CN116715753A - 一种保持单链构象的类肽胶原杂交肽的制备方法与应用 - Google Patents
一种保持单链构象的类肽胶原杂交肽的制备方法与应用 Download PDFInfo
- Publication number
- CN116715753A CN116715753A CN202310533389.5A CN202310533389A CN116715753A CN 116715753 A CN116715753 A CN 116715753A CN 202310533389 A CN202310533389 A CN 202310533389A CN 116715753 A CN116715753 A CN 116715753A
- Authority
- CN
- China
- Prior art keywords
- collagen
- peptide
- peptoid
- hybrid peptide
- chp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 102
- 102000008186 Collagen Human genes 0.000 title claims abstract description 91
- 108010035532 Collagen Proteins 0.000 title claims abstract description 91
- 229920001436 collagen Polymers 0.000 title claims abstract description 91
- 108010043958 Peptoids Proteins 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 65
- 239000003929 acidic solution Substances 0.000 claims abstract description 9
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 7
- UVJWCNFWEYPYSP-SNKMQCGQSA-N 2-aminoacetic acid;(2s,4r)-4-hydroxypyrrolidine-2-carboxylic acid;(2s)-pyrrolidine-2-carboxylic acid Chemical compound NCC(O)=O.OC(=O)[C@@H]1CCCN1.O[C@H]1CN[C@H](C(O)=O)C1 UVJWCNFWEYPYSP-SNKMQCGQSA-N 0.000 claims abstract description 3
- 150000001413 amino acids Chemical group 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 11
- 230000001575 pathological effect Effects 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 238000011503 in vivo imaging Methods 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 238000006467 substitution reaction Methods 0.000 claims description 3
- 239000012670 alkaline solution Substances 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 abstract description 13
- 238000002474 experimental method Methods 0.000 abstract description 5
- 238000009396 hybridization Methods 0.000 abstract description 5
- 230000006378 damage Effects 0.000 abstract description 3
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 230000037369 collagen remodeling Effects 0.000 abstract description 2
- 208000015951 Cytophagic histiocytic panniculitis Diseases 0.000 description 70
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 27
- 229920001184 polypeptide Polymers 0.000 description 25
- 102000004196 processed proteins & peptides Human genes 0.000 description 25
- 108010010803 Gelatin Proteins 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 16
- 239000008273 gelatin Substances 0.000 description 16
- 229920000159 gelatin Polymers 0.000 description 16
- 235000019322 gelatine Nutrition 0.000 description 16
- 235000011852 gelatine desserts Nutrition 0.000 description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 10
- 239000000523 sample Substances 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 235000013930 proline Nutrition 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000002073 fluorescence micrograph Methods 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 208000010125 myocardial infarction Diseases 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000011580 nude mouse model Methods 0.000 description 3
- 210000002027 skeletal muscle Anatomy 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- FPIRBHDGWMWJEP-UHFFFAOYSA-N 1-hydroxy-7-azabenzotriazole Chemical compound C1=CN=C2N(O)N=NC2=C1 FPIRBHDGWMWJEP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000007821 HATU Substances 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 150000003148 prolines Chemical class 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- WOXFMYVTSLAQMO-UHFFFAOYSA-N 2-Pyridinemethanamine Chemical group NCC1=CC=CC=N1 WOXFMYVTSLAQMO-UHFFFAOYSA-N 0.000 description 1
- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 1
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- -1 Fmoc amino acid Chemical class 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 239000007987 MES buffer Substances 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 101100163901 Rattus norvegicus Asic2 gene Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- HOPSCVCBEOCPJZ-UHFFFAOYSA-N carboxymethyl(trimethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC(O)=O HOPSCVCBEOCPJZ-UHFFFAOYSA-N 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002983 circular dichroism Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 108010025899 gelatin film Proteins 0.000 description 1
- 150000002333 glycines Chemical class 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- UGNWTBMOAKPKBL-UHFFFAOYSA-N tetrachloro-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(Cl)=C(Cl)C1=O UGNWTBMOAKPKBL-UHFFFAOYSA-N 0.000 description 1
- 230000003685 thermal hair damage Effects 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000005829 trimerization reaction Methods 0.000 description 1
Classifications
-
- 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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及蛋白质工程技术领域,公开了一种保持单链构象的类肽胶原杂交肽的制备方法与应用。该制备方法,其特征在于,所述方法包括以下步骤:(1)将胶原杂交肽的甘氨酸‑脯氨酸‑羟脯氨酸三联体中的脯氨酸替换成强偏顺式肽键类肽残基,制得类肽胶原杂交肽;(2)将所述类肽胶原杂交肽溶解在酸性溶液中,制得保持单链构象的类肽胶原杂交肽溶液。本发明N2pic3‑CHP具有比传统GPO序列CHP更强的三螺旋杂交能力。荧光成像实验证明该CHP在pH3.0下可以不用预先加热处理就可以保持单链状态,并可以与具有活跃胶原重塑或严重胶原破坏的疾病的变性胶原杂交。
Description
技术领域
本发明涉及蛋白质工程技术领域,具体涉及一种保持单链构象的类肽胶原杂交肽的制备方法与应用。
背景技术
胶原蛋白是脊椎动物中含量最丰富的蛋白质,也是细胞外基质的主要成分。它为细胞、组织提供支架的同时,也在细胞生长、粘附、迁移等过程发挥了重要作用。人体胶原的合成和降解处于动态平衡中,一旦平衡被打破会导致各种疾病,如癌症、骨质疏松、关节炎和纤维化。
胶原蛋白分子是由Gly-Xaa-Yaa三联体重复氨基酸序列组成,其中脯氨酸(Pro,P)和羟脯氨酸(Hyp,O)分别往往出现在Xaa和Yaa位置。三螺旋是胶原蛋白独特的二级结构,它通过链间氢键将三条链交织在一起。病理状态下,胶原三螺旋分子会被基质金属蛋白酶酶解发生断裂。断裂的胶原分子在体温下不稳定自发发生解旋。胶原杂交肽(CollagenHybridizing Peptide,CHP)作为一种分子探针,它可以和解旋的胶原蛋白链杂交通过链间氢键重新形成三螺旋结构。这与PCR反应中引物与解旋DNA链杂交的方式十分类似。正常的胶原三螺旋分子缺乏结合位点,所以CHP不会和正常的胶原分子结合。荧光标记的CHP可以靶向各种病理状态下破坏的胶原蛋白分子,是评估病理相关的胶原破坏状态的一个强有力工具。
然而,传统GPO序列的CHP探针特别容易自身三聚成三螺旋结构。一旦CHP自身三聚成三螺旋,它就失去了与破坏胶原分子杂交的能力。因此,在使用CHP前,需要在85度下加热CHP溶液来生成单链CHP。虽然预先加热的方案可以迅速将多肽溶液降至室温以避免组织热损伤,但加热过程不利于活体内的应用,且由于CHP在室温下容易自身三聚而无法确定单体浓度,从而难以对成像结果进行定量分析。因此,迫切需要开发一种新的保持单链状态的CHP探针。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此,本发明提出一种保持单链构象的类肽胶原杂交肽的制备方法与应用。该方法不仅使三螺旋的类肽胶原杂交肽变性解旋保持单链构象,还能保持类肽胶原杂交肽的胶原杂交能力。
本发明的第一方面提供一种保持单链构象的类肽胶原杂交肽的制备方法。
具体的,所述方法包括以下步骤:
(1)将胶原杂交肽的甘氨酸-脯氨酸-羟脯氨酸三联体中的脯氨酸替换成强偏顺式肽键类肽残基,制得类肽胶原杂交肽;
(2)将所述类肽胶原杂交肽溶解在酸性溶液中,制得保持单链构象的类肽胶原杂交肽溶液。
优选的,步骤(1)中,所述胶原杂交肽的氨基酸序列为Ac-(GPO)n-NH2,n为7-12。
优选的,步骤(1)中,所述强偏顺式肽键类肽残基包括N-(2-氨甲基吡啶)取代甘氨酸(N2pic)。
优选的,步骤(1)中,所述强偏顺式肽键类肽残基替换的个数为3-9个。
进一步优选的,步骤(1)中,所述强偏顺式肽键类肽残基替换的个数为3个。
优选的,步骤(2)中,所述酸性溶液的pH为1.0-4.0。
进一步优选的,所述酸性溶液包括盐酸溶液、硫酸溶液、硝酸溶液、醋酸溶液中的至少一种。
更进一步优选的,所述酸性溶液的浓度为1-3mM;例如1mM、2mM、3mM。
更进一步优选的,所述酸性溶液为1mM盐酸溶液。
更进一步优选的,所述酸性溶液的pH为3.0。
本发明的第二方面提供一种类肽胶原杂交肽。
具体的,所述类肽胶原杂交肽的氨基酸序列为Ac-(GPO)1(GN2picO)3(GPO)3-NH2。
优选的,将所述类肽胶原杂交肽置于中性或碱性的溶液进行激活,激活后的类肽胶原杂交肽恢复胶原杂交能力。
优选的,所述溶液的pH为7.4-14.0。
进一步优选的,所述溶液包括磷酸缓冲盐溶液。
更进一步优选的,磷酸缓冲盐溶液为1×PBS溶液。
本发明的第三方面提供一种类肽胶原杂交肽在制备检测试剂盒上的应用。
本发明的第四方面提供一种类肽胶原杂交肽在制备靶向变性胶原蛋白生物传感器、病理组织染色、活体成像上的应用。
具体的,所述类肽胶原杂交肽用于构建靶向变性胶原蛋白生物传感器、病理组织染色、活体成像。
相对于现有技术,本发明的有益效果如下:
本发明设计合成一种含有三个N2pic类肽残基的CHP探针(N2pic3-CHP)。N2pic在质子化状态下(酸性条件)强烈倾向形成顺式肽键,在非质子化状态下(中性或碱性)倾向于形成反式肽键。N2pic3-CHP探针可以在酸性条件下(pH=3.0)保持单链状态,不折叠成三螺旋。本发明N2pic3-CHP具有比传统GPO序列CHP更强的三螺旋杂交能力。荧光成像实验证明该CHP在pH3.0下可以不用预先加热处理就可以保持单链状态,并可以与具有活跃胶原重塑或严重胶原破坏的疾病的变性胶原杂交。
附图说明
图1为不同处理方式的多肽与明胶结合结果图;
图2为不同处理方式的多肽与明胶结合结果图;
图3为注射不同多肽后活体小鼠的近红外荧光图像;
图4为注射不同多肽后去除皮肤裸鼠的近红外荧光图像;
图5为注射不同多肽后心梗小鼠心脏的近红外荧光图像。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案,现列举以下实施例进行说明。需要指出的是,以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明,均可从常规商业途径得到,或者可以通过现有已知方法得到。
胶原杂交肽Pro-CHP(氨基酸序列为Ac-(GPO)7-NH2)的合成方法如下:(1)溶胀树脂:以合成0.01mmol多肽为例,称取27mg的Rink酰胺树脂(载样量:0.37mmol/g)置于多肽固相合成管中,加入1mL的DMF,室温摇晃至少半小时。(2)脱Fmoc保护基和偶联氨基酸:将树脂抽干,加入0.7mL 20%的哌啶DMF溶液,室温摇晃20分钟。反应完后,用10mL DMF洗涤树脂,重复三次后抽干溶剂。然后将Fmoc氨基酸(50μmol),HATU(50μmol),HOAT(50μmol)和DIEA(75μmol)的DMF(0.4mL)混合溶液加入到树脂中,室温摇晃不少于3小时。所有氨基酸偶联和脱保护反应均通过茚三酮或四氯苯醌验色反应进行监测。(3)乙酰化封端:将乙酸(50μmol),HATU(50μmol),HOAT(50μmol)和DIEA(75μmol)的DMF(0.4mL)混合溶液加入到树脂中,室温摇晃不少于3小时。(4)剪切树脂:将乙酰化封端的树脂(10μmol)分别用DMF和DCM洗涤三次并抽干溶剂。加入1mL的TFA/TIS/H2O=95:2.5:2.5的混合溶液,室温下搅拌三小时。然后,把剪切液转移到50mL离心管中,用氮气吹干TFA。加入5mL冰乙醚,有白色絮状物析出。将离心管放到离心机中离心(4℃,4000rpm,4min)。离心完,去掉上清液。重复冰乙醚沉淀操作一次。将带有沉淀物的离心管放到通风橱中,使乙醚挥发。待乙醚挥发完全后,加入5mL纯水溶解多肽粗产物,并用微孔滤膜(0.45μm)过滤除去不溶物。(5)多肽通过高效液相色谱法(HPLC)纯化。流动相A液为0.1% TFA的纯水溶液,B液为0.1% TFA的乙腈溶液。液相梯度程序为:乙腈浓度由5%(5min)-35%(25min)-5%(25.1min)-5%(30min),流速:4mL/min,柱温:85℃。收集的产物通过冻干机冻干成絮状物。
实施例1
一种保持单链构象的类肽胶原杂交肽的制备方法。
将胶原杂交肽Pro-CHP(氨基酸序列为Ac-(GPO)7-NH2)的3个的脯氨酸替换成3个强偏顺式肽键类肽残基N2pic,制得类肽胶原杂交肽N2pic3-CHP(氨基酸序列为Ac-(GPO)1(GN2picO)3(GPO)3-NH2)。将N2pic3-CHP溶解在1mM盐酸溶液中,得到pH 3.0的N2pic3-CHP溶液。
对比例1
一种类肽胶原杂交肽的制备方法。
将胶原杂交肽Pro-CHP(氨基酸序列为Ac-(GPO)7-NH2)溶解在1mM盐酸溶液中,得到pH 3.0的Pro-CHP溶液。
对比例2
一种类肽胶原杂交肽的制备方法。
将胶原杂交肽Pro-CHP(氨基酸序列为Ac-(GPO)7-NH2)的1个的脯氨酸替换成1个强偏顺式肽键类肽残基N2pic,制得类肽胶原杂交肽N2pic1-CHP(氨基酸序列为Ac-(GPO)3(GN2picO)1(GPO)3-NH2)。将N2pic1-CHP溶解在1mM盐酸溶液中,得到pH 3.0的N2pic1-CHP溶液。
对比例3
一种类肽胶原杂交肽的制备方法。
将胶原杂交肽Pro-CHP(氨基酸序列为Ac-(GPO)7-NH2)的2个的脯氨酸替换成2个强偏顺式肽键类肽残基N2pic,制得类肽胶原杂交肽N2pic2-CHP(氨基酸序列为Ac-(GPO)2(GN2picO)2(GPO)3-NH2)。将N2pic2-CHP溶解在1mM盐酸溶液中,得到pH 3.0的N2pic2-CHP溶液。
对比例4
一种保持单链构象的类肽胶原杂交肽的制备方法。
将胶原杂交肽Pro-CHP(氨基酸序列为Ac-(GPO)7-NH2)的7个的脯氨酸替换成7个强偏顺式肽键类肽残基N2pic,制得类肽胶原杂交肽N2pic7-CHP(氨基酸序列为Ac-(GN2picO)7-NH2)。将N2pic7-CHP溶解在1mM盐酸溶液中,得到pH 3.0的N2pic7-CHP溶液。
对比例5
一种类肽胶原杂交肽的制备方法。
将胶原杂交肽Pro-CHP(氨基酸序列为Ac-(GPO)7-NH2)的3个的脯氨酸替换成3个强偏顺式肽键类肽残基N2pic,但设置不同排列顺序的氨基酸序列(序列为Ac-(GPO)1(N2picOG)3(GPO)3-NH2),制得类肽胶原杂交肽N2pic3-sCHP。将N2pic3-sCHP溶解在1mM盐酸溶液中,得到pH 3.0的N2pic3-sCHP溶液。
对比例6
一种类肽胶原杂交肽的制备方法。
与实施例1相比,不同在于对比例6制得类肽胶原杂交肽N2pic3-CHP(氨基酸序列为Ac-(GPO)1(GN2picO)3(GPO)3-NH2)溶解在1×PBS溶液中,得到pH 7.4的N2pic3-CHP溶液。
对比例7
一种保持单链构象的类肽胶原杂交肽的制备方法。
与对比例6相比,不同在于对比例7将得到pH 7.4的N2pic3-CHP溶液在85℃下加热5min,然后在冰水中冷却20秒。
1.圆二色光谱检测(CD):
将多肽样品装入光径为1mm的石英比色皿中,通过圆二色光谱仪(JASCO,J-1500)测定得到多肽样品的CD信号。测试前,将实施例1和对比例1-4制得的多肽溶液在85℃下加热5min后放入4℃冰箱孵育至少48小时;将实施例1和对比例1-4制得的多肽溶液用1×PBS溶液稀释pH为7.4,并在85℃下加热5min后放入4℃冰箱孵育至少48小时。各多肽溶液用参数(扫描速度:20nm/min;数据间距:0.1nm;带宽:5nm;数字积分时间:16s)扫描CD光谱。热变性曲线是多肽溶液在0.5℃/min加热速率下测量4-80℃的225nm下的CD信号值。根据肽的长度和浓度,将原始CD信号归一化为平均残基椭圆度。平均残基椭圆度(MRE,[θ])用公式[θ]=(θ×m)/(c×l×n)计算,其中θ是测量的椭圆度(mdeg),m是分子量(g/mol),c是浓度(mg/mL),l是比色管的路径长度(mm),n是肽中氨基酸残基的数量。使用JASCO Spectra Manager软件对热变性曲线求导,得到导数曲线,将导数曲线最低处的温度定义为熔解温度(Tm)。
表1各多肽在不同溶剂中的热稳定性检测结果
由表1可知,在pH 3.0的1mM盐酸溶液中,N2pic1-CHP和N2pic2-CHP都会自聚形成稳定的三螺旋。当N2pic类肽残基增加到3个,N2pic3-CHP在pH 3.0的1mM盐酸溶液中无法自聚形成三螺旋。同样,N2pic7-CHP在pH 3.0的1mM盐酸溶液中无法自聚形成三螺旋。但是在pH 7.4的PBS溶液,N2pic1-CHP、N2pic2-CHP、N2pic3-CHP和N2pic7-CHP都可以形成稳定的三螺旋。说明N2pic3-CHP和N2pic7-CHP在pH 3.0时可保持单链构象,当pH调节到7.4时,N2pic3-CHP和N2pic7-CHP可恢复其胶原杂交能力,并且Tm值高于Pro-CMP的Tm值。
2.明胶结合检测:
将猪明胶(Sigma,V900863-100G)70℃加热溶解于1×PBS(10%w/v)中,每孔取约6μL的明胶溶液于96孔板中,然后在4℃孵育10min使明胶得以凝胶化。将含有2mM NHS和10mMEDC的MES缓冲溶液(0.1M,pH 4.7,100μL/孔)加入到明胶孔中,在室温下摇晃过夜。交联后的明胶膜用1×PBS洗涤10min,重复三次。在明胶结合实验前,将Cy5标记的N2pic3-CHP、N2pic7-CHP、Pro-CHP和N2pic3-sCHP分别用1mM盐酸溶液和1×PBS溶液溶解配成50μM母液。所有多肽在使用前都配成1×PBS的工作液,浓度为10μM,每孔50μL。其中“加热组”的肽溶液在85℃下加热5min,然后在冰水中冷却20秒后加入明胶孔中。“不加热”组的多肽溶液配成1×PBS溶液后在室温下直接加入明胶孔中。4℃孵育过夜后,用1×PBS洗涤明胶孔3次,用酶标仪测定荧光(Ex:646nm,Em:670nm)。
由图1可知,与Cy5-Pro-CHP类似,储存在PBS溶液中的Cy5-N2pic3-CHP在没有加热处理的情况下处于三聚状态不能与明胶(热变性胶原蛋白)结合。与PBS储存液相反,储存在1mM HCl溶液中的Cy5-N2pic3-CHP保持单链状态,其结合能力强于加热的Cy5-Pro-CHP。残基组成相同但序列不同的对照肽N2pic3-sCHP由于缺乏三螺旋折叠能力而无法结合明胶。这些结果说明Cy5-N2pic3-CHP被酸溶液处理后,其在使用前配成1×PBS的工作液在无需加热的情况下即可与明胶杂交,且杂交能力高于加热的Cy5-Pro-CHP。
由图2可知,除Cy5-Pro-CHP外,所有多肽都保存在盐酸溶液中(pH=3.0)。Cy5-N2pic3-CHP在没有加热处理的情况下可以结合明胶(热变性胶原蛋白),结合能力高于加热后的Cy5-Pro-CHP,也高于Cy5-N2pic7-CHP。氨基酸组成相同但序列不同的对照肽N2pic3-sCHP由于缺乏三螺旋能力而无法结合明胶。
3.裸鼠活体成像检测:
给正常雌性BALB/c裸鼠(8-12周龄)尾静脉注射前,把实施例1和对比例5-7制备多肽溶液用Cy5标记成为探针,再将探针配成1×PBS溶液(剂量:1nmol),在室温下直接尾静脉注射。90分钟后,用IVIS光谱成像仪(激发光:620nm,发射光:670nm,PerkinElmer LuminaIII)对小鼠进行荧光成像。然后,处死小鼠并去除它们的皮肤以允许对深层组织进行成像。本实验重复三次,每次得到的结果都一致。
由图3可知,静脉注射1nmol的Cy5-N2pic3-CHP或Cy5-N2pic3-sCHP的裸鼠近红外荧光图像结果显示储存在1mM盐酸溶液中的Cy5-N2pic3-CHP组保持在单链状态,可以活体靶向骨肌系统中重塑的胶原蛋白,说明实施例1制备的多肽溶液N2pic3-CHP在无需加热的情况下也具备活体靶向骨肌系统中重塑的胶原蛋白的能力。而对比例6制备的多肽溶液储存在1×PBS溶液中,其Cy5-N2pic3-CHP处于三聚状态,而对比例7制备的多肽溶液虽然储存在1×PBS溶液中,但经过加热后小鼠也具备活体靶向骨肌系统中重塑的胶原蛋白的能力,说明不被酸溶液处理的多肽溶液只有在加热处理后才能靶向重塑的胶原蛋白。由图4可知,去除皮肤后裸鼠的近红外荧光图像结果与活体荧光成像图结果一致。
4.离体心脏荧光成像检测:
小鼠在手术结扎左冠状动脉后7-14天用于体内试验(心梗小鼠模型)。将实施例1制备的N2pic3-CHP和对比例5制备的N2pic3-sCHP用Cy5标记后,配成1×PBS溶液(剂量:4nmol),然后快速注射到小鼠的尾静脉中。一小时后,处死小鼠,并分别用0.02%肝素的PBS溶液(m/V)和PBS溶液灌注心脏以去除血液。对离体小鼠心脏在Z视野下荧光成像,激发波长620nm,发射波长670nm。
由图5可知,与假手术组(sham)相比,尾静脉注射Cy5-N2pic3-CHP(4nmol)一小时后,心梗模型组小鼠(MI)的心脏具有明显的荧光信号。同是心梗小鼠,注射相同剂量的Cy5-N2pic3-sCHP无明显荧光信号,说明实施例1制备的N2pic3-CHP可与心梗小鼠心脏中的破坏胶原杂交。
Claims (10)
1.一种保持单链构象的类肽胶原杂交肽的制备方法,其特征在于,所述方法包括以下步骤:
(1)将胶原杂交肽的甘氨酸-脯氨酸-羟脯氨酸三联体中的脯氨酸替换成强偏顺式肽键类肽残基,制得类肽胶原杂交肽;
(2)将所述类肽胶原杂交肽溶解在酸性溶液中,制得保持单链构象的类肽胶原杂交肽溶液。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,所述胶原杂交肽的氨基酸序列为Ac-(GPO)n-NH2,n为7-12。
3.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,所述强偏顺式肽键类肽残基包括N2pic。
4.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,所述强偏顺式肽键类肽残基替换的个数为3-9个。
5.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,所述酸性溶液的pH为1.0-4.0。
6.一种类肽胶原杂交肽,其特征在于,所述类肽胶原杂交肽的氨基酸序列为Ac-(GPO)1(GN2picO)3(GPO)3-NH2。
7.根据权利要求6所述的类肽胶原杂交肽,其特征在于,将所述类肽胶原杂交肽置于中性或碱性的溶液进行激活,激活后的类肽胶原杂交肽恢复胶原杂交能力。
8.根据权利要求6所述的类肽胶原杂交肽,其特征在于,所述溶液的pH为7.4-14.0。
9.权利要求6所述的类肽胶原杂交肽在制备检测试剂盒上的应用。
10.权利要求6所述的类肽胶原杂交肽在制备靶向变性胶原蛋白生物传感器、病理组织染色、活体成像上的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310533389.5A CN116715753B (zh) | 2023-05-11 | 2023-05-11 | 一种保持单链构象的类肽胶原杂交肽的制备方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310533389.5A CN116715753B (zh) | 2023-05-11 | 2023-05-11 | 一种保持单链构象的类肽胶原杂交肽的制备方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116715753A true CN116715753A (zh) | 2023-09-08 |
CN116715753B CN116715753B (zh) | 2024-03-19 |
Family
ID=87874198
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310533389.5A Active CN116715753B (zh) | 2023-05-11 | 2023-05-11 | 一种保持单链构象的类肽胶原杂交肽的制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116715753B (zh) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5668294A (en) * | 1995-04-21 | 1997-09-16 | University Of Georgia Research Foundation Inc. | Metal resistance sequences and transgenic plants |
CN107530454A (zh) * | 2015-01-30 | 2018-01-02 | 犹他大学研究基金会 | 二聚胶原杂交肽和使用方法 |
CN110129029A (zh) * | 2019-02-14 | 2019-08-16 | 兰州大学 | 一种电荷排斥作用诱导的单链胶原多肽功能探针及其制备方法 |
US20200308253A1 (en) * | 2017-10-18 | 2020-10-01 | Swansea University | Single alpha chain collagens |
CN111909245A (zh) * | 2020-08-05 | 2020-11-10 | 兰州大学 | 一种包含芳香族氨基酸的胶原靶向多肽探针,制备方法及其应用 |
US20230087644A1 (en) * | 2020-01-02 | 2023-03-23 | Lanzhou University | Probe for Specifically Detecting Pathological Collagens, and Preparation Method Therefor and Use Thereof |
CN115925988A (zh) * | 2022-09-14 | 2023-04-07 | 中山大学附属第五医院 | 一种变性胶原靶向抗菌肽及其制备方法与应用 |
-
2023
- 2023-05-11 CN CN202310533389.5A patent/CN116715753B/zh active Active
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5668294A (en) * | 1995-04-21 | 1997-09-16 | University Of Georgia Research Foundation Inc. | Metal resistance sequences and transgenic plants |
CN107530454A (zh) * | 2015-01-30 | 2018-01-02 | 犹他大学研究基金会 | 二聚胶原杂交肽和使用方法 |
US20180000960A1 (en) * | 2015-01-30 | 2018-01-04 | University Of Utah Research Foundation | Dimeric collagen hybridizing peptides and methods of using |
US20200308253A1 (en) * | 2017-10-18 | 2020-10-01 | Swansea University | Single alpha chain collagens |
CN110129029A (zh) * | 2019-02-14 | 2019-08-16 | 兰州大学 | 一种电荷排斥作用诱导的单链胶原多肽功能探针及其制备方法 |
CN111057539A (zh) * | 2019-02-14 | 2020-04-24 | 兰州大学 | 一种电荷排斥作用诱导的单链胶原多肽探针及制备方法 |
US20230087644A1 (en) * | 2020-01-02 | 2023-03-23 | Lanzhou University | Probe for Specifically Detecting Pathological Collagens, and Preparation Method Therefor and Use Thereof |
CN111909245A (zh) * | 2020-08-05 | 2020-11-10 | 兰州大学 | 一种包含芳香族氨基酸的胶原靶向多肽探针,制备方法及其应用 |
CN115925988A (zh) * | 2022-09-14 | 2023-04-07 | 中山大学附属第五医院 | 一种变性胶原靶向抗菌肽及其制备方法与应用 |
Non-Patent Citations (3)
Title |
---|
JEONGMIN HWANG ET AL.: "Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide", 《ACTA BIOMATERIALIA》, vol. 53, pages 268 * |
JULIAN L. KESSLER ET AL.: "Enrichment of Collagen Fragments Using Dimeric Collagen Hybridizing Peptide for Urinary Collagenomics", 《J PROTEOME RES.》, vol. 19, no. 8, pages 2926 * |
魏文宇 等: "新型荧光环肽探针用于病变胶原蛋白的靶向检测", 《中国科学》, vol. 51, no. 4, pages 485 - 492 * |
Also Published As
Publication number | Publication date |
---|---|
CN116715753B (zh) | 2024-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU723268B2 (en) | Improved solid-phase peptide synthesis and agent for use in such synthesis | |
AU2006314708B2 (en) | Production of recombinant collagen like proteins | |
CA1220735A (en) | Molecular cloning and characterization of a further gene sequence coding for human relaxin | |
JP4423542B2 (ja) | 抗菌性ポリペプチド及びその利用 | |
JP2000503324A (ja) | 固体支持体上でのポリピロール及びポリイミダゾールカルボキサミドの合成方法 | |
WO2006020594A2 (en) | Cloning and expression of recombinant adhesive protein mefp-1 of the blue mussel, mytilus edulis | |
JPS5985292A (ja) | ヒト−レラキシン遺伝子 | |
Yang et al. | Biosynthesis and characterization of a non-repetitive polypeptide derived from silk fibroin heavy chain | |
Shah et al. | Structural consequences of D‐amino acids in collagen triple‐helical peptides | |
CN116715753B (zh) | 一种保持单链构象的类肽胶原杂交肽的制备方法与应用 | |
CN111973578A (zh) | C188-9、Venetoclax、Bumetanide在纤维化疾病药物中的应用 | |
EP1776463A2 (en) | Cloning and expression of recombinant adhesive protein mefp-2 of the blue mussel mytilus edulis | |
US10174083B2 (en) | Aza-amino acid collagen compounds | |
JP5743370B2 (ja) | 改善されたフォールディングの空気ガス処理によってインスリンを抽出する方法 | |
US5179082A (en) | Method for blocking platelet adhesion to collagen | |
JP5601437B2 (ja) | コラーゲン様構造を有するポリペプチド | |
WO2022016751A1 (zh) | 一种子宫内膜异位症分子诊疗制剂及其制备方法和应用 | |
US20070054298A1 (en) | Methods for enzyme-mediated coupling of oligomers | |
CN111607004A (zh) | 一种在胰蛋白酶酶切过程中选择性保护酶切位点的方法 | |
CN114075266A (zh) | 一种用于光热治疗的多肽组装单体及其制备方法和应用 | |
JP3129523B2 (ja) | 新規ペプチド及びその製造方法 | |
JP2007521316A (ja) | ペプチド合成方法 | |
Hidaka et al. | Advanced Insulin Synthesis by One‐pot/Stepwise Disulfide Bond Formation Enabled by S‐Protected Cysteine Sulfoxide | |
JP3770659B2 (ja) | 新規ペプチド及びその製造方法 | |
JPH02275899A (ja) | ポリペプチド合成 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |