CN115124557A - 基于苝酰亚胺的fret乏氧酶荧光探针、其制备方法和应用 - Google Patents
基于苝酰亚胺的fret乏氧酶荧光探针、其制备方法和应用 Download PDFInfo
- Publication number
- CN115124557A CN115124557A CN202210510225.6A CN202210510225A CN115124557A CN 115124557 A CN115124557 A CN 115124557A CN 202210510225 A CN202210510225 A CN 202210510225A CN 115124557 A CN115124557 A CN 115124557A
- Authority
- CN
- China
- Prior art keywords
- hypoxic
- fret
- reaction
- enzyme
- perylene
- 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.)
- Pending
Links
- 206010021143 Hypoxia Diseases 0.000 title claims abstract description 36
- 230000001146 hypoxic effect Effects 0.000 title claims abstract description 31
- 238000002866 fluorescence resonance energy transfer Methods 0.000 title claims abstract description 23
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 22
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 22
- CSHWQDPOILHKBI-UHFFFAOYSA-N peryrene Natural products C1=CC(C2=CC=CC=3C2=C2C=CC=3)=C3C2=CC=CC3=C1 CSHWQDPOILHKBI-UHFFFAOYSA-N 0.000 title claims abstract description 21
- 125000002080 perylenyl group Chemical group C1(=CC=C2C=CC=C3C4=CC=CC5=CC=CC(C1=C23)=C45)* 0.000 title claims abstract description 18
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 11
- -1 perylene imide Chemical class 0.000 claims abstract description 10
- 102000016680 Dioxygenases Human genes 0.000 claims abstract description 3
- 108010028143 Dioxygenases Proteins 0.000 claims abstract description 3
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 28
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 206010028980 Neoplasm Diseases 0.000 claims description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- QPQKUYVSJWQSDY-CCEZHUSRSA-N 4-(phenylazo)aniline Chemical class C1=CC(N)=CC=C1\N=N\C1=CC=CC=C1 QPQKUYVSJWQSDY-CCEZHUSRSA-N 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- 230000007954 hypoxia Effects 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 150000002460 imidazoles Chemical class 0.000 claims description 3
- 238000003333 near-infrared imaging Methods 0.000 claims description 3
- CRWVOXFUXPYTRK-UHFFFAOYSA-N pent-4-yn-1-ol Chemical compound OCCCC#C CRWVOXFUXPYTRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 3
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 3
- 229960005055 sodium ascorbate Drugs 0.000 claims description 3
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 3
- GTDPSWPPOUPBNX-UHFFFAOYSA-N ac1mqpva Chemical compound CC12C(=O)OC(=O)C1(C)C1(C)C2(C)C(=O)OC1=O GTDPSWPPOUPBNX-UHFFFAOYSA-N 0.000 claims description 2
- 150000003973 alkyl amines Chemical class 0.000 claims description 2
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 14
- 125000000751 azo group Chemical group [*]N=N[*] 0.000 abstract description 10
- 239000000463 material Substances 0.000 abstract description 10
- 230000004044 response Effects 0.000 abstract description 7
- 102100022365 NAD(P)H dehydrogenase [quinone] 1 Human genes 0.000 abstract description 5
- 108010066657 azoreductase Proteins 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 230000009977 dual effect Effects 0.000 abstract description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 15
- 230000007246 mechanism Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 101000734336 Arabidopsis thaliana Protein disulfide isomerase-like 1-2 Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 102000004960 NAD(P)H dehydrogenase (quinone) Human genes 0.000 description 1
- 108020000284 NAD(P)H dehydrogenase (quinone) Proteins 0.000 description 1
- 102000004459 Nitroreductase Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108091007187 Reductases Proteins 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- JIBVNTPFRXKPSQ-UHFFFAOYSA-N dodecan-6-amine Chemical compound CCCCCCC(N)CCCCC JIBVNTPFRXKPSQ-UHFFFAOYSA-N 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108020001162 nitroreductase Proteins 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- KJOLVZJFMDVPGB-UHFFFAOYSA-N perylenediimide Chemical compound C=12C3=CC=C(C(NC4=O)=O)C2=C4C=CC=1C1=CC=C2C(=O)NC(=O)C4=CC=C3C1=C42 KJOLVZJFMDVPGB-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- 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
-
- 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
-
- 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
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
-
- 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
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1055—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with other heteroatoms
-
- 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
- C09K2211/1059—Heterocyclic compounds characterised by ligands containing three nitrogen atoms as heteroatoms
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Materials Engineering (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本案涉及一种基于苝酰亚胺的FRET乏氧酶荧光探针、其制备方法及应用，该材料以苝酰亚胺为主体发光单元、偶氮衍生物作为修饰基团、BODIPY作为分子内能量给体，构筑分子内FRET分子，具有如下结构式：
其中，R1为H或‑N((CH2)mCH3)2，m为2～10的整数；R2为长链烷基。本发明所制备的乏氧酶材料首次把偶氮基团修饰到苝酰亚胺衍生物上，通过简要的步骤引入到苝酰亚胺上、制备了光稳定性好、对乏氧酶选择性识别明显、抗干扰能力强的系列乏氧酶苝酰亚胺类荧光材料，针对细胞中的偶氮还原酶有强烈的吸收和荧光响应，可实现对肿瘤细胞中的乏氧酶实现双重信号检测。
Description
技术领域
本发明涉及荧光探针技术领域，具体涉及一种基于苝酰亚胺的FRET乏氧酶荧光探针、其制备方法和应用。
背景技术
荧光成像技术由于具有可视化、原位、无损等优点，广泛应用于细胞及活体生物成像分析。常用的荧光探针中，小分子荧光探针因其具有容易合成与修饰、光谱易调节、荧光量子产率高、生物相容性良好等优点，可通过各种不同的原理(比如PET机理、ICT机理和FRET机理)构建灵敏度高、选择性好、响应快的荧光探针，从而实现对特定目标物质的响应性识别。
乏氧是绝大部分实体瘤中的普遍现象，乏氧可以诱导加速生物还原反应和导致细胞内还原酶的表达，如醌还原酶，偶氮还原酶(NZR)以及硝基还原酶。其中，偶氮还原酶是最具代表性的，这种酶催化还原偶氮为氨基反应被证实为一种有效的定位和乏氧成像原理。目前基于这NZR机理的识别研究有所报道，然而，相关研究大都基于罗丹明和花氰类染料的单分子荧光响应，而基于分子内FRET兼具偶氮识别机理的研究相对欠缺。
发明内容
针对现有技术中的不足之处，本发明提供了一种分子内FRET兼具偶氮识别的荧光探针，该荧光探针可用于对肿瘤乏氧近红外成像。
为实现上述目的，本发明提供如下技术方案：
一种基于苝酰亚胺的FRET乏氧酶荧光探针，该材料以苝酰亚胺为主体发光单元、偶氮衍生物作为修饰基团、BODIPY作为分子内能量给体，构筑分子内FRET分子，具有如下结构式：
其中，R1为H或-N((CH2)mCH3)2，m为2～10的整数；R2为长链烷基。
本发明第二目的是提供一种如上所述的基于苝酰亚胺的FRET乏氧酶荧光探针的制备方法，包括如下步骤：
S1：向反应瓶中加入摩尔比为1:1的1-溴苝四甲二酸酐、氨基化合物，乙醇做溶剂升温到85℃油浴锅中反应48小时，反应结束后，分离提纯得中间体1；
S2：向反应瓶中加入中间体1和4-氨基偶氮苯衍生物，加入冰醋酸/咪唑，升温到125-135℃反应8小时，反应结束后，分离提纯得中间体2；
S3：氮气保护下，将中间体2、5-羟基戊炔和碳酸钾溶解在乙二醇单甲醚中加热回流12小时，待反应冷却到室温后将水加入反应液中，有大量黄色固体生成，用二氯甲烷/石油醚柱层析得中间体3；
S4：向反应瓶中加入中间体3、带有叠氮基团的BODIPY衍生物、抗坏血酸钠和硫酸铜溶液，滴加三乙胺，室温下搅拌48h，反应结束后，用二氯甲烷/甲醇分离提纯得最终产物，即得。
进一步地，所述氨基化合物为C4～C20的长链烷基胺；所述4-氨基偶氮苯衍生物的4’号位为H或-N((CH2)mCH3)2基团。
本发明第三目的提供如上所述的基于苝酰亚胺的FRET乏氧酶荧光探针在对肿瘤乏氧近红外成像中的应用。
本发明首次选择苝酰亚胺衍生物作为发光主体，通过化学修饰在苝的酰胺引入偶氮基团以调整分子的发光性能，在苝的弯位引入BODIPY能量受体基团，构筑具有分子内FRET性质的乏氧酶识别基团，通过开关响应和FRET双重作用实现对乏氧酶的精准识别。探针分子与乏氧酶作用，偶氮基团被氧化，从而影响分子主体发光强度发生强烈的变化，通过这种选择性识别的变化进而表达细胞或者组织内乏氧酶的浓度，从而实现对生物体内的癌变情况的诊断。
与现有技术相比，本发明的有益效果是：本发明所制备的乏氧酶材料首次把偶氮基团修饰到苝酰亚胺衍生物上，通过简要的步骤引入到苝酰亚胺上、制备了光稳定性好、对乏氧酶选择性识别明显、抗干扰能力强的系列乏氧酶苝酰亚胺类荧光材料，针对细胞中的偶氮还原酶有强烈的吸收和荧光响应，可实现对肿瘤细胞中的乏氧酶实现双重信号检测。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为探针分子PDI-8的紫外吸收谱图。
图2为探针分子PDI-8对乏氧酶的识别增强荧光光谱图。
图3为探针分子PDI-8对乏氧酶的选择性识别柱状图(1-Na+、2-K+、3-Ca2+、4-Mg2+、5-H202、6-GSH、7-DTT、8-Arg、9-Try、10-Cys、11-His、12-Leu、13-Glu、14-SOD、15-HSA、16-BSA、17-GOx、18-AZR)。
图4为探针分子PDI-8用于肝细胞的荧光成像图(4a-探针分子与正常细胞的成像图，4b-非乏氧条件下探针分子与癌细胞的成像图，4c-乏氧条件下探针分子与乏氧细胞成像图)。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
此外，下面所描述的本发明不同实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互结合。
实施例1：
步骤1：
氮气保护下，向250mL两口瓶中，加入化合物1-溴苝四甲酸酐PDI-1 3.2g(12mmol)，6-氨基十二烷1.8g(12mmol)，无水乙醇120ml，85℃油浴锅中反应24小时。通过薄层层析板检测反应进程，反应结束后减压去除溶剂，将反应液倒入150ml冰水中，用稀盐酸调节pH至弱酸性，减压抽滤，乙醇重结晶，得化合物PDI-2，8.3g，产率：60％。δ8.95(s,1H),8.35-8.29(d,J＝9.0Hz,3H),8.20-8.16(d,J＝9.0Hz,3H),3.24(m,12H),1.54-1.30(m,8H),0.92(m,6H)。
步骤2
氮气保护下，将化合物PDI-2(4g，6.3mmol)、4-氨基偶氮苯(1.9g，7mmol)，溶于100ml冰醋酸/咪唑中然后缓慢升温到125-130℃持续保温36小时，色谱检测跟踪反应进程，反应结束后将反应液倒入50ml冰水中，大量红色固体析出，减压抽滤、干燥，用二氯甲烷/甲醇做流动相柱层析得一红色固体PDI-3 4.6g，产率：79％。1H NMR(300MHz,CDCl3):δ8.95(s,1H),8.35-8.29(d,J＝9.0Hz,3H),8.20-8.16(d,J＝9.0Hz,3H),8.02-7.96(d,J＝9.0Hz,4H),7.36-7.33(d,J＝9.0Hz,4H),4.26(m,2H),3.24(m,1H),2.88-1.42(m,24H),1.44-1.30(m,8H),1.24(m,12H)。
步骤3：
氮气保护下，将化合物PDI-3(5g,5.4mmol)、5-羟基戊炔1g,11mmol)、碳酸钾(4g)溶解在100mL乙二醇单甲醚中加热回流12小时。待反应冷却到室温后将100mL水加入反应液中，有大量黄色固体生成，二氯甲烷/石油醚柱层析得一红色固体3g。产率：62％。1H NMR(300MHz,CDCl3):δ8.95(s,1H),8.35-8.29(d,J＝9.0Hz,3H),8.20-8.16(d,J＝9.0Hz,3H),8.02-7.96(d,J＝9.0Hz,4H),7.36-7.33(d,J＝9.0Hz,4H),4.26(m,2H),4.16(m,2H),3.24(m,1H),2.93(s,1H),2.46(m,2H),1.88-1.42(m,26H),1.44-1.30(m,8H),1.24(m,12H)。
步骤4：
氮气保护下，向含有化合物BODIPY-1(1.1mmol,0.5g)、PDI-4(0.9mmol,0.8g)、抗坏血酸钠(0.023mmol,0.0045g)和CuSO4(0.019mmol,0.003g)的氯仿/乙醇/H2O(v/v/v＝8:2:1)溶液滴加三乙胺1mL，室温搅拌48小时。反应完成后向减压除去溶剂，抽滤得红色固体，用二氯甲烷/甲醇(1:5)柱层析PDI-51.1g，产率：90％。1H NMR(300MHz,CDCl3):δ8.95(s,1H),8.35-8.29(d,J＝9.0Hz,3H),8.20-8.16(d,J＝9.0Hz,3H),8.02-7.96(d,J＝9.0Hz,4H),7.52-7.50(d,J＝6.0Hz,2H),7.36-7.33(d,J＝9.0Hz,4H),6.93-6.91(d,J＝6.0Hz,2H),6.02(s,2H),4.45-4.05(m,4H),4.26(m,2H),2.88-1.42(m,30H),1.64(s,6H),1.44-1.30(m,8H),1.24(m,12H)。
实施例2：
得一红色固体PDI-3 3.0g，产率：78％。1H NMR(300MHz,CDCl3):δ8.95(s,1H),8.35-8.29(d,J＝9.0Hz,3H),8.20-8.16(d,J＝9.0Hz,3H),8.02-7.96(d,J＝9.0Hz,4H),7.36-7.33(d,J＝9.0Hz,4H),4.26(m,2H),3.24(m,1H),2.88-1.42(m,22H),1.44-1.30(m,4H),1.24(m,6H)。
步骤2：
有大量黄色固体生成。产率：84％。1H NMR(300MHz,CDCl3):δ8.95(s,1H),8.35-8.29(d,J＝9.0Hz,3H),8.20-8.16(d,J＝9.0Hz,3H),8.02-7.96(d,J＝9.0Hz,4H),7.36-7.33(d,J＝9.0Hz,4H),4.26(m,2H),4.16(m,2H),3.24(m,1H),2.93(s,1H),2.46(m,2H),1.88-1.42(m,22H),1.44-1.30(m,4H),1.24(m,6H)。
步骤3：
得红色固体PDI-5 4.6g,产率：90％。1H NMR(300MHz,CDCl3):δ8.95(s,1H),8.35-8.29(d,J＝9.0Hz,3H),8.20-8.16(d,J＝9.0Hz,3H),8.02-7.96(d,J＝9.0Hz,4H),7.52-7.50(d,J＝6.0Hz,2H),7.36-7.33(d,J＝9.0Hz,4H),6.93-6.91(d,J＝6.0Hz,2H),6.02(s,2H),4.45-4.05(m,4H),4.26(m,2H),2.88-1.42(m,22H),1.64(s,6H),1.44-1.30(m,4H),1.24(m,6H)。
对实施例1制得的材料进行荧光和紫外光谱测试：将材料配置成10-5～10-6mol/L浓度的二氯甲烷溶液，采用紫外吸收光谱仪和荧光光谱仪分别测定材料是吸收光谱和发射光谱，如图1-2所示。
本发明设计的反应条件温和、反应高效、操作简便、适用性广，为丰富和开发红外乏氧酶检测提供了新的思路。
实施例3：荧光识别实验：
2μg/mL的荧光探针PDI-8在10mM PBS(pH＝7.4)溶液中分别与金属离子(Na+、K+、Ca2+、Mg2+，2mM)、氧化还原分子(AA、H2O2、GSH、DTT，2mM)、氨基酸(Arg、Try、Cys、His、Leu、Glu，2mM)和蛋白质(BSA、HSA，2mg/mL；SOD、GOx，2μg/mL)以及2μM的AZR在37℃反应半小时，然后检测其荧光变化情况。从图3可以发现，除AZR外，其它溶液的荧光信号都没有发生明显变化。结果表明，探针CHR-C对乏氧相关偶氮还原酶具有特异响应能力。
实施例4：荧光细胞成像实验：
本发明进一步考察了PDI-8用于细胞荧光成像的研究。35mm的培养皿接种癌细胞后，分别在1％氧气和常氧环境中孵育24小时，然后与2μg/mL探针PDI-8反应一段时间并用AZR进行细胞核染色，最后用共聚焦荧光显微镜进行成像研究，以未用探针处理的细胞作为对照，激发波长为405nm。如图4所示，不管常氧还是乏氧状态的细胞，当没有与探针PDI-8反应时，405nm激光照射后不能观测到红光(图4a)；乏氧状态的细胞当没有与探针结合时，只有很暗的红光图4b)；而当乏氧状态下的细胞与乏氧探针反应后再用激光进行照射时，则能观察到很明显的红色荧光(图4c)。结果表明本案构建的探针确实能够用于乏氧成像。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的图例。
Claims (5)
1.一种基于苝酰亚胺的FRET乏氧酶荧光探针，其特征在于，该材料以苝酰亚胺为主体发光单元、偶氮衍生物作为修饰基团、BODIPY作为分子内能量给体，构筑分子内FRET分子，具有如下结构式：
其中，R1为H或-N((CH2)mCH3)2，m为2～10的整数；R2为长链烷基。
2.如权利要求1所述的基于苝酰亚胺的FRET乏氧酶荧光探针的制备方法，其特征在于，包括如下步骤：
S1：向反应瓶中加入摩尔比为1:1的1-溴苝四甲二酸酐、氨基化合物，乙醇做溶剂升温到85℃油浴锅中反应48小时，反应结束后，分离提纯得中间体1；
S2：向反应瓶中加入中间体1和4-氨基偶氮苯衍生物，加入冰醋酸/咪唑，升温到125-135℃反应8小时，反应结束后，分离提纯得中间体2；
S3：氮气保护下，将中间体2、5-羟基戊炔和碳酸钾溶解在乙二醇单甲醚中加热回流12小时，待反应冷却到室温后将水加入反应液中，有大量黄色固体生成，用二氯甲烷/石油醚柱层析得中间体3；
S4：向反应瓶中加入中间体3、带有叠氮基团的BODIPY衍生物、抗坏血酸钠和硫酸铜溶液，滴加三乙胺，室温下搅拌48h，反应结束后，用二氯甲烷/甲醇分离提纯得最终产物，即得。
3.如权利要求2所述的基于苝酰亚胺的FRET乏氧酶荧光探针的制备方法，其特征在于，所述氨基化合物为C4～C20的长链烷基胺。
4.如权利要求2所述的基于苝酰亚胺的FRET乏氧酶荧光探针的制备方法，其特征在于，所述4-氨基偶氮苯衍生物的4’号位为为H或-N((CH2)mCH3)2基团。
5.如权利要求1所述的基于苝酰亚胺的FRET乏氧酶荧光探针在对肿瘤乏氧近红外成像中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210510225.6A CN115124557A (zh) | 2022-05-11 | 2022-05-11 | 基于苝酰亚胺的fret乏氧酶荧光探针、其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210510225.6A CN115124557A (zh) | 2022-05-11 | 2022-05-11 | 基于苝酰亚胺的fret乏氧酶荧光探针、其制备方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115124557A true CN115124557A (zh) | 2022-09-30 |
Family
ID=83376865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210510225.6A Pending CN115124557A (zh) | 2022-05-11 | 2022-05-11 | 基于苝酰亚胺的fret乏氧酶荧光探针、其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115124557A (zh) |
-
2022
- 2022-05-11 CN CN202210510225.6A patent/CN115124557A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhou et al. | An APN-activated NIR photosensitizer for cancer photodynamic therapy and fluorescence imaging | |
| CN107603269B (zh) | 一类基于萘酰亚胺的荧光染料、其制备方法及应用 | |
| CN111303102B (zh) | 一种硝基还原酶响应的乏氧探针化合物及其制备与应用 | |
| CN110498799B (zh) | 一种荧光探针及其制备方法和用途 | |
| WO2015097262A1 (en) | Fluorescent red emitting functionalizable ph probes | |
| CN110498758B (zh) | 用于识别谷胱甘肽的近红外荧光探针及其制备和应用 | |
| Yang et al. | Rosamine with pyronine-pyridinium skeleton: unique mitochondrial targetable structure for fluorescent probes | |
| CN113200940B (zh) | 一种Aβ斑块响应型荧光探针及其制备和应用 | |
| Wu et al. | Crown-ether-bridging bis-diphenylacrylonitrile macrocycle: The effective fluorescence sensor for oxytetracycline | |
| CN108299438B (zh) | pH响应性近红外荧光探针化合物及其制备方法和应用 | |
| CN111732581A (zh) | 一种检测次氯酸/次氯酸钠的荧光探针及制备方法和应用 | |
| Ni et al. | Convenient construction of fluorescent markers for lipid droplets with 1, 8-naphthalimide unit | |
| WO2013131235A1 (zh) | 一类以萘为母体的双光子荧光探针、其制备方法及应用 | |
| Zhu et al. | Modulation of ICT and PET processes in boranil derivatives: a ratiometric fluorescent probe for imaging of cysteine | |
| Shen et al. | A mitochondria-oriented fluorescent probe for ultrafast and ratiometric detection of HSO 3− based on naphthalimide–hemicyanine | |
| CN114591632B (zh) | 一类氮杂吲哚-半花菁染料、其合成方法及应用 | |
| Maiti et al. | A benzothiazole-based dual reaction site fluorescent probe for the selective detection of hydrazine in water and live cells | |
| CN108409642A (zh) | 一种继发性识别铜离子和Hcy的双光子荧光探针及其制备方法 | |
| CN111073634B (zh) | 基于硝基还原、硫氮转位的硝基还原酶荧光探针及其制备方法 | |
| Yang et al. | A near-infrared fluorescent probe for the discrimination of cysteine in pure aqueous solution and imaging of cysteine in hepatocellular carcinoma cells with facile cell-compatible ability | |
| CN117603203A (zh) | 一种比率荧光探针及其制备方法和应用 | |
| CN109608495B (zh) | 一种检测hno的化合物及其制备方法和应用 | |
| US11639360B2 (en) | Oxazine compound and application thereof | |
| CN115124557A (zh) | 基于苝酰亚胺的fret乏氧酶荧光探针、其制备方法和应用 | |
| CN116375692A (zh) | 一种用于检测半胱氨酸的近红外荧光分子探针及其制备方法和试剂盒 |
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 |