CN113088281A - 检测β-淀粉样蛋白的荧光探针及其制备方法和应用 - Google Patents
检测β-淀粉样蛋白的荧光探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN113088281A CN113088281A CN202110272723.7A CN202110272723A CN113088281A CN 113088281 A CN113088281 A CN 113088281A CN 202110272723 A CN202110272723 A CN 202110272723A CN 113088281 A CN113088281 A CN 113088281A
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- beta
- amyloid beta
- probe
- detecting amyloid
- 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
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 43
- 102000013455 Amyloid beta-Peptides Human genes 0.000 title claims abstract description 30
- 108010090849 Amyloid beta-Peptides Proteins 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000000178 monomer Substances 0.000 claims abstract description 20
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 239000000835 fiber Substances 0.000 claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 78
- 239000000523 sample Substances 0.000 claims description 46
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- 210000004556 brain Anatomy 0.000 claims description 17
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 claims description 16
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 14
- UMLWXYJZDNNBTD-UHFFFAOYSA-N alpha-dimethylaminoacetophenone Natural products CN(C)CC(=O)C1=CC=CC=C1 UMLWXYJZDNNBTD-UHFFFAOYSA-N 0.000 claims description 12
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 10
- 230000002776 aggregation Effects 0.000 claims description 9
- 238000004220 aggregation Methods 0.000 claims description 9
- 238000011065 in-situ storage Methods 0.000 claims description 8
- 238000010898 silica gel chromatography Methods 0.000 claims description 8
- 150000001263 acyl chlorides Chemical class 0.000 claims description 7
- 229940125904 compound 1 Drugs 0.000 claims description 7
- 229940098779 methanesulfonic acid Drugs 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 5
- 238000003384 imaging method Methods 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 210000003462 vein Anatomy 0.000 claims description 4
- 238000009987 spinning Methods 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims 1
- 239000012615 aggregate Substances 0.000 abstract description 22
- 230000004044 response Effects 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 230000004931 aggregating effect Effects 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- JPBGLQJDCUZXEF-UHFFFAOYSA-N chromenylium Chemical class [O+]1=CC=CC2=CC=CC=C21 JPBGLQJDCUZXEF-UHFFFAOYSA-N 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 20
- 239000000243 solution Substances 0.000 description 20
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- JADVWWSKYZXRGX-UHFFFAOYSA-M thioflavine T Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C1=[N+](C)C2=CC=C(C)C=C2S1 JADVWWSKYZXRGX-UHFFFAOYSA-M 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000012043 crude product Substances 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 238000001704 evaporation Methods 0.000 description 6
- 239000012074 organic phase Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 208000024827 Alzheimer disease Diseases 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010029350 Neurotoxicity Diseases 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 206010044221 Toxic encephalopathy Diseases 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000007135 neurotoxicity Effects 0.000 description 3
- 231100000228 neurotoxicity Toxicity 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 238000010175 APPswe/PSEN1dE9 Methods 0.000 description 2
- 102000002659 Amyloid Precursor Protein Secretases Human genes 0.000 description 2
- 108010043324 Amyloid Precursor Protein Secretases Proteins 0.000 description 2
- 230000006933 amyloid-beta aggregation Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 1
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 1
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 210000005257 cortical tissue Anatomy 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 230000000971 hippocampal effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
-
- 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"
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Neurosurgery (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Physics & Mathematics (AREA)
- Pharmacology & Pharmacy (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Neurology (AREA)
- Hospice & Palliative Care (AREA)
- Optics & Photonics (AREA)
- Psychiatry (AREA)
- General Chemical & Material Sciences (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
技术领域
本发明涉及生物医药技术领域,具体涉及检测β-淀粉样蛋白的荧光探针及其制备方法和应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
淀粉样前体蛋白通过β-分泌酶、γ-分泌酶裂解产生含有38-43个氨基酸残基的β-淀粉样蛋白(Aβ)单体。Aβ单体自身不稳定,易于发生错误折叠而聚合形成大的聚集体或纤维,从而沉积在大脑皮层以及海马区形成Aβ斑块,产生神经毒性。早期研究表明,不溶性Aβ斑块产生神经毒性,与阿尔兹海默病的发生发展密切相关。近期研究表明,可溶性的Aβ单体和低聚体同样具有较高的神经毒性。目前发展的大多数荧光探针只能检测聚集程度高的Aβ斑块和纤维,而无法检测聚集程度低的Aβ单体和低聚体,也不能抑制Aβ单体聚集形成斑块或纤维。
发明内容
为了解决现有技术中存在的问题,本发明的目的是提供两种基于苯并吡喃鎓骨架的检测Aβ的荧光探针及其制备方法和应用,两种荧光探针分别命名为BP-2、BP-3。探针BP-2和BP-3均能够快速、高亲和性地响应Aβ单体、聚集体和纤维,对阿尔兹海默病小鼠脑部的Aβ斑块进行荧光成像检测;此外,两种探针均能够抑制β-淀粉样蛋白单体聚集形成斑块或纤维,具有对β-淀粉样蛋白相关疾病的潜在治疗效果。
为了实现上述目的,本发明的技术方案如下所述:
在本发明的第一方面,提供一种检测β-淀粉样蛋白的荧光探针,所述荧光探针的结构式如下:
在本发明的第二方面,提供一种第一方面所述荧光探针在抑制β-淀粉样蛋白单体聚集形成斑块或纤维的产品中的应用。
本发明的第三方面,提供了一种对活体动物脑部Aβ斑块进行荧光成像检测的方法,所述方法包括:
小鼠禁食后尾静脉注射探针BP-2或BP-3,用小动物活体成像系统或双光子激光共聚焦显微镜对小鼠脑部进行原位荧光成像检测;或者取出小鼠脑部做成组织切片,采用激光共聚焦显微镜进行荧光成像检测。
本发明的具体实施方式具有以下有益效果:
(1)探针的最大荧光发射波长约为650nm,位于近红外区,能够消除生物自发荧光的干扰,并具有较大的组织穿透深度,更适宜用于活体动物脑部的荧光成像;
(2)探针能够透过血脑屏障,可用于原位成像活体动物脑部的Aβ;
(3)探针对不同聚集状态的Aβ(包括单体、聚集体和纤维)均具有较高的亲和力,检测的底物范围广;
(4)探针能够抑制β-淀粉样蛋白单体聚集形成斑块或纤维,具有对β-淀粉样蛋白相关疾病的潜在治疗效果。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1是本发明所述荧光探针BP-2(图1a)和BP-3(图1b)对不同浓度Aβ聚集体的荧光响应光谱图,横坐标为波长(nm),纵坐标为荧光发射强度(a.u.)。
图2是本发明所述荧光探针BP-2(图2a)和BP-3(图2b)在650nm处的荧光强度与Aβ聚集体浓度之间的线性关系,横坐标为Aβ聚集体浓度(μM),纵坐标为650nm处的荧光发射强度(a.u.)。
图3是本发明所述荧光探针BP-2(图3a)和BP-3(图3b)对不同聚集状态的Aβ(单体、聚集体和纤维)的荧光响应光谱图;横坐标为波长(nm),纵坐标为荧光发射强度(a.u.)。
图4是本发明所述荧光探针BP-2(图4a)和BP-3(图4b)与Aβ聚集体之间的亲和力测试,横坐标为探针的浓度(μM),纵坐标为650nm处的荧光发射强度(a.u.)。
图5是本发明所述荧光探针对Aβ聚集的抑制作用荧光成像图。
图6是本发明所述荧光探针对17月龄APP/PS1基因型小鼠脑组织切片中Aβ斑块的荧光成像图。
图7是本发明所述荧光探针对15月龄APP/PS1基因型小鼠脑部Aβ斑块的原位荧光成像图。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本申请使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
本发明的一种实施方式中,提供了一种检测β-淀粉样蛋白的荧光探针,所述荧光探针的结构式如下:
在一种具体的实施方式中,上述荧光探针BP-2的制备方法,包括以下步骤:
将化合物BP-1溶于二氯甲烷溶液中,依次加入DMF溶液和草酰氯,得酰氯产物;酰氯产物的二氯甲烷溶液加进甲醇溶液中,即得BP-2;
优选的,草酰氯用二氯甲烷稀释后再滴加,得到的酰氯产物要快速旋干;
优选的,所述制备方法中还包括采用硅胶柱层析法进行提纯的步骤;
优选的,所述二氯甲烷溶液和DMF溶液均为超干溶液。
在一种具体的实施方式中,上述荧光探针BP-3的制备方法,包括以下步骤:将化合物1与N,N-二甲氨基苯乙酮溶于强酸溶剂中,然后加入高氯酸,即得BP-3。
优选的,所述强酸选自浓硫酸或甲烷磺酸,进一步优选为甲烷磺酸;
优选的,所述化合物1与N,N-二甲氨基苯乙酮的摩尔比为1:1.2;
所述荧光探针BP-2和BP-3的制备方法反应过程如下:
本发明的一种实施方式中,提供了一种上述荧光探针在抑制β-淀粉样蛋白单体聚集形成斑块或纤维的产品中的应用。
本发明的一种实施方式中,提供了一种对活体动物脑部Aβ斑块进行荧光成像检测的方法,所述方法包括:
小鼠禁食后尾静脉注射探针BP-2或BP-3,用小动物活体成像系统或双光子激光共聚焦显微镜对小鼠脑部进行原位荧光成像检测;或者取出小鼠脑部做成组织切片,采用激光共聚焦显微镜进行荧光成像检测。
优选的,所述禁食的时间为12h以上。
下面结合具体的实施例对本发明作进一步的解释和说明。
实施例1
荧光探针BP-2和BP-3的合成:
化合物BP-1(110mg,0.2mmol)溶于2mL超干二氯甲烷,向混合溶液中滴加2滴超干DMF,冰水浴下逐滴加入草酰氯(249μL,2mmol,用1mL超干二氯甲烷稀释),滴加完毕,将反应缓慢升至室温并反应2小时。反应结束后,快速旋干。
向反应瓶中加入5mL甲醇溶液,将上述旋干的反应液用5mL超干二氯甲烷溶解后,逐滴加进甲醇溶液中,室温反应10min。反应结束后,减压蒸发掉溶剂,得到的粗产物用硅胶柱层析法进行分离提纯,洗脱剂为二氯甲烷:甲醇=40:1(v:v),得到110.6mg探针BP-2(深紫色产物,产率为98%)。
氮气保护下,化合物1(178.7mg,0.664mmol)溶于5mL甲烷磺酸,加入N,N-二甲氨基苯乙酮(130mg,0.797mmol),100℃反应过夜,反应结束后冷却至室温,淬灭。加入1.5mL75%的高氯酸反应10min,用二氯甲烷和饱和氯化钠溶液萃取,用无水硫酸钠干燥有机相,过滤并减压蒸发掉有机相,得到的粗产物用硅胶柱层析法进行分离提纯,洗脱剂为二氯甲烷:甲醇=100:1(v:v),得到40mg探针BP-3(蓝紫色固体,产率为12%)。
核磁及质谱表征:
探针BP-2:1H NMR(400MHz,DMSO-d6):δ8.32(d,J=9.2Hz,2H),8.19(d,J=7.8Hz,1H),7.92(s,1H),7.88(t,J=7.6Hz,1H),7.79(t,J=7.7Hz,1H),7.57(d,J=7.6Hz,1H),7.32(d,J=2.0Hz,1H),7.16(dd,J=9.5,2.1Hz,1H),7.08(d,J=9.4Hz,1H),6.93(d,J=9.3Hz,2H),3.69-3.55(m,7H),3.19(s,6H),1.22(t,J=6.9Hz,6H)ppm.13C NMR(100MHz,DMSO-d6):δ166.26,165.56,160.31,157.45,154.83,154.04,136.06,133.06,131.15,130.55,130.34,130.12,128.91,128.65,115.82,114.85,114.08,112.50,108.84,96.37,52.78,52.47,44.99,12.43ppm.HRMS-ESI(m/z):calculated for C29H31N2O3 +[M]+455.2329,found 455.2347.
探针BP-3:1H NMR(400MHz,DMSO-d6):δ8.32(d,J=9.3Hz,2H),7.88(s,1H),7.73-7.66(m,5H),7.61(d,J=9.5Hz,1H),7.29(d,J=2.5Hz,1H),7.23(dd,J=9.5,2.5Hz,1H),6.90(d,J=9.4Hz,2H),3.63(q,J=6.9Hz,4H),3.17(s,6H),1.23(t,J=6.9Hz,6H)ppm.13CNMR(100MHz,DMSO-d6):δ166.39,158.34,158.00,154.73,153.91,134.62,131.13,131.02,129.50,129.45,129.05,115.76,114.97,112.80,112.33,108.74,96.62,44.96,12.45ppm.HRMS-ESI(m/z):calculated for C27H29N2O+[M]+397.2274;found 397.2281.
实施例2
荧光探针BP-2和BP-3的合成:
化合物BP-1(100mg,0.18mmol)溶于2mL超干二氯甲烷,向混合溶液中滴加2滴超干DMF,冰水浴下逐滴加入草酰氯(224μL,1.8mmol,用1mL超干二氯甲烷稀释),滴加完毕,将反应缓慢升至室温并反应2小时。反应结束后,快速旋干。
向反应瓶中加入4.5mL甲醇溶液,将上述旋干的反应液用3mL超干二氯甲烷溶解后,逐滴加进甲醇溶液中,室温反应15min。反应结束后,减压蒸发掉溶剂,得到的粗产物用硅胶柱层析法进行分离提纯,洗脱剂为二氯甲烷:甲醇=40:1(v:v),得到99.74mg探针BP-2(深紫色产物,产率为97.2%)。
氮气保护下,化合物1(100mg,0.372mmol)溶于3mL甲烷磺酸,加入N,N-二甲氨基苯乙酮(75mg,0.460mmol),100℃反应过夜,反应结束后冷却至室温,淬灭。加入1.5mL 75%的高氯酸反应10min,用二氯甲烷和饱和氯化钠溶液萃取,用无水硫酸钠干燥有机相,过滤并减压蒸发掉有机相,得到的粗产物用硅胶柱层析法进行分离提纯,洗脱剂为二氯甲烷:甲醇=100:1(v:v),得到20.11mg探针BP-3(蓝紫色固体,产率为12%)。
实施例3
荧光探针BP-2和BP-3的合成:
化合物BP-1(200mg,0.36mmol)溶于3mL超干二氯甲烷,向混合溶液中滴加2滴超干DMF,冰水浴下逐滴加入草酰氯(450μL,3.6mmol,用1mL超干二氯甲烷稀释),滴加完毕,将反应缓慢升至室温并反应2小时。反应结束后,快速旋干。
向反应瓶中加入5mL甲醇溶液,将上述旋干的反应液用5mL超干二氯甲烷溶解后,逐滴加进甲醇溶液中,室温反应15min。反应结束后,减压蒸发掉溶剂,得到的粗产物用硅胶柱层析法进行分离提纯,洗脱剂为二氯甲烷:甲醇=40:1(v:v),得到202.35mg探针BP-2(深紫色产物,产率为98.6%)。
氮气保护下,化合物1(200mg,0.744mmol)溶于4mL甲烷磺酸,加入N,N-二甲氨基苯乙酮(150mg,0.920mmol),100℃反应过夜,反应结束后冷却至室温,淬灭。加入2mL 75%的高氯酸反应10min,用二氯甲烷和饱和氯化钠溶液萃取,用无水硫酸钠干燥有机相,过滤并减压蒸发掉有机相,得到的粗产物用硅胶柱层析法进行分离提纯,洗脱剂为二氯甲烷:甲醇=100:1(v:v),得到48.3mg探针BP-3(蓝紫色固体,产率为13.1%)。
效果实验
探针对Aβ聚集体的荧光响应实验
用1.0μM的探针BP-2对0-20μM的Aβ聚集体进行荧光响应测试,得到探针BP-2的荧光强度和Aβ聚集体浓度变化的关系;实验条件:λex/λem=615/650nm,50mM PBS,pH 7.4,室温下立即检测。同样的用1.0μM的探针BP-3对0-20μM的Aβ聚集体进行荧光响应测试。测试效果如图1,横坐标为波长(nm),纵坐标为荧光强度(a.u.)。可以看出,随着Aβ聚集体浓度的增大,探针BP-2(图1a)和探针BP-3(图1b)在650nm处的荧光强度逐渐增强,说明本发明所述两种探针可以用于Aβ聚集体的检测。此外,如图2所示,探针BP-2(图2a)和探针BP-3(图2b)在650nm处的荧光强度与Aβ聚集体的浓度有良好的线性关系,根据公式3σ/k计算出二者的检测限分别为168nM和80nM。
采用与上述相同的实验方法研究了探针对不同聚集状态Aβ的荧光响应。如图3所示,探针BP-2(图3a)和探针BP-3(图3b)对不同聚集状态的Aβ,包括单体、聚集体以及纤维均有明显的荧光增强。
探针对Aβ聚集体的亲和力测试
分别向5μM的Aβ聚集体中滴加不同浓度的探针BP-2或BP-3,采集650nm处的荧光强度,如图4所示。经计算得到探针BP-2(图4a)和BP-3(图4b)的Kd值分别为0.310μM和0.270μM。表明探针BP-2和BP-3与Aβ聚集体具有较高的亲和力。
探针对Aβ聚集的抑制作用
实验组中分别加入20μM的BP-2或BP-3或硫磺素T(ThT),以及40μM的Aβ单体,对照组中加入等体积的DMSO和40μM的Aβ单体。在摇床中37℃下震荡孵育3天。在对照组中分别加入20μM的BP-2或BP-3或ThT,用激光共聚焦显微镜进行荧光成像。如图5所示,探针BP-2和BP-3的实验组(Exp)中Aβ聚集体明显小于相应的对照组(Con),而ThT的实验组与对照组中Aβ聚集体大小无明显区别。表明探针BP-2和BP-3均能够抑制Aβ单体聚集形成斑块,而ThT没有抑制效果。
探针对小鼠脑切片中Aβ斑块的荧光成像
分别用5μM的BP-2或BP-3对17月龄的APP/PS1基因型小鼠的脑组织切片进行染色,10min后吸去探针,用去离子水冲洗,再用5μM的ThT进行染色,10min后冲洗,用激光共聚焦显微镜进行成像。如图6所示,探针BP-2和BP-3的染色斑块均与ThT的染色斑块完全重叠,说明本发明所述两种探针均能对阿尔茨海默病小鼠脑切片中Aβ斑块进行荧光成像检测。
探针BP-3对小鼠脑部Aβ斑块的原位荧光成像
将15月龄的基因型小鼠禁食12h以上,尾静脉注射探针BP-3 100μL(8mg/kg体重),去除头部皮层组织,裸露出头骨,用双光子激光共聚焦显微镜对不同深度脑组织中Aβ斑块进行原位荧光成像,如图7所示,说明本发明所述探针能够原位成像阿尔茨海默病小鼠脑部的Aβ斑块。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
3.如权利要求2所述的检测β-淀粉样蛋白的荧光探针,其特征在于:荧光探针BP-2的制备方法包括如下步骤:
将化合物BP-1溶于二氯甲烷溶液中,依次加入DMF溶液和草酰氯,得酰氯产物;酰氯产物的二氯甲烷溶液加进甲醇溶液中,即得BP-2。
4.如权利要求3所述的检测β-淀粉样蛋白的荧光探针,其特征在于:所述草酰氯用二氯甲烷稀释后再滴加,得到的酰氯产物要快速旋干;
优选的,所述二氯甲烷溶液和DMF溶液均为超干溶液。
5.如权利要求3所述的检测β-淀粉样蛋白的荧光探针,其特征在于:所述制备方法中还包括采用硅胶柱层析法进行提纯的步骤。
6.如权利要求2所述的检测β-淀粉样蛋白的荧光探针,其特征在于:荧光探针BP-3的制备方法包括如下步骤:
将化合物1与N,N-二甲氨基苯乙酮溶于强酸溶剂中,然后加入高氯酸,即得BP-3。
7.如权利要求6所述的检测β-淀粉样蛋白的荧光探针,其特征在于:所述强酸选自浓硫酸或甲烷磺酸,优选为甲烷磺酸;
优选的,所述化合物1与N,N-二甲氨基苯乙酮的摩尔比为1:1.2。
8.权利要求1或2所述检测β-淀粉样蛋白的荧光探针在抑制β-淀粉样蛋白单体聚集形成斑块或纤维方面的应用。
9.权利要求1或2所述检测β-淀粉样蛋白的荧光探针对活体动物脑部Aβ斑块进行荧光成像检测的应用。
10.如权利要求9所述的应用,其特征在于:小鼠禁食后尾静脉注射探针BP-2或BP-3,用小动物活体成像系统或双光子激光共聚焦显微镜对小鼠脑部进行原位荧光成像检测;或者取出小鼠脑部做成组织切片,采用激光共聚焦显微镜进行荧光成像检测;
优选的,所述禁食的时间为12h以上。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110272723.7A CN113088281B (zh) | 2021-03-13 | 2021-03-13 | 检测β-淀粉样蛋白的荧光探针及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110272723.7A CN113088281B (zh) | 2021-03-13 | 2021-03-13 | 检测β-淀粉样蛋白的荧光探针及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113088281A true CN113088281A (zh) | 2021-07-09 |
CN113088281B CN113088281B (zh) | 2022-07-29 |
Family
ID=76667592
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110272723.7A Expired - Fee Related CN113088281B (zh) | 2021-03-13 | 2021-03-13 | 检测β-淀粉样蛋白的荧光探针及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113088281B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112525868A (zh) * | 2019-09-19 | 2021-03-19 | 吉林大学 | 喹哪啶红及其衍生物在制备检测淀粉样纤维的试剂盒中的应用 |
CN113773831A (zh) * | 2021-08-25 | 2021-12-10 | 湖北大学 | 一种检测淀粉样蛋白聚集体的荧光探针及其制备方法和应用 |
CN114874198A (zh) * | 2022-06-14 | 2022-08-09 | 黄淮学院 | 一种基于罗丹明-铜配合物的Aβ荧光探针及其应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110208064A1 (en) * | 2008-07-31 | 2011-08-25 | Ran Chongzhao | Curcumin Derivatives for Amyloid-Beta Plaque Imaging |
CN102279270A (zh) * | 2011-04-27 | 2011-12-14 | 商丘师范学院 | 利用聚集诱导发光监测β淀粉样蛋白聚集过程的方法 |
WO2017211220A1 (zh) * | 2016-06-07 | 2017-12-14 | 四川大学 | 用于荧光标记β-淀粉样斑块和/或神经纤维缠结的化合物、制备方法和应用以及一种药物组合物 |
CN109946280A (zh) * | 2019-03-29 | 2019-06-28 | 南京工业大学 | 一种用比例计量型荧光探针定量检测可溶性β淀粉样蛋白的方法 |
CN111777603A (zh) * | 2020-06-15 | 2020-10-16 | 山东师范大学 | 一种可同时检测β淀粉样蛋白沉积斑块和过氧亚硝酸根的荧光探针及其制备方法和应用 |
CN111875560A (zh) * | 2020-07-09 | 2020-11-03 | 山东师范大学 | 一种比率型双光子荧光探针及其制备方法和应用 |
-
2021
- 2021-03-13 CN CN202110272723.7A patent/CN113088281B/zh not_active Expired - Fee Related
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110208064A1 (en) * | 2008-07-31 | 2011-08-25 | Ran Chongzhao | Curcumin Derivatives for Amyloid-Beta Plaque Imaging |
CN102279270A (zh) * | 2011-04-27 | 2011-12-14 | 商丘师范学院 | 利用聚集诱导发光监测β淀粉样蛋白聚集过程的方法 |
WO2017211220A1 (zh) * | 2016-06-07 | 2017-12-14 | 四川大学 | 用于荧光标记β-淀粉样斑块和/或神经纤维缠结的化合物、制备方法和应用以及一种药物组合物 |
CN109946280A (zh) * | 2019-03-29 | 2019-06-28 | 南京工业大学 | 一种用比例计量型荧光探针定量检测可溶性β淀粉样蛋白的方法 |
CN111777603A (zh) * | 2020-06-15 | 2020-10-16 | 山东师范大学 | 一种可同时检测β淀粉样蛋白沉积斑块和过氧亚硝酸根的荧光探针及其制备方法和应用 |
CN111875560A (zh) * | 2020-07-09 | 2020-11-03 | 山东师范大学 | 一种比率型双光子荧光探针及其制备方法和应用 |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112525868A (zh) * | 2019-09-19 | 2021-03-19 | 吉林大学 | 喹哪啶红及其衍生物在制备检测淀粉样纤维的试剂盒中的应用 |
CN113773831A (zh) * | 2021-08-25 | 2021-12-10 | 湖北大学 | 一种检测淀粉样蛋白聚集体的荧光探针及其制备方法和应用 |
CN114874198A (zh) * | 2022-06-14 | 2022-08-09 | 黄淮学院 | 一种基于罗丹明-铜配合物的Aβ荧光探针及其应用 |
CN114874198B (zh) * | 2022-06-14 | 2023-08-25 | 黄淮学院 | 一种基于罗丹明-铜配合物的Aβ荧光探针及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN113088281B (zh) | 2022-07-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113088281B (zh) | 检测β-淀粉样蛋白的荧光探针及其制备方法和应用 | |
CN112028915B (zh) | 一种荧光探针及其合成及应用 | |
CN108059638B (zh) | 一类标记淀粉样蛋白的荧光探针及其制备方法与应用 | |
CN108191789B (zh) | 一种吩噻嗪衍生物、其制备方法和应用 | |
EP2778161B1 (en) | Two-photon fluorescent probe using naphthalene as matrix and preparation method and use thereof | |
CN109180556B (zh) | 一种部花菁类荧光化合物及其制备方法和应用 | |
CN112574239B (zh) | 3-噻唑烯基氟化硼络合二吡咯甲川类化合物及其制备方法和用途 | |
CN113651834A (zh) | 基于双噻吩并苯衍生物的荧光探针及其在细胞脂滴成像中的应用 | |
CN106867512A (zh) | 一种检测抗生物素蛋白的比率型荧光探针及其合成方法和应用 | |
CN111675696A (zh) | 一种基于三氮唑活性分子的光亲合探针分子及其制备方法和应用 | |
CN114315880B (zh) | 一类基于氟硼配合物的近红外二区荧光和光声双模态成像试剂 | |
CN114426534B (zh) | 一种检测铜离子的可逆荧光探针及其制备方法 | |
CN113666937B (zh) | 一种用于检测锌离子的近红外荧光探针及其制备方法和应用 | |
WO2023097820A1 (zh) | 黄酮醇类化合物及制备方法与其在检测生物硫醇中的应用 | |
CN111777603B (zh) | 一种可同时检测β淀粉样蛋白沉积斑块和过氧亚硝酸根的荧光探针及其制备方法和应用 | |
CN114656447A (zh) | 一类基于高时空分辨率的近红外荧光、磁共振Aβ双模式成像探针及其制备方法和应用 | |
CN105985770A (zh) | 一种硫化氢荧光探针的制备及应用 | |
CN108047145A (zh) | 与Tau蛋白具有亲和力的2-芳基喹喔啉类化合物及其制备方法与应用 | |
Sun et al. | Designing enhanced and ratiometric probes for detecting OCl− based on substituents influencing the fluorescence of HBT and their application in strips and bioimaging | |
CN111704570A (zh) | 具有七甲川花菁结构的近红外反应型荧光探针及其制备方法和应用 | |
CN114380856B (zh) | 用于脑部硫化氢检测的硅罗丹明衍生物及制备方法与应用 | |
CN110563702B (zh) | 近红外荧光化合物与制备方法及其检测亚铁离子的应用 | |
CN113880821B (zh) | 一类双光子荧光探针对癫痫脑内次氯酸特征成像的荧光探针设计及其合成方法 | |
CN114478612B (zh) | 一种基于硅罗丹明的脑部次氯酸检测荧光探针及其制备方法和应用 | |
CN113527347B (zh) | 近红外荧光标记脂肪酸及其制备方法 |
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 | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220729 |
|
CF01 | Termination of patent right due to non-payment of annual fee |