 30 minutes from the king resin, Fmoc protection was removed with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then washed 6 times with DMF to prepare HD-Lys(Boc)-king resin.
2)称取Fmoc-D-Ala-OH0.5mmol、HOBt0.5mmol加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入80μl DIC(0.5mmol)活化后，加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时，此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点，从而制备得到Fmoc-D-Ala-D-Lys(Boc)-王树脂。2) Weigh Fmoc-D-Ala-OH 0.5mmol and HOBt0.5mmol into a mixed solution of DCM and DMF in a volume ratio of 1:1. After adding 80μl DIC (0.5mmol) to the ice bath, activate the above resin. In the reaction column, after reacting at room temperature for 2 hours, the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete, and the reaction needs to be further reacted for 1 hour. The standard is applicable to the subsequent amino acid coupling to determine the end point of the reaction by the ninhydrin method, thereby preparing Fmoc-D-Ala-D-Lys(Boc)-king resin.
3)重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，依次完成Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-HomoArg(Pbf)-OH、Fmoc-Cys(Trt)-OH的偶联。偶联完毕，将肽树脂用DMF洗涤3次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到0.782g粗肽树脂。3) repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D- Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc) -OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH , Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-HomoArg ( Coupling of Pbf)-OH, Fmoc-Cys(Trt)-OH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH, and dried to give 0.782 g of crude peptide resin.
4)裂解：称取0.782g全保护的肽树脂，加入到25mL的三口圆底烧瓶中，按TFA：苯甲硫醚：苯甲醚＝95：3：2的体积比配置裂解液10mL，将裂解液加入上述树脂中，室温反应2小时，过滤，用少量TFA洗涤裂解后的树脂3次，合并滤液，浓缩，将浓缩后的液体加入到冰乙醚中沉淀1小时，离心，无水乙醚离心洗涤6次，真空干燥，得到粗肽261.7mg。4) Cleavage: Weigh 0.782 g of fully protected peptide resin, add to a 25 mL three-neck round bottom flask, and arrange 10 mL of lysate according to the volume ratio of TFA: thioanisole: anisole = 95:3:2. The lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA, the filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for precipitation for 1 hour, centrifuged, and dried with diethyl ether. It was washed 6 times and dried under vacuum to give 261.7 mg of crude peptide.
5)液相环化：将粗肽用250ml水溶解后，用氨水调节PH至8后，加入 双氧水50μl，反应2h后，用醋酸调节PH至6。5) Liquid phase cyclization: After dissolving the crude peptide in 250 ml of water, adjust the pH to 8 with ammonia water, and then add 50 μl of hydrogen peroxide, after reacting for 2 h, the pH was adjusted to 6 with acetic acid.
6)纯化，冻干：而后通过C8柱2次纯化、转盐、冷冻干燥后得到目标产物(22.7mg，8.73％)，用MALDI-TOF分析产物，发现质子化的分子离子峰的m/z值为2598.5088(理论量为2598)。6) Purification, lyophilization: then purified by C8 column twice, transferred to salt, and lyophilized to obtain the target product (22.7 mg, 8.73%). The product was analyzed by MALDI-TOF, and the protonated molecular ion peak m/z was found. The value is 2598.5088 (theoretical amount is 2598).
实施例三Embodiment 3
实施例三首先制备得到了链状的SEQ36：KGGG Homo-RARE-GG-KLAKLAKKLAKLAK脂肪组织靶向多肽，其中的第11位到第24位氨基酸均为D型氨基酸；然后通过环化，将其中第1位的侧链的Lys和第8位的Glu的侧链成一个酰胺键的环，其制备过程如下：In the third embodiment, a chained SEQ36:KGGG Homo-RARE-GG-KLAKLAKKLAKLAK adipose tissue targeting polypeptide was prepared, wherein the 11th to the 24th amino acids were D-form amino acids; The side chain of Lys at position 1 and the side chain of Glu at position 8 form an amide bond ring, and the preparation process is as follows:
1)称取0.1mmol取代度为0.45mmol/g的Fmoc-D-Lys(Boc)-王树脂，加入固相反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-D-Lys(Boc)-王树脂30分钟后，用DMF：哌啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，制备得到H-D-Lys(Boc)-王树脂。1) Weigh 0.1 mmol of Fmoc-D-Lys(Boc)-Wang resin with a degree of substitution of 0.45 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-D-Lys (Boc) with DMF. After 30 minutes from the king resin, Fmoc protection was removed with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then washed 6 times with DMF to prepare HD-Lys(Boc)-king resin.
2)称取Fmoc-D-Ala-OH 0.5mmol、0.068g HOBt 0.5mmol加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入80μl DIC(0.5mmol)活化后，加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时，此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点。从而制备得到Fmoc-D-Ala-D-Lys(Boc)-王树脂。2) Weigh Fmoc-D-Ala-OH 0.5mmol, 0.068g HOBt 0.5mmol and add a mixture of DCM and DMF in a volume ratio of 1:1. Add 80μl DIC (0.5mmol) to the ice bath and activate it. In the reaction column of the resin, after reacting at room temperature for 2 hours, the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; when the resin develops color, the reaction is incomplete, and the reaction needs to be further reacted for 1 hour. This criterion is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method. Thus, Fmoc-D-Ala-D-Lys(Boc)-king resin was prepared.
3)重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，依次完成Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Glu(OAll)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-HomoArg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Lys(Dde)-OH的偶联。3) repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D- Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc) -OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Glu(OAll)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-HomoArg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Lys(Dde)- Coupling of OH.
4)固相环化：偶联完毕，采用5倍量Pd(PPh3)4脱去Glu上的烯丙基并采用3％水合肼DMF溶液10ml脱去Lys上的Dde，并采用10倍量的PyBop和 15倍量的DIEA反应过夜。环化完成后，将肽树脂用DMF洗涤3次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到0.725g粗肽树脂。4) Solid phase cyclization: After coupling, 5 times Pd(PPh 3 ) 4 was used to remove the allyl group on Glu and 10 ml of 3% hydrazine hydrate DMF solution was used to remove Dde from Lys, and 10 times of the amount was used. PyBop was reacted with 15 times the amount of DIEA overnight. After the cyclization was completed, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH and dried to give 0.725 g of crude peptide resin.
5)裂解：称取0.725g全保护的肽树脂，加入到25mL的三口圆底烧瓶中，按TFA：苯甲硫醚：苯甲醚＝95：3：2的体积比配置裂解液10mL，将裂解液加入上述树脂中，室温反应2小时，过滤，用少量TFA洗涤裂解后的树脂3次，合并滤液，浓缩，将浓缩后的液体加入到冰乙醚中沉淀1小时，离心，无水乙醚离心洗涤6次，真空干燥，得到粗肽236.5mg。5) Lysis: Weigh 0.725 g of fully protected peptide resin, add to a 25 mL three-neck round bottom flask, and arrange 10 mL of lysate according to the volume ratio of TFA: thioanisole: anisole = 95:3:2. The lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA, the filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for precipitation for 1 hour, centrifuged, and dried with diethyl ether. It was washed 6 times and dried under vacuum to give 236.5 mg of crude peptide.
6)纯化，冻干：将粗肽用250ml水溶解后。而后通过C18柱2次纯化、转盐、冷冻干燥后得到目标产物(22.6mg，9.24％)，用MALDI-TOF分析产物，发现质子化的分子离子峰的m/z值为2445.3248(理论量为2445)。6) Purification, lyophilization: The crude peptide was dissolved in 250 ml of water. Then, the target product (22.6 mg, 9.24%) was obtained by C2 column twice purification, salt transfer and lyophilization, and the product was analyzed by MALDI-TOF. The m/z value of the protonated molecular ion peak was found to be 2445.3248 (theoretical amount was 2445).
实施例四Embodiment 4
实施例四制备得到了链状的SEQ37：CRGGRAKDC-GG-KLAKLAKRLAKLAK脂肪组织靶向多肽，其中的第12位到第25位氨基酸均为D型氨基酸；然后通过环化，将其中第1位的Cys的侧链和第9位的Cys的侧链成一个二硫键的环，其制备过程如下：Example 4 prepared a chained SEQ37: CRGGRAKDC-GG-KLAKLAKRLAKLAK adipose tissue targeting polypeptide, wherein the 12th to 25th amino acids are D-type amino acids; then, by cyclization, the first position of Cys The side chain and the side chain of Cys at position 9 form a disulfide bond ring, and the preparation process is as follows:
1)称取0.1mmol取代度为0.45mmol/g的Fmoc-D-Lys(Boc)-王树脂，加入固相反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-D-Lys(Boc)-王树脂30分钟后，用DMF：哌啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，从而制备得到H-D-Lys(Boc)-王树脂。1) Weigh 0.1 mmol of Fmoc-D-Lys(Boc)-Wang resin with a degree of substitution of 0.45 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-D-Lys (Boc) with DMF. After 30 minutes from the king resin, Fmoc protection was removed with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then washed 6 times with DMF to prepare HD-Lys(Boc)-king resin.
2)称取Fmoc-D-Ala-OH 0.5mmol、HOBt 0.5mmol加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入80μl DIC(0.5mmol)活化后，加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时，此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点。从而制备得到Fmoc-D-Ala-D-Lys(Boc)-王树脂。2) Weighing 0.5 mmol of Fmoc-D-Ala-OH and 0.5 mmol of HOBt into a mixed solution of DCM and DMF in a volume ratio of 1:1, adding 80 μl of DIC (0.5 mmol) to the ice bath, and adding the above resin-containing In the reaction column, after reacting at room temperature for 2 hours, the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete, and the reaction needs to be further reacted for 1 hour. The standard is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method. Thus, Fmoc-D-Ala-D-Lys(Boc)-king resin was prepared.
3)重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，依次完成Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、 Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Cys(Trt)-OH的偶联。偶联完毕，将肽树脂用DMF洗涤3次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到0.762g粗肽树脂。3) repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D- Leu-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc -D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH , coupling of Fmoc-Cys(Trt)-OH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH, and dried to give 0.762 g of crude peptide resin.
4)裂解：称取0.762g全保护的肽树脂，加入到25mL的三口圆底烧瓶中，按TFA：苯甲硫醚：苯甲醚＝95：3：2的体积比配置裂解液10mL，将裂解液加入上述树脂中，室温反应2小时，过滤，用少量TFA洗涤裂解后的树脂3次，合并滤液，浓缩，将浓缩后的液体加入到冰乙醚中沉淀1小时，离心，无水乙醚离心洗涤6次，真空干燥，得到粗肽267.4mg。4) Cleavage: Weigh 0.762 g of fully protected peptide resin, add to a 25 mL three-neck round bottom flask, and arrange 10 mL of lysate according to the volume ratio of TFA: thioanisole: anisole = 95:3:2. The lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA, the filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for precipitation for 1 hour, centrifuged, and dried with diethyl ether. It was washed 6 times and dried under vacuum to give 267.4 mg of crude peptide.
5)液相环化：将粗肽用250ml水溶解后，用氨水调节PH至8后，加入双氧水50μl，反应2h后，用醋酸调节PH至6。5) Liquid phase cyclization: After dissolving the crude peptide in 250 ml of water, the pH was adjusted to 8 with aqueous ammonia, 50 μl of hydrogen peroxide was added, and after reacting for 2 hours, the pH was adjusted to 6 with acetic acid.
6)纯化，冻干：通过C18柱2次纯化、转盐、冷冻干燥后得到目标产物(24.2mg，9.27％)，用MALDI-TOF分析产物，发现质子化的分子离子峰的m/z值为2610.5373(理论量为2610)。6) Purification, lyophilization: Purification by two times on a C18 column, salt transfer, lyophilization to obtain the target product (24.2 mg, 9.27%), and the product was analyzed by MALDI-TOF, and the m/z value of the protonated molecular ion peak was found. It is 2610.5373 (theoretical amount is 2610).
实施例五：Embodiment 5:
实施例五首先制备得到了链状的SEQ38：C Homo-RGGRAKDC-GG-KLAKLAKRLAKLAK脂肪组织靶向多肽，其中的第12位到第25位氨基酸均为D型氨基酸；然后通过环化，将其中第1位的Cys的侧链和第9位的Cys的侧链成一个二硫键的环，其制备过程如下：In the fifth embodiment, a chained SEQ38:C Homo-RGGRAKDC-GG-KLAKLAKRLAKLAK adipose tissue targeting polypeptide is prepared, wherein the amino acids 12 to 25 are D-form amino acids; The side chain of Cys at position 1 and the side chain of Cys at position 9 form a disulfide bond ring, and the preparation process is as follows:
1)称取0.1mmol取代度为0.45mmol/g的Fmoc-D-Lys(Boc)-王树脂，加入固相反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-D-Lys(Boc)-王树脂30分钟后，用DMF：哌啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，从而制备得到H-D-Lys(Boc)-王树脂。1) Weigh 0.1 mmol of Fmoc-D-Lys(Boc)-Wang resin with a degree of substitution of 0.45 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-D-Lys (Boc) with DMF. After 30 minutes from the king resin, Fmoc protection was removed with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then washed 6 times with DMF to prepare HD-Lys(Boc)-king resin.
2)称取Fmoc-D-Ala-OH 0.5mmol、0.068g HOBt 0.5mmol加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入80μl DIC(0.5mmol)活化后， 加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时，此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点，从而制备得到Fmoc-D-Ala-D-Lys(Boc)-王树脂。2) Weigh Fmoc-D-Ala-OH 0.5mmol, 0.068g HOBt 0.5mmol, add a mixture of DCM and DMF in a volume ratio of 1:1, and add 80μl DIC (0.5mmol) to activate in an ice water bath. Adding to the reaction column containing the above resin, reacting at room temperature for 2 hours, and determining the end point of the reaction by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete and needs to be further The reaction was carried out for 1 hour, and the criterion was applied to the subsequent amino acid coupling to determine the end point of the reaction by the ninhydrin method, thereby preparing Fmoc-D-Ala-D-Lys(Boc)-king resin.
3)重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，依次完成Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-HomoArg(Pbf)-OH、Fmoc-Cys(Trt)-OH的偶联。偶联完毕，将肽树脂用DMF洗涤3次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到0.805g粗肽树脂。3) repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D- Leu-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc) -OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH , Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-HomoArg ( Coupling of Pbf)-OH, Fmoc-Cys(Trt)-OH. After the coupling was completed, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH and dried to give 0.805 g of crude peptide resin.
4)裂解：称取0.805g全保护的肽树脂，加入到25mL的三口圆底烧瓶中，按TFA:苯甲硫醚:苯甲醚＝95:3:2的体积比配置裂解液10mL，将裂解液加入上述树脂中，室温反应2小时，过滤，用少量TFA洗涤裂解后的树脂3次，合并滤液，浓缩，将浓缩后的液体加入到冰乙醚中沉淀1小时，离心，无水乙醚离心洗涤6次，真空干燥，得到粗肽284.2mg。4) Cleavage: Weigh 0.805 g of fully protected peptide resin, add to a 25 mL three-neck round bottom flask, and arrange 10 mL of lysate according to the volume ratio of TFA: thioanisole: anisole = 95:3:2. The lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA, the filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for precipitation for 1 hour, centrifuged, and dried with diethyl ether. It was washed 6 times and dried under vacuum to give 284.2 mg of crude peptide.
5)液相环化：将粗肽用250ml水溶解后，用氨水调节PH至8后，加入双氧水50μl，反应2h后，用醋酸调节PH至6。5) Liquid phase cyclization: After dissolving the crude peptide in 250 ml of water, the pH was adjusted to 8 with aqueous ammonia, 50 μl of hydrogen peroxide was added, and after reacting for 2 hours, the pH was adjusted to 6 with acetic acid.
6)纯化，冻干：通过C18柱2次纯化、转盐、冷冻干燥后得到目标产物(23.6mg，8.73％)，用MALDI-TOF分析产物，发现质子化的分子离子峰的m/z值为2626.4573(理论量为2626)。6) Purification, lyophilization: Purification by C18 column twice, salt transfer, freeze-drying to obtain the target product (23.6 mg, 8.73%), and the product was analyzed by MALDI-TOF, and the m/z value of the protonated molecular ion peak was found. It is 2626.4573 (theoretical quantity is 2626).
实施例六Embodiment 6
实施例六制备得到了链状的SEQ39：KGGG Homo-RARE-GG-KLAKLAKRLAKLAK脂肪组织靶向多肽，其中的第11位到第24位氨基酸均为D型氨基酸；然后通过环化，将其中第1位的Lys的侧链和第8位的Glu的侧链的成一个酰胺键的环，其制备过程如下： Example 6 prepared a chained SEQ39:KGGG Homo-RARE-GG-KLAKLAKRLAKLAK adipose tissue targeting polypeptide, wherein the 11th to 24th amino acids are D-type amino acids; then, by cyclization, the first of them The side chain of the Lys and the side chain of the Glu of the 8th position form an amide bond ring, and the preparation process is as follows:
1)称取0.1mmol取代度为0.45mmol/g的Fmoc-D-Lys(Boc)-王树脂，加入固相反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-D-Lys(Boc)-王树脂30分钟后，用DMF:哌啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，从而制备得到H-D-Lys(Boc)-王树脂。1) Weigh 0.1 mmol of Fmoc-D-Lys(Boc)-Wang resin with a degree of substitution of 0.45 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-D-Lys (Boc) with DMF. After 30 minutes from the king resin, Fmoc protection was removed with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then washed 6 times with DMF to prepare HD-Lys(Boc)-king resin.
2)称取Fmoc-D-Ala-OH 0.5mmol、0.068g HOBt 0.5mmol加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入80μl DIC(0.5mmol)活化后，加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时，此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点。从而制备得到Fmoc-D-Ala-D-Lys(Boc)-王树脂。2) Weigh Fmoc-D-Ala-OH 0.5mmol, 0.068g HOBt 0.5mmol and add a mixture of DCM and DMF in a volume ratio of 1:1. Add 80μl DIC (0.5mmol) to the ice bath and activate it. In the reaction column of the resin, after reacting at room temperature for 2 hours, the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; when the resin develops color, the reaction is incomplete, and the reaction needs to be further reacted for 1 hour. This criterion is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method. Thus, Fmoc-D-Ala-D-Lys(Boc)-king resin was prepared.
3)重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，依次完成Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Arg(Pbf)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Glu(OAll)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Ala-OH、Fmoc-HomoArg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Lys(Dde)-OH的偶联。3) repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D- Leu-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc) -OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Glu(OAll)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-HomoArg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Lys(Dde)- Coupling of OH.
4)固相环化：偶联完毕，采用5倍量Pd(PPh3)4脱去Glu上的烯丙基并采用3％水合肼DMF溶液10ml脱去Lys上的Dde，并采用10倍量的PyBop和15倍量的DIEA反应过夜。环化完成后，将肽树脂用DMF洗涤3次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到0.736g粗肽树脂。4) Solid phase cyclization: After coupling, 5 times Pd(PPh 3 ) 4 was used to remove the allyl group on Glu and 10 ml of 3% hydrazine hydrate DMF solution was used to remove Dde from Lys, and 10 times of the amount was used. PyBop was reacted with 15 times the amount of DIEA overnight. After the cyclization was completed, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH and dried to give 0.736 g of crude peptide resin.
5)裂解：称取0.736g全保护的肽树脂，加入到25mL的三口圆底烧瓶中，按TFA：苯甲硫醚：苯甲醚＝95：3：2的体积比配置裂解液10mL，将裂解液加入上述树脂中，室温反应2小时，过滤，用少量TFA洗涤裂解后的树脂3次，合并滤液，浓缩，将浓缩后的液体加入到冰乙醚中沉淀1小时，离心，无水乙醚离心洗涤6次，真空干燥，得到粗肽230.9mg。5) Cleavage: Weigh 0.736 g of fully protected peptide resin, add to a 25 mL three-neck round bottom flask, and arrange 10 mL of lysate according to the volume ratio of TFA: thioanisole: anisole = 95:3:2. The lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA, the filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for precipitation for 1 hour, centrifuged, and dried with diethyl ether. It was washed 6 times and dried under vacuum to give 230.9 mg of crude peptide.
6)纯化，冻干：将粗肽用250ml水溶解后，而后通过C18柱2次纯化、转盐、冷冻干燥后得到目标产物(21.0mg，9.24％)，用MALDI-TOF分析产物， 发现质子化的分子离子峰的m/z值为2473.3188(理论量为2473)。6) Purification, lyophilization: The crude peptide was dissolved in 250 ml of water, and then purified twice by a C18 column, transferred to a salt, and lyophilized to give the object product (21.0 mg, 9.24%), and the product was analyzed by MALDI-TOF. The m/z value of the protonated molecular ion peak was found to be 2473.3188 (theoretical amount was 2473).
实施例七Example 7
实施例七首先制备得到了SEQ40：CRGGRAKDC-G-(PEG)10-CH2CH2-G-KLAKLAKKLAKLAK脂肪组织靶向多肽，其中的第12位到第25位氨基酸均为D型氨基酸；然后通过环化，将其中第1位的Cys的侧链和第9位的Cys的侧链成一个二硫键的环，其制备过程如下：Example 7 First, SEQ40: CRGGRAKDC-G-(PEG)10-CH 2 CH 2 -G-KLAKLAKKLAKLAK adipose tissue targeting polypeptide was prepared, wherein the 12th to 25th amino acids were D-form amino acids; Cyclization, wherein the side chain of the Cys at position 1 and the side chain of Cys at position 9 form a disulfide bond ring, and the preparation process is as follows:
1)称取0.1mmol取代度为0.45mmol/g的Fmoc-D-Lys(Boc)-王树脂，加入固相反应柱中，用DMF洗涤1次，用DMF溶胀Fmoc-D-Lys(Boc)-王树脂30分钟后，用DMF：哌啶体积比为4:1的混合溶液脱去Fmoc保护，然后用DMF洗涤6次，从而制备得到H-D-Lys(Boc)-王树脂。1) Weigh 0.1 mmol of Fmoc-D-Lys(Boc)-Wang resin with a degree of substitution of 0.45 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-D-Lys (Boc) with DMF. After 30 minutes from the king resin, Fmoc protection was removed with a mixed solution of DMF: piperidine in a volume ratio of 4:1, and then washed 6 times with DMF to prepare HD-Lys(Boc)-king resin.
2)称取Fmoc-D-Ala-OH 0.5mmol、HOBt 0.5mmol加入体积比为1:1的DCM和DMF混合溶液，冰水浴下加入80μl DIC(0.5mmol)活化后，加入上述装有树脂的反应柱中，室温下反应2小时后，以茚三酮法检测判断反应终点，如果树脂无色透明，则表示反应完全；树脂显色，则表示反应不完全，需要再反应1小时，此判断标准适用于后续氨基酸偶联中以茚三酮法检测判断反应终点。从而制备得到Fmoc-D-Ala-D-Lys(Boc)-王树脂。2) Weighing 0.5 mmol of Fmoc-D-Ala-OH and 0.5 mmol of HOBt into a mixed solution of DCM and DMF in a volume ratio of 1:1, adding 80 μl of DIC (0.5 mmol) to the ice bath, and adding the above resin-containing In the reaction column, after reacting at room temperature for 2 hours, the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete; if the resin is colored, the reaction is incomplete, and the reaction needs to be further reacted for 1 hour. The standard is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method. Thus, Fmoc-D-Ala-D-Lys(Boc)-king resin was prepared.
3)重复上述脱除Fmoc保护和加入相应氨基酸偶联的步骤，依次完成Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-D-Ala-OH、Fmoc-D-Leu-OH、Fmoc-D-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-NH-PEG10-CH2CH2COOH、Fmoc-Gly-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Cys(Trt)-OH的偶联。偶联完毕，将肽树脂用DMF洗涤3次，DCM洗涤3次，MeOH洗涤3次，DCM洗涤3次，MeOH洗涤3次，抽干得到0.837g粗肽树脂。3) repeat the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D- Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc) -OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-NH-PEG10-CH 2 CH 2 COOH, Fmoc- Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly- Coupling of OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH. After the coupling was completed, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH and dried to give 0.837 g of crude peptide resin.
4)裂解：称取0.837g全保护的肽树脂，加入到25mL的三口圆底烧瓶中，按TFA：苯甲硫醚：苯甲醚＝95：3：2的体积比配置裂解液10mL，将裂解液 加入上述树脂中，室温反应2小时，过滤，用少量TFA洗涤裂解后的树脂3次，合并滤液，浓缩，将浓缩后的液体加入到冰乙醚中沉淀1小时，离心，无水乙醚离心洗涤6次，真空干燥，得到粗肽305.2mg。4) Cleavage: Weigh 0.837g of fully protected peptide resin, add it to a 25mL three-neck round bottom flask, and arrange 10ml of lysate according to the volume ratio of TFA: thioanisole: anisole = 95:3:2. Lysate Adding to the above resin, reacting at room temperature for 2 hours, filtering, washing the cracked resin 3 times with a small amount of TFA, combining the filtrate, concentrating, adding the concentrated liquid to ice diethyl ether for precipitation for 1 hour, centrifuging, and washing with anhydrous diethyl ether. After drying in vacuo, 305.2 mg of crude peptide was obtained.
5)液相环化：将粗肽用250ml水溶解后，用氨水调节PH至8后，加入双氧水50μl，反应2h后，用醋酸调节PH至6。5) Liquid phase cyclization: After dissolving the crude peptide in 250 ml of water, the pH was adjusted to 8 with aqueous ammonia, 50 μl of hydrogen peroxide was added, and after reacting for 2 hours, the pH was adjusted to 6 with acetic acid.
6)纯化、冻干：通过C18柱2次纯化、转盐、冷冻干燥后得到目标产物(19.7mg，6.37％)，用MALDI-TOF分析产物，发现质子化的分子离子峰的m/z值为3093.5676(理论量为3093)。6) Purification and lyophilization: The target product (19.7 mg, 6.37%) was obtained by two-time purification on a C18 column, salt-transfer, and lyophilization. The product was analyzed by MALDI-TOF, and the m/z value of the protonated molecular ion peak was found. Is 3093.5676 (theoretical amount is 3093).
(二)动物实验(2) Animal experiment
1.实验目的1. Experimental purpose
选取C57BL/6小鼠通过饲喂高脂饮食建造肥胖模型，观察脂肪组织靶向多肽对肥胖症小鼠的治疗效果，筛选出最佳治疗效果的脂肪组织靶向多肽和给药剂量，给其他相关实验研究提供参考依据。C57BL/6 mice were selected to build an obesity model by feeding a high-fat diet to observe the therapeutic effect of adipose tissue-targeted peptides on obese mice, and to screen out the best therapeutic effect of adipose tissue-targeting peptides and doses. Relevant experimental research provides a reference basis.
2.试验系统及选择的理由2. Test system and reasons for selection
选用C57BL/6小鼠作为实验动物，是因为该啮齿类动物背景资料丰富，是国内外最常采用的啮齿类实验系统，且有利于与同类试验结果进行比较。C57BL/6 mice were selected as experimental animals because the rodent has rich background information and is the most commonly used rodent experimental system at home and abroad, and is conducive to comparison with similar test results.
3.检疫与驯化3. Quarantine and domestication
C57BL/6小鼠，购自山东弘立医学动物实验研究有限公司。实验用动物送达本公司后由检疫人员核对签收，并由检疫人员负责适应性饲养3d，每天对动物的行为动作、刺激反应、肌肉紧张力、呼吸、二便、外生繁系统、皮肤粘膜及眼等进行观察。C57BL/6 mice were purchased from Shandong Hongli Medical Animal Experimental Research Co., Ltd. After the experimental animals are delivered to the company, they are checked by the quarantine personnel, and the quarantine personnel are responsible for the adaptive feeding for 3 days. The daily behaviors, irritation, muscle tension, breathing, feces, exogenous system, skin and mucous membranes of the animals Observe with eyes and so on.
4.试验动物的识别方法4. Identification method of test animals
检疫期采用尾部划线法标记，正式试验采用苦味酸涂抹法标记动物编号，并在笼具上标明实验编号、动物组别及给药剂量。The quarantine period is marked by the tail line method. In the formal test, the animal number is marked by picric acid smearing, and the experiment number, animal group and dosage are indicated on the cage.
5.实验动物饲养管理5. Laboratory animal feeding management
5.1环境条件5.1 Environmental conditions
[动物使用许可证号：SYXK(鲁)20110012][Animal use license number: SYXK (Lu) 20110012]
饲养环境：SPF级动物室； Breeding environment: SPF class animal room;
温度设定：20～25℃；Temperature setting: 20 ~ 25 ° C;
湿度设定：40～70％；Humidity setting: 40~70%;
换气次数：10～15次/h；Number of air changes: 10 to 15 times / h;
照明时间：12h(早8：00点到晚8：00开灯)；Lighting time: 12h (lights from 8:00 am to 8:00 pm);
5.2饲养条件5.2 Feeding conditions
笼具：小鼠饲养盒，苏州市通安医学设备厂生产，生产许可证号：SCXK(苏)2003-2006；Cage: mouse breeding box, produced by Suzhou Tongan Medical Equipment Factory, production license number: SCXK (Su) 2003-2006;
放置动物数：5只/盒；Number of animals placed: 5 / box;
笼具交换：2次/周；Cage exchange: 2 times / week;
粪便处理方法：随垫料一起更换，2次/周；Manure treatment method: replace with the litter, 2 times / week;
清扫及消毒方法：检疫期间隔日用布蘸取消毒液擦拭笼架及地面。实验期间，每次实验结束后清扫地面及工作台，并用布蘸取消毒液擦拭(消毒液：1％新洁尔灭、1％84消毒液、0.2％过氧乙酸，每周更换一次)。Cleaning and disinfection methods: Wipe the cage and the ground with a cloth to remove the venom. During the experiment, clean the ground and the workbench after each experiment, and wipe off the venom with a cloth (disinfectant: 1% nevi, 1% 84 disinfectant, 0.2% peracetic acid, once a week).
6.试验动物饲料6. Test animal feed
名称：高脂肪食料TD97366(25.4％脂肪，21.79％蛋白质，38.41％碳化合物)Name: High fat foodstuff TD97366 (25.4% fat, 21.79% protein, 38.41% carbon compound)
有效期：常温下3个月Validity period: 3 months under normal temperature
生产单位：Harlan TekladProduction unit: Harlan Teklad
保存条件：储存于专用的饲料间，低温、干燥和卫生Storage conditions: stored in dedicated feed, low temperature, dry and sanitary
饲料检测：每批饲料均有生产商提供的质量合格证明Feed testing: Each batch of feed has a quality certificate from the manufacturer.
给料方法：自由摄食Feeding method: free feeding
7.试验动物饮用水7. Test animal drinking water
种类：反渗透水Type: reverse osmosis water
给水方法：通过饮水瓶设置的饮水嘴自由摄取Water supply method: Free access to the drinking fountain set by the drinking water bottle
水质检测：每年检测一次，检测依据参考国家标准GB5749-2006。Water quality testing: once a year, the test is based on the reference national standard GB5749-2006.
8.供试品及溶媒的配制8. Preparation of test materials and solvents
供试品为实施例一到实施例五制备得到的脂肪组织靶向多肽，溶媒为灭菌注射用水。The test article is the adipose tissue-targeting polypeptide prepared in the first embodiment to the fifth embodiment, and the solvent is sterile water for injection.
其配置方法为：在超净工作台中，用电子天平称取不同编号的脂肪组织靶 向多肽，将其置于一灭菌容器中，并加入无菌注射用水溶解，使其终浓度为1g/L，现用现配。The configuration method is: using an electronic balance to weigh different numbers of adipose tissue targets in a clean bench To the polypeptide, it is placed in a sterile container and dissolved in sterile water for injection to a final concentration of 1 g/L.
实施例五靶向多肽储存液用PBS稀释为不同剂量。Example 5 The targeting polypeptide stock solution was diluted to different doses with PBS.
其中，相应的实验标签上标有实验编号、供试品编号、配制浓度、配制人、复核人以及配制时间。Among them, the corresponding experimental label is marked with the experiment number, the test sample number, the preparation concentration, the formulator, the reviewer, and the preparation time.
9.实验方法9. Experimental methods
9.1小鼠试验前喂养9.1 mice pre-test feeding
C57BL/6雄性小鼠在抗肥胖试验前用高脂肪食料TD97366(25.4％脂肪，21.79％蛋白质，38.41％碳化合物，购于Harlan Teklad)喂养，使其在试验前平均体重达到50g。C57BL/6 male mice were fed a high fat diet TD97366 (25.4% fat, 21.79% protein, 38.41% carbon compound, purchased from Harlan Teklad) prior to the anti-obesity test, giving an average body weight of 50 g before the test.
9.2给药剂量与分组9.2 Dosage and grouping
9.2.1脂肪组织靶向多肽样品筛选9.2.1 Screening of adipose tissue targeting peptide samples
将成模小鼠随机分为六组，每组10只小鼠，具体给药剂量与分组见表1。Mice were randomly divided into six groups of 10 mice each. The specific doses and groupings are shown in Table 1.
表1脂肪组织靶向多肽对肥胖小鼠模型给药剂量与分组Table 1 Adipose tissue targeting polypeptides administered dose and grouping in obese mouse models
Figure PCTCN2015098741-appb-000001
Figure PCTCN2015098741-appb-000001
其中，给药途径采用颈背部皮下注射的方式，其给药途径与临床药物的给药途径保持一致。给药时间为每天上午的9：30～10：30之间。Among them, the route of administration is a subcutaneous injection of the neck and the side, and the route of administration is consistent with the route of administration of the clinical drug. The administration time is between 9:30 and 10:30 every morning.
9.2.2给药剂量筛选9.2.2 Dosage screening
选取最好的减重靶向多肽(实施例五制备得到的脂肪组织靶向多肽)，将成模小鼠按给予不同的给药途径和不同的给药剂量，随机分为不同的实验组，每组10只小鼠，具体给药剂量与分组见表2。The best weight loss targeting polypeptide (the adipose tissue targeting polypeptide prepared in Example 5) was selected, and the mice were randomly divided into different experimental groups according to different administration routes and different administration doses. Group 10 mice, the specific dose and grouping are shown in Table 2.
表2实施例五脂肪组织靶向多肽对肥胖小鼠模型给药剂量与分组Table 2 Example 5 Adipose tissue targeting polypeptides administered dose and grouping in obese mouse models
Figure PCTCN2015098741-appb-000002
Figure PCTCN2015098741-appb-000002
Figure PCTCN2015098741-appb-000003
Figure PCTCN2015098741-appb-000003
其中，给药途径部分IP代表腹腔注射，IM代表肌肉注射，给药时间为每天上午的9：30～10：30之间。Among them, the administration route part IP represents intraperitoneal injection, and IM represents intramuscular injection, and the administration time is between 9:30 and 10:30 every morning.
10.各种指标的检测和数据处理10. Detection and data processing of various indicators
分别于给药前以及给药后不同的实验点，测量小鼠的体重。对实验小鼠每天给药一次，同时每三天一个间隔进行体重测定。同时采用EXCEL2013和SPSS13.0软件对数据进行录入和统计分析；小鼠体重采用均数(
Figure PCTCN2015098741-appb-000004
)±标准差(SD或S)表示。The body weight of the mice was measured before the administration and at different experimental points after the administration. Experimental mice were dosed once a day while weight measurements were taken every three days. At the same time, the data were entered and statistically analyzed using EXCEL2013 and SPSS13.0 software; the mouse body weight was averaged (
Figure PCTCN2015098741-appb-000004
) ± standard deviation (SD or S).
实验过程中并未发现影响实验研究可靠性和造成研究工作偏离实验方案的异常情况。No abnormalities affecting the reliability of the experimental study and the abnormalities that caused the research work to deviate from the experimental scheme were found during the experiment.
11.结果11. Results
11.1多肽样品筛选11.1 Screening of peptide samples
对小鼠给予实施例一到实施例五的脂肪组织靶向多肽，分别于不同的时间测定小鼠的体重变化，其中实验结果见表3。The adipose tissue-targeting polypeptides of Examples 1 to 5 were administered to mice, and the body weight changes of the mice were measured at different times, and the experimental results are shown in Table 3.
表3多肽样品筛选小鼠体重下降值(g)Table 3 polypeptide sample screening mouse weight loss value (g)
Figure PCTCN2015098741-appb-000005
Figure PCTCN2015098741-appb-000005
从表3的实验结果可以看出，小鼠给予不同实施例制备得到的脂肪组织靶向多肽供试品，实施例一到实施例五的小鼠体重都出现了明显的下降，在给药 后第15天之后，除了实施例二的小鼠体重下降1.62g，其他小鼠体重普遍下降3～5g，表明实施例一到实施例五的靶向多肽具有明显的治疗效果，可以降低小鼠的体重，缓解小鼠肥胖症状。而且小鼠体重的变化随时间逐步下降，表现出明显的时间依赖性。It can be seen from the experimental results in Table 3 that the mice were administered the adipose tissue-targeting polypeptide prepared in the different examples, and the weights of the mice of Example 1 to Example 5 were significantly decreased. After the 15th day, except for the mice of Example 2, the body weight decreased by 1.62 g, and the weight of other mice generally decreased by 3 to 5 g, indicating that the targeted polypeptides of Examples 1 to 5 have obvious therapeutic effects and can reduce the mice. The weight of the body relieves the symptoms of obesity in mice. Moreover, the change in body weight of mice gradually decreased with time, showing a significant time dependence.
11.2给药剂量筛选11.2 Dosing dose screening
选取实施例五的靶向多肽供试品，然后用无菌水稀释成不同的浓度，分别采用不同的给药途径，包括腹腔注射(IP)和肌肉注射(IM)，然后于不同的时间测定小鼠给药前和给药后的体重变化，其中小鼠给药后相较于给药前体重减少值见表4。The target polypeptide of Example 5 was selected and then diluted with sterile water to different concentrations, using different routes of administration, including intraperitoneal (IP) and intramuscular (IM), and then determined at different times. The change in body weight before and after administration of the mice, wherein the weight loss after administration of the mice compared to the weight before administration is shown in Table 4.
表4实施例五脂肪组织靶向多肽给药小鼠体重下降值(g)Table 4 Example 5 Adipose tissue targeting polypeptide administered to mice weight loss value (g)
Figure PCTCN2015098741-appb-000006
Figure PCTCN2015098741-appb-000006
从表4可以看出，无论腹腔注射还是肌肉注射脂肪组织靶向多肽，小鼠体重均随着给药浓度的增加，表现出明显的体重下降；而且体重下降值均表现出明显的时间依赖性。尤其是在给药剂量为10mg/kg和15mg/kg时，小鼠表现出非常明显的减重效果。As can be seen from Table 4, regardless of intraperitoneal injection or intramuscular injection of adipose tissue-targeting polypeptide, the body weight of the mice showed significant weight loss with increasing concentration; and the weight loss values showed significant time-dependent . Especially at doses of 10 mg/kg and 15 mg/kg, the mice showed a very significant weight loss effect.
12.结论12. Conclusion
从动物实验数据可以看出，小鼠给予实施例一到实施例五制备得到的脂肪组织靶向多肽供试品，小鼠体重都出现了明显的下降，表明实施例一到实施例五的靶向多肽具有明显的治疗效果，可以降低小鼠的体重，缓解小鼠肥胖症状。而且小鼠体重的变化随时间逐步下降，表现出明显的时间依赖性。样品筛选结果表明实施例五的靶向多肽表现出最好的抗肥胖治疗效果。同时剂量筛选结果显示实施例五的靶向多肽在给药剂量为10mg/kg和15mg/kg时减重效果明显，可以用于缓解小鼠肥胖，降低小鼠体重，而且有剂量效应。 It can be seen from the animal experimental data that the mice were given the adipose tissue-targeting polypeptides prepared in the first to the fifth examples, and the body weight of the mice showed a significant decrease, indicating the targets of Examples 1 to 5. It has a significant therapeutic effect on the polypeptide, can reduce the body weight of the mouse, and alleviate the symptoms of obesity in the mouse. Moreover, the change in body weight of mice gradually decreased with time, showing a significant time dependence. The sample screening results indicated that the targeted polypeptide of Example 5 exhibited the best anti-obesity therapeutic effect. At the same time, the dose screening results showed that the target polypeptide of Example 5 had obvious weight loss effect at the doses of 10 mg/kg and 15 mg/kg, and could be used for relieving obesity in mice, reducing body weight of mice, and having a dose effect.
最后说明的是，以上实施例仅为本发明的较佳实施例，仅用以说明本发明的技术方案，并不用于限制本发明，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，凡在本发明的精神和原则之内所做的修改、等同替换和改进等，均包含在本发明的保护范围之内。 The above embodiments are merely preferred embodiments of the present invention, and are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention, although the present invention has been described in detail by the above preferred embodiments. However, it should be understood by those skilled in the art that the modifications, equivalents, and improvements made within the spirit and scope of the present invention are included in the scope of the present invention.
Figure PCTCN2015098741-appb-000007
Figure PCTCN2015098741-appb-000007
Figure PCTCN2015098741-appb-000008
Figure PCTCN2015098741-appb-000008
Figure PCTCN2015098741-appb-000009
Figure PCTCN2015098741-appb-000009
Figure PCTCN2015098741-appb-000010
Figure PCTCN2015098741-appb-000010
Figure PCTCN2015098741-appb-000011
Figure PCTCN2015098741-appb-000011
Figure PCTCN2015098741-appb-000012
Figure PCTCN2015098741-appb-000012
Figure PCTCN2015098741-appb-000013
Figure PCTCN2015098741-appb-000013
Figure PCTCN2015098741-appb-000014
Figure PCTCN2015098741-appb-000014
Figure PCTCN2015098741-appb-000015
Figure PCTCN2015098741-appb-000015
Figure PCTCN2015098741-appb-000016
Figure PCTCN2015098741-appb-000016
Figure PCTCN2015098741-appb-000017
Figure PCTCN2015098741-appb-000017
Figure PCTCN2015098741-appb-000018
Figure PCTCN2015098741-appb-000018
Figure PCTCN2015098741-appb-000019
Figure PCTCN2015098741-appb-000019
Figure PCTCN2015098741-appb-000020
Figure PCTCN2015098741-appb-000020
Figure PCTCN2015098741-appb-000021
Figure PCTCN2015098741-appb-000021
Figure PCTCN2015098741-appb-000022
Figure PCTCN2015098741-appb-000022
Figure PCTCN2015098741-appb-000023
Figure PCTCN2015098741-appb-000023
Figure PCTCN2015098741-appb-000024
Figure PCTCN2015098741-appb-000024
Figure PCTCN2015098741-appb-000025
Figure PCTCN2015098741-appb-000025
Figure PCTCN2015098741-appb-000026
Figure PCTCN2015098741-appb-000026
Figure PCTCN2015098741-appb-000027
Figure PCTCN2015098741-appb-000027
Figure PCTCN2015098741-appb-000028
Figure PCTCN2015098741-appb-000028
Figure PCTCN2015098741-appb-000029
Figure PCTCN2015098741-appb-000029
Figure PCTCN2015098741-appb-000030
Figure PCTCN2015098741-appb-000030
Figure PCTCN2015098741-appb-000031
Figure PCTCN2015098741-appb-000031
Figure PCTCN2015098741-appb-000032
Figure PCTCN2015098741-appb-000032
Figure PCTCN2015098741-appb-000033
Figure PCTCN2015098741-appb-000033

Claims (19)

  1. 一种脂肪组织靶向多肽，其特征在于：所述的脂肪组织靶向多肽包括细胞靶向多肽肽段、细胞凋亡多肽肽段以及连接细胞靶向多肽肽段和细胞凋亡多肽肽段的桥链。An adipose tissue targeting polypeptide, characterized in that the adipose tissue targeting polypeptide comprises a cell targeting polypeptide peptide segment, an apoptotic polypeptide peptide segment, and a cell-targeting polypeptide peptide segment and an apoptotic polypeptide peptide segment. Bridge chain.
  2. 根据权利要求1所述的脂肪组织靶向多肽，其特征在于，其中细胞靶向多肽肽段选自以下序列中的一种：SEQ1，SEQ2，SEQ3，SEQ4，SEQ5，SEQ6，SEQ7，SEQ8，SEQ9，SEQ10，SEQ11，SEQ12，SEQ13，SEQ14，SEQ15，SEQ16，SEQ17，SEQ18，SEQ19，SEQ20，SEQ21，SEQ22，SEQ23，SEQ24，SEQ25，SEQ26，SEQ27，SEQ28，SEQ29，SEQ30；The adipose tissue-targeting polypeptide of claim 1, wherein the cell-targeting polypeptide peptide is selected from one of the following sequences: SEQ1, SEQ2, SEQ3, SEQ4, SEQ5, SEQ6, SEQ7, SEQ8, SEQ9 , SEQ10, SEQ11, SEQ12, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ, SEQ.
    其中所述的细胞靶向多肽肽段中的氨基酸独立地为甘氨酸和D型或者L型氨基酸，优选为甘氨酸和L型氨基酸。The amino acids in the peptides of the cell-targeting polypeptides described herein are independently glycine and a D- or L-form amino acid, preferably a glycine and an L-form amino acid.
  3. 根据权利要求1或2所述的脂肪组织靶向多肽，其特征在于，所述的细胞靶向多肽肽段中的氨基酸为天然或非天然氨基酸，所述的天然氨基酸优选为经保护基团修饰的天然氨基酸；所述的保护基团优选为叔丁氧羰基、氧叔丁基、三苯甲基、2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基、烯丙基、N-1-(4,4-二甲基-2,6-二氧环亚己基-1)乙基中的一种或几种。The adipose tissue-targeting polypeptide according to claim 1 or 2, wherein the amino acid in the peptide of the cell-targeting polypeptide is a natural or unnatural amino acid, and the natural amino acid is preferably modified by a protective group. Natural amino acid; the protecting group is preferably tert-butoxycarbonyl, oxy-tert-butyl, trityl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl One or more of allyl, N-1-(4,4-dimethyl-2,6-dioxacyclohexylidene-1)ethyl.
  4. 根据权利要求1-3任一项所述的脂肪组织靶向多肽，其特征在于：所述的细胞凋亡多肽肽段选自细胞凋亡因子或下列所述的肽链片段中的一种：SEQ31，SEQ32；The adipose tissue-targeting polypeptide according to any one of claims 1 to 3, wherein the apoptotic polypeptide peptide is selected from the group consisting of an apoptotic factor or one of the following peptide chain fragments: SEQ31, SEQ32;
    其中所述肽链片段中的氨基酸独立地为D型氨基酸或者L型氨基酸；优选为氨基酸全部为D型氨基酸或者L型氨基酸；进一步优选为氨基酸全部为D型氨基酸。The amino acid in the peptide chain fragment is independently a D-form amino acid or an L-form amino acid; preferably, all of the amino acids are D-form amino acids or L-form amino acids; further preferably, the amino acids are all D-form amino acids.
  5. 根据权利要求1-3任一项所述的脂肪组织靶向多肽，其特征在于：所述的细胞凋亡多肽肽段选自细胞凋亡因子或肽链片段SEQ33；The adipose tissue-targeting polypeptide according to any one of claims 1 to 3, wherein the apoptotic polypeptide peptide segment is selected from the group consisting of an apoptotic factor or a peptide chain fragment SEQ33;
    其中所述肽链片段中的氨基酸独立地为甘氨酸和D型或者L型氨基酸；优选为甘氨酸和D型氨基酸。Wherein the amino acid in the peptide chain fragment is independently a glycine and a D-form or an L-form amino acid; preferably a glycine and a D-form amino acid.
  6. 根据权利要求4或5所述的脂肪组织靶向多肽，其特征在于，所述的肽链片段中的氨基酸为天然或非天然氨基酸；所述的天然氨基酸优选为经保护 基团修饰的天然氨基酸；所述的保护基团优选为叔丁氧羰基、氧叔丁基、三苯甲基、2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基、烯丙基、N-1-(4,4-二甲基-2,6-二氧环亚己基-1)乙基中的一种或几种。The adipose tissue-targeting polypeptide according to claim 4 or 5, wherein the amino acid in the peptide chain fragment is a natural or unnatural amino acid; the natural amino acid is preferably protected a group-modified natural amino acid; the protecting group is preferably tert-butoxycarbonyl, oxy-tert-butyl, trityl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5 One or more of a sulfonyl group, an allyl group, and an N-1-(4,4-dimethyl-2,6-dioxocyclohexylene-1)ethyl group.
  7. 根据权利要求4-6任一项所述的脂肪组织靶向多肽，其特征在于，所述的细胞凋亡因子选自以下中的一种：白细胞介素-1、白细胞介素-2、白细胞介素-5、白细胞介素-10、白细胞介素-11、白细胞介素-12、白细胞介素-18、干扰素-γ，干扰素-α、干扰素-β、肿瘤坏死因子-α、巨噬细胞集落刺激因子。The adipose tissue-targeting polypeptide according to any one of claims 4 to 6, wherein the apoptotic factor is selected from the group consisting of interleukin-1, interleukin-2, and white blood cells. Interleukin-5, interleukin-10, interleukin-11, interleukin-12, interleukin-18, interferon-γ, interferon-α, interferon-β, tumor necrosis factor-α, Macrophage colony stimulating factor.
  8. 根据权利要求1-7任一项所述的脂肪组织靶向多肽，其特征在于，所述的桥链为2～10个的天然或非天然氨基酸片段，优选为2～6个氨基酸组成的片段，更优选为2个氨基酸片段；进一步优选为包含甘氨酸或丙氨酸。The adipose tissue-targeting polypeptide according to any one of claims 1 to 7, wherein the bridge chain is 2 to 10 natural or unnatural amino acid fragments, preferably a fragment of 2 to 6 amino acids. More preferably, it is a 2 amino acid fragment; it is further preferable to contain glycine or alanine.
  9. 根据权利要求1-8任一项所述的脂肪组织靶向多肽，其特征在于，所述的脂肪组织靶向多肽为链状或者是环状脂肪组织靶向多肽；所述的环状脂肪组织靶向多肽优选为分子内成环的方式。The adipose tissue targeting polypeptide according to any one of claims 1-8, wherein the adipose tissue targeting polypeptide is a chain or a circular adipose tissue targeting polypeptide; the cyclic adipose tissue The targeting polypeptide is preferably in a manner of intramolecular ring formation.
  10. 根据权利要求9所述的脂肪组织靶向多肽，其特征在于，所述的成环方式为化学键，选自C-N、C-O、C＝N、C-C、C＝C、C-S、S-S、CO-NH、CO-S键中的一种或几种以上，优选为S-S、CO-NH或CO-S键。The adipose tissue-targeting polypeptide according to claim 9, wherein the ring-forming method is a chemical bond selected from the group consisting of CN, CO, C=N, CC, C=C, CS, SS, CO-NH, One or more of the CO-S bonds are preferably SS, CO-NH or CO-S bonds.
  11. 根据权利要求1-10任一项所述的脂肪组织靶向多肽，其特征在于，所述的脂肪组织靶向多肽采用聚乙二醇修饰，所述的脂肪组织靶向多肽的C端、N端或侧链的任一位置与聚乙二醇进行结合，优选为脂肪组织靶向多肽的桥链与聚乙二醇进行结合。The adipose tissue-targeting polypeptide according to any one of claims 1 to 10, wherein the adipose tissue-targeting polypeptide is modified with polyethylene glycol, and the adipose tissue-targeting polypeptide C-terminal, N- Any position of the end or side chain is bound to polyethylene glycol, preferably a bridge of the adipose tissue targeting polypeptide is bound to the polyethylene glycol.
  12. 根据权利要求11所述的脂肪组织靶向多肽，其特征在于，所述聚乙二醇选自PEG10，PEG100，PEG200，PEG300，PEG400，PEG500，PEG600，PEG700，PEG800，PEG900，PEG1000，PEG1100，PEG1200，PEG1300，PEG1400，PEG1500，PEG1600，PEG1700，PEG1800，PEG2000，PEG3000，PEG4000，PEG10000，PEG20000，PEG40000中的一种或几种以上；优选为PEG10，PEG100，PEG200，PEG300，PEG400，PEG500中的一种或几种以上；更优选为PEG10，PEG100，PEG200中的一种或几种以上。The adipose tissue targeting polypeptide according to claim 11, wherein the polyethylene glycol is selected from the group consisting of PEG10, PEG100, PEG200, PEG300, PEG400, PEG500, PEG600, PEG700, PEG800, PEG900, PEG1000, PEG1100, PEG1200 One or more of PEG1300, PEG1400, PEG1500, PEG1600, PEG1700, PEG1800, PEG2000, PEG3000, PEG4000, PEG10000, PEG20000, PEG40000; preferably one of PEG10, PEG100, PEG200, PEG300, PEG400, PEG500 Or more than several; more preferably one or more of PEG10, PEG100, and PEG200.
  13. 一种脂肪组织靶向多肽的制备方法，其特征在于，包括如下步骤：A method for preparing an adipose tissue targeting polypeptide, comprising the steps of:
    (1)采用固相合成法，依照脂肪组织靶向多肽的连接次序在偶联剂的作 用下，依次进行氨基酸的偶联，合成具有N端保护的链状脂肪组织靶向多肽；(1) using solid phase synthesis, according to the order of attachment of adipose tissue targeting polypeptides in the coupling agent The amino acid is coupled in sequence to synthesize a chain adipose tissue targeting polypeptide having N-terminal protection;
    (2)裂解去除N端保护的基团，得到链状的脂肪组织靶向多肽。(2) Cleavage to remove the N-terminal protected group to obtain a chain adipose tissue targeting polypeptide.
  14. 根据权利要求13所述的制备方法，其特征在于，所述偶联剂包括缩合剂和反应溶剂，所述缩合剂选自以下几种组合中的一种：(1)N，N′-二异丙基碳化二亚胺和1-羟基苯并三唑，The preparation method according to claim 13, wherein the coupling agent comprises a condensing agent and a reaction solvent, and the condensing agent is selected from one of the following combinations: (1) N, N'-di Isopropylcarbodiimide and 1-hydroxybenzotriazole,
    (2)六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、1-羟基苯并三唑和N，N′-二异丙基乙胺，(2) benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxybenzotriazole and N,N'-diisopropylethylamine,
    (3)2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯和N，N′-二异丙基乙胺，(3) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N'-diisopropylethylamine,
    (4)六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、1-羟基苯并三唑和N-甲基吗啡啉，(4) benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxybenzotriazole and N-methylmorpholine,
    (5)2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯和N-甲基吗啡啉；(5) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N-methylmorpholine;
    所述反应溶剂选自N，N′-二甲基甲酰胺、二氯甲烷、N-甲基吡咯烷酮、二甲基亚砜中的一种或几种以上。The reaction solvent is one or more selected from the group consisting of N,N'-dimethylformamide, dichloromethane, N-methylpyrrolidone, and dimethyl sulfoxide.
  15. 根据权利要求13或14所述的制备方法，其特征在于，所述的制备方法还包括将步骤(2)得到的链状脂肪组织靶向多肽进行环化过程，得到环状脂肪组织靶向多肽；所述的环化过程包括液相环化和固相环化。The preparation method according to claim 13 or 14, wherein the preparation method further comprises the step of cyclizing the chain adipose tissue targeting polypeptide obtained in the step (2) to obtain a cyclic adipose tissue targeting polypeptide. The cyclization process includes liquid phase cyclization and solid phase cyclization.
  16. 根据权利要求15所述的制备方法，其特征在于，所述的液相环化包括如下步骤：The method according to claim 15, wherein said liquid phase cyclization comprises the following steps:
    (1)链状脂肪组织靶向多肽在溶剂中进行溶解，(1) a chain adipose tissue targeting polypeptide is dissolved in a solvent,
    (2)调节溶液成碱性后加入氧化剂进行环化，(2) adjusting the solution to be alkaline and then adding an oxidizing agent to cyclize.
    (3)调节溶液成酸性后得到环状脂肪组织靶向多肽；(3) adjusting the solution to be acidic to obtain a cyclic adipose tissue targeting polypeptide;
    所述的固相环化为将链状脂肪组织靶向多肽加入氧化剂得到环状脂肪组织靶向多肽。The solid phase cyclization is to add a chain adipose tissue targeting polypeptide to an oxidizing agent to obtain a cyclic adipose tissue targeting polypeptide.
  17. 一种药物组合物，该药物组合物包含治疗有效量的游离形式或可药用盐形式的权利要求1-12任一项所述的脂肪组织靶向多肽作为活性成分，以及一种或多种药用载体物质和/或稀释剂。A pharmaceutical composition comprising a therapeutically effective amount of the adipose tissue-targeting polypeptide of any one of claims 1 to 12 as an active ingredient, in one or more forms, in free form or in a pharmaceutically acceptable salt form, and one or more Pharmaceutically acceptable carrier materials and/or diluents.
  18. 权利要求1-12任一项所述的脂肪组织靶向多肽或权利要求17所述的 药物组合物在制备治疗和/或预防减肥或其他与减肥相关疾病药物方面的用途。The adipose tissue targeting polypeptide of any one of claims 1 to 12 or the method of claim 17. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment and/or prevention of weight loss or other diseases associated with weight loss.
  19. 权利要求1-12任一项所述的脂肪组织靶向多肽或权利要求17所述的药物组合物在治疗和/或预防减肥或其他与减肥相关疾病方面的用途。 Use of the adipose tissue targeting polypeptide of any of claims 1-12 or the pharmaceutical composition of claim 17 for the treatment and/or prevention of weight loss or other weight loss related diseases.
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