CN114478435B - 一种基于查尔酮的溶酶体荧光探针及其制备方法与应用 - Google Patents
一种基于查尔酮的溶酶体荧光探针及其制备方法与应用 Download PDFInfo
- Publication number
- CN114478435B CN114478435B CN202210259082.6A CN202210259082A CN114478435B CN 114478435 B CN114478435 B CN 114478435B CN 202210259082 A CN202210259082 A CN 202210259082A CN 114478435 B CN114478435 B CN 114478435B
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- hca
- chalcone
- lysosome
- organic solvent
- 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.)
- Active
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 61
- 210000003712 lysosome Anatomy 0.000 title claims abstract description 49
- 230000001868 lysosomic effect Effects 0.000 title claims abstract description 45
- DQFBYFPFKXHELB-UHFFFAOYSA-N Chalcone Natural products C=1C=CC=CC=1C(=O)C=CC1=CC=CC=C1 DQFBYFPFKXHELB-UHFFFAOYSA-N 0.000 title claims abstract description 23
- 235000005513 chalcones Nutrition 0.000 title claims abstract description 23
- DQFBYFPFKXHELB-VAWYXSNFSA-N trans-chalcone Chemical compound C=1C=CC=CC=1C(=O)\C=C\C1=CC=CC=C1 DQFBYFPFKXHELB-VAWYXSNFSA-N 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims abstract description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 230000002132 lysosomal effect Effects 0.000 claims description 18
- 239000003960 organic solvent Substances 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 15
- VEFLKXRACNJHOV-UHFFFAOYSA-N 1,3-dibromopropane Chemical compound BrCCCBr VEFLKXRACNJHOV-UHFFFAOYSA-N 0.000 claims description 14
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 10
- 238000010992 reflux Methods 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 8
- 238000010898 silica gel chromatography Methods 0.000 claims description 8
- 239000003513 alkali Substances 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
- AETKQQBRKSELEL-UHFFFAOYSA-N (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one Natural products OC1=CC=CC=C1C(=O)C=CC1=CC=CC=C1 AETKQQBRKSELEL-UHFFFAOYSA-N 0.000 claims description 4
- AETKQQBRKSELEL-ZHACJKMWSA-N 2'-hydroxychalcone Chemical compound OC1=CC=CC=C1C(=O)\C=C\C1=CC=CC=C1 AETKQQBRKSELEL-ZHACJKMWSA-N 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 150000007529 inorganic bases Chemical class 0.000 claims 2
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 21
- 230000005284 excitation Effects 0.000 abstract description 7
- 238000012632 fluorescent imaging Methods 0.000 abstract description 7
- 230000002378 acidificating effect Effects 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000005481 NMR spectroscopy Methods 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 8
- 238000001228 spectrum Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 description 4
- 229910000024 caesium carbonate Inorganic materials 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001506 fluorescence spectroscopy Methods 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 229910000104 sodium hydride Inorganic materials 0.000 description 2
- 239000012312 sodium hydride Substances 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000187398 Streptomyces lividans Species 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
- C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
- C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
- C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
-
- 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
-
- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- 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
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
-
- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
-
- 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
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种查尔酮的溶酶体荧光探针及其制备方法与应用,本发明属于有机分子荧光探针技术领域,将2’‑羟基查尔酮HCA和1,3‑二溴丙烷加入有机溶剂中反应,经硅胶柱色谱分离获得中间体Br‑HCA,将中间体Br‑HCA和吗啉加入有机溶剂中反应,经硅胶柱色谱分离获得溶酶体荧光探针Lyso‑HCA。该荧光探针制备方法简单、快速、成本低,制得的荧光探针背景干扰低、检测灵敏度高,应用于细胞荧光成像的激发能力强,该探针荧光对酸碱性环境稳定性好,对溶酶体荧光成像的特异性好。该探针为细胞溶酶体荧光成像研究提供了具有广阔应用前景的工具。
Description
技术领域
本发明属于有机分子荧光探针技术领域,涉及一种基于查尔酮的溶酶体荧光探针及其制备方法与应用。
背景技术
溶酶体是一种存在于真核细胞中的重要细胞器,含有多种酸性水解酶,是细胞分解各种外源和内源大分子物质的主要场所,还参与细胞自噬、分化和凋亡等过程,另外还与多种遗传疾病和肿瘤的发生密切相关。因此,监测活细胞中溶酶体的变化具有重大意义。荧光成像法是研究细胞中溶酶体形貌、分布及动态变化过程的有力工具,它具有特异性好、实时、可视化等众多优点。一些商品化的有机分子类荧光探针已广泛应用细胞内溶酶体的靶向荧光成像,但目前很多商品化溶酶体荧光探针的结构复杂,制备过程繁琐,成本高。因此,仍需要进一步开发结构简单、低成本、高性能的溶酶体荧光探针。
发明内容
本发明的目的在于解决现有技术中的问题,提供一种基于查尔酮的溶酶体荧光探针及其制备方法与应用。
为达到上述目的,本发明采用以下技术方案予以实现:
一种基于查尔酮的溶酶体荧光探针,所述探针结构式如下所示:
一种基于查尔酮的溶酶体荧光探针的制备方法,包括如下步骤:
步骤1)将2’-羟基查尔酮HCA和1,3-二溴丙烷加入有机溶剂中混合均匀,在碱性条件下加热回流搅拌,过滤除去固体残渣,减压蒸馏除去溶剂,粗产品经硅胶柱色谱分离获得中间体Br-HCA;
步骤2)将中间体Br-HCA和吗啉加入有机溶剂中混合均匀,在碱性条件下加热回流,减压蒸馏除去溶剂,粗产品经硅胶柱色谱分离获得溶酶体荧光探针Lyso-HCA。
进一步的,步骤1)中,反应原料HCA与1,3-二溴丙烷的摩尔比为1:2~10。
进一步的,步骤1)中,所述有机溶剂使用N,N-二甲基甲酰胺、四氢呋喃或乙腈,反应原料HCA、1,3-二溴丙烷和有机溶剂的摩尔比为1:2~10:60~1000。
进一步的,步骤2)中,所述有机溶剂使用N,N-二甲基甲酰胺、四氢呋喃或乙腈,中间体Br-HCA、吗啉和有机溶剂的摩尔比为1:2~10:60~1000。
进一步的,步骤1)中,所述碱性条件使用无机碱为氢氧化钠、氢氧化钾、碳酸钠、碳酸钾、碳酸铯或氢化钠,且反应原料HCA、1,3-二溴丙烷和碱的摩尔比为1:2~10:1~10。
进一步的,步骤2)中,所述碱性条件使用无机碱为氢氧化钠、氢氧化钾、碳酸钠、碳酸钾、碳酸铯或氢化钠,且反应原料Br-HCA、吗啉和碱的摩尔比为1:1~5:1~10。
进一步的,步骤1)和步骤2)中,反应温度为60-120℃,反应时间为4~24h。
基于查尔酮的溶酶体荧光探针在细胞溶酶体靶向荧光成像中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明提供了一种基于查尔酮分子的溶酶体荧光探针,该荧光探针包括具有荧光性能的查尔酮分子和溶酶体定位功能的吗啉基团,查尔酮分子具有分子内共轭的推-拉电子效应,结构简单,具有较大的斯托克斯位移,荧光性能优异;而弱碱性的吗啉基团可以和弱酸性的溶酶体内环境相互作用,从而实现该荧光探针在细胞溶酶体内的特异性定位与荧光检测。
该荧光探针制备方法简单、快速、成本低;斯托克斯位移约70-110nm,相比常见商品化溶酶体荧光染料的10-20nm显著增大,具有背景干扰低、检测灵敏度高等优点;荧光最大激发波长420nm与共聚焦荧光显微镜常用的405nm激光器匹配程度高,应用于细胞荧光成像的激发能力强;在较宽的pH范围内荧光强度比较稳定,表明该探针荧光对酸碱性环境稳定性好,不会受到细胞溶酶体酸性变化的影响;该探针与商品化溶酶体荧光探针复染成像的共定位系数可达0.947,说明其对溶酶体荧光成像的特异性好。该探针为细胞溶酶体荧光成像研究提供了具有广阔应用前景的工具。
附图说明
为了更清楚的说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,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相关的附图。
图1为本发明的实施例1所合成中间体Br-HCA的核磁共振氢谱图。
图2为本发明的实施例1所合成溶酶体荧光探针Lyso-HCA的核磁共振氢谱图。
图3为本发明的实施例1所合成溶酶体荧光探针Lyso-HCA的核磁共振碳谱图。
图4为本发明的实施例1所合成溶酶体荧光探针Lyso-HCA的高分辨质谱图。
图5为本发明的实施例1所合成溶酶体荧光探针Lyso-HCA在不同溶剂中的荧光光谱图。
图6为本发明的实施例1所合成溶酶体荧光探针Lyso-HCA荧光强度与溶液pH的关系曲线图。
图7为本发明的实施例1所合成溶酶体荧光探针Lyso-HCA与市售溶酶体染料Lyso-tracker Red细胞共定位荧光成像图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中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。通常在此处附图中描述和示出的本发明实施例的组件可以以各种不同的配置来布置和设计。
因此,以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。基于本发明中的实施例,本领域普通技术人员在没有作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。
应注意到:相似的标号和字母在下面的附图中表示类似项,因此,一旦某一项在一个附图中被定义,则在随后的附图中不需要对其进行进一步定义和解释。
下面结合附图对本发明做进一步详细描述:
本发明提供的一种基于查尔酮的溶酶体荧光探针的制备方法,包括如下步骤:
步骤1)将HCA和1,3-二溴丙烷加入有机溶剂中混合均匀,反应原料HCA与1,3-二溴丙烷的摩尔比为1:2~10,反应原料HCA、1,3-二溴丙烷和有机溶剂的摩尔比为1:2~10:60~1000,在碱性条件下,加热到60-120℃,回流搅拌反应4~24h,反应原料HCA、1,3-二溴丙烷和碱的摩尔比为1:2~10:1~10,过滤除去固体残渣,减压蒸馏除去溶剂,粗产品经硅胶柱色谱分离获得中间体Br-HCA,反应式如下:
步骤2)将中间体Br-HCA和吗啉加入有机溶剂中混合均匀,中间体Br-HCA、吗啉和有机溶剂的摩尔比为1:2~10:60~1000,在碱性条件下加热到60-120℃回流,反应原料Br-HCA、吗啉和碱的摩尔比为1:1~5:1~10,反应4~24h,减压蒸馏除去溶剂,粗产品经硅胶柱色谱分离获得溶酶体荧光探针Lyso-HCA,反应式如下:
实施例1:
一种基于查尔酮分子的溶酶体荧光探针的制备方法,包括如下步骤:
步骤(1)中间体Br-HCA的制备
2’-羟基查尔酮(HCA,0.668g,2.5mmol)与1,3-二溴丙烷(1.010g,5mmol)溶于15mL的N,N-二甲基甲酰胺中,加入无水碳酸钾(0.518g,3.75mmol)。升温至80℃下回流反应8h。停止反应,然后用乙酸乙酯萃取,萃取后合并有机相,用无水硫酸镁干燥,过滤,减压蒸馏,粗产品经硅胶柱层析分离得到化合物Br-HCA,产率51%。
步骤(2)溶酶体荧光探针Lyso-HCA的制备
将化合物Br-HCA(39mg,0.1mmol)与吗啉(43mg,0.5mmol)加入到100mL圆底烧瓶中,后加入5mL乙腈,向溶液中加入无水碳酸钾(20mg,0.15mmol)粉末,温度升至100℃回流反应12h。停止反应并冷却,减压旋蒸除去溶剂乙腈,将粗产物进行柱色谱分离,洗脱剂CH2Cl2:CH3OH=40:1。得到橙黄色固体产物Lyso-HCA,产率56%。
实施例2:
一种基于查尔酮分子的溶酶体荧光探针的制备方法,包括如下步骤:
步骤(1)中间体Br-HCA的制备
2’-羟基查尔酮HCA(0.267g,1mmol)与1,3-二溴丙烷(1.010g,5mmol)溶于8mL的N,N-二甲基甲酰胺中,加入碳酸铯(0.652g,2mmol)。升温至80℃下回流反应12h。停止反应,然后用乙酸乙酯萃取,萃取后合并有机相,用无水硫酸镁干燥,过滤,减压蒸馏,粗产品经硅胶柱层析分离得到化合物Br-HCA,产率58%。
步骤(2)溶酶体荧光探针Lyso-HCA的制备
将化合物Br-HCA(78mg,0.2mmol)与吗啉(70mg,0.8mmol)加入到100mL圆底烧瓶中,后加入5mL乙腈,向溶液中加入碳酸铯(130mg,0.4mmol)粉末,升温至80℃回流反应24h。停止反应并冷却,减压旋蒸除去溶剂乙腈,将粗产物进行柱色谱分离,洗脱剂CH2Cl2:CH3OH=40:1。得到橙黄色固体产物Lyso-HCA,产率60%。
1.对实施例1制备的溶酶体荧光探针进行结构表征:
中间体Br-HCA的核磁共振氢谱如如图1所示,氢的数量和峰的耦合裂分情况与分子结构相符,表明所合成中间体Br-HCA的结构正确。1H NMR(400MHz,CDCl3)δ:7.64–7.41(m,5H),7.19–6.99(m,3H),6.69(d,J=8.9Hz,2H),4.21(t,J=5.7Hz,2H),3.56(t,J=6.2Hz,2H),3.06(s,6H),2.34-2.25(m,2H)。
溶酶体荧光探针Lyso-HCA的核磁共振氢谱如图2所示,氢的数量和峰的耦合裂分情况与分子结构相符,表明所合成探针分子的结构正确。1H NMR(400MHz,CDCl3)δ:7.61-7.41(m,5H),7.17(d,J=15.7Hz,1H),7.07-6.97(m,2H),6.68(d,J=8.7Hz,2H),4.11(t,J=6.0Hz,2H),3.66-3.59(m,4H),3.04(s,6H),2.50-2.44(m,2H),2.30(s,4H),1.97(dd,J=13.8,6.5Hz,2H)。
溶酶体荧光探针Lyso-HCA的核磁共振碳谱如图3所示,碳峰的数量与分子结构相符,表明所合成探针分子的结构正确。13C NMR(100MHz,CDCl3)δ:193.55,157.10,151.86,144.50,132.11,130.27,130.16,122.75,122.54,120.74,112.38,111.83,66.92,66.66,55.62,53.56,40.18,26.44。
溶酶体荧光探针Lyso-HCA的高分辨质谱如图4所示,HRMS(ESI)m/z:C24H30N2O3[M+H]+测量值395.2320,与预测值395.2335基本一致,表明所合成探针分子的结构正确。
2.溶酶体荧光探针Lyso-HCA在不同溶剂中的荧光光谱测试:
取实施例1制备的溶酶体荧光探针Lyso-HCA测试其在不同溶剂中的荧光光谱,具体操作步骤如下:所用溶剂包括甲苯、乙酸乙酯、四氢呋喃、二氯甲烷、乙醇、乙腈、二甲基亚砜、水。以荧光光谱仪进行测试,测试浓度为10μmol/L。结果如图5所示,在420nm波长的激发下,探针的最大荧光发射波长约480-540nm,波长随溶剂极性的增大发生显著红移,体现了该探针典型的分子内电荷转移特征,其中在二甲基亚砜中荧光最强,在540nm处有较强的荧光发射,斯托克斯位移可达110nm。
3.溶酶体荧光探针Lyso-HCA在不同pH溶液中的荧光光谱测试:
取实施例1制备的溶酶体荧光探针Lyso-HCA在不同pH溶液中测定荧光光谱,得到探针的荧光强度与pH关系如图6所示。具体操作步骤如下:不同pH的溶液通过向PBS缓冲溶液中分别加入NaOH和HCl来配制得到,具体pH值由pH计测定。测试时将不同pH的溶液和二甲基亚砜混合(v:v=1:1)作为测试溶剂。用二甲基亚砜配制5mmol/L的探针母液,探针母液加入测试溶剂得浓度为10μmol/L的测试溶液。图6所示为探针在不同pH条件下530nm处的荧光强度与pH关系图。结果显示在pH范围从3到12时,探针的荧光强度没有较大差异,表明该探针荧光对酸碱性环境稳定性好,不会受到细胞溶酶体酸性变化的影响。
4.溶酶体荧光探针Lyso-HCA在细胞溶酶体荧光成像中的应用:
将实施例1制备的溶酶体荧光探针Lyso-HCA应用于细胞溶酶体荧光成像,具体操作步骤如下:探针母液使用二甲基亚砜配制,浓度为5mmol/L。实验所用HeLa细胞在DMEM培养基(1%青链霉素混合液双抗,10%胎牛血清)在37℃、饱和湿度、含5%的CO2的二氧化碳培养箱中培养,提前24h传代至细胞培养皿内贴壁培养准备进行荧光成像实验。取2μL探针母液加入到2mL培养基中,充分震荡均匀得到探针浓度为5μmol/L的细胞染色液,吸去细胞培养皿中的培养基,加入细胞染色液孵育30min,再加入市售溶酶体染料Lyso-Tracker Red(浓度0.1μmol/L)孵育10min,后用PBS洗三次,将细胞培养皿置于激光共聚焦荧光显微镜下进行观察和荧光成像,Lyso-HCA的激发波长为405nm,检测波长450-550nm,市售溶酶体染料Lyso-Tracker Red的激发波长为561nm,检测波长575-675nm。结果如图7所示,图中(a)为Lyso-HCA的荧光成像、(b)为Lyso-Tracker Red的荧光成像、(c)为细胞眀场成像、(d)为荧光成像中划线区域的荧光强度分布曲线。由图可以看出荧光探针Lyso-HCA与市售溶酶体荧光染料Lyso-Tracker Red在细胞内的重合性好,并得到其Pearson’s共定位系数为0.947,说明荧光探针Lyso-HCA成功定位于细胞内溶酶体,实现了溶酶体的特异性荧光成像。
综上所述,该荧光探针制备方法简单、快速、成本低,制得的荧光探针具有较大的斯托克斯位移,背景干扰低、检测灵敏度高,应用于细胞荧光成像的激发能力强,该探针荧光对酸碱性环境稳定性好,对溶酶体荧光成像的特异性好。该探针为细胞溶酶体荧光成像研究提供了具有广阔应用前景的工具。
以上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 (9)
1.一种基于查尔酮的溶酶体荧光探针,其特征在于,所述探针结构式如下所示:
。
2.一种基于查尔酮的溶酶体荧光探针的制备方法,其特征在于,包括如下步骤:
步骤1)将2’-羟基查尔酮(HCA)和1,3-二溴丙烷加入有机溶剂中混合均匀,在碱性条件下加热回流搅拌,过滤除去固体残渣,减压蒸馏除去溶剂,粗产品经硅胶柱色谱分离获得中间体Br-HCA;
步骤2)将中间体Br-HCA和吗啉加入有机溶剂中混合均匀,在碱性条件下加热回流,减压蒸馏除去溶剂,粗产品经硅胶柱色谱分离获得溶酶体荧光探针Lyso-HCA。
3.根据权利要求2所述的基于查尔酮的溶酶体荧光探针的制备方法,其特征在于,步骤1)中,反应原料HCA与1,3-二溴丙烷的摩尔比为1:(2~10)。
4.根据权利要求2所述的基于查尔酮的溶酶体荧光探针的制备方法,其特征在于,步骤1)中,所述有机溶剂使用N,N-二甲基甲酰胺、四氢呋喃或乙腈,反应原料HCA、1,3-二溴丙烷和有机溶剂的摩尔比为1:(2~10):(60~1000)。
5.根据权利要求2所述的基于查尔酮的溶酶体荧光探针的制备方法,其特征在于,步骤2)中,所述有机溶剂使用N,N-二甲基甲酰胺、四氢呋喃或乙腈,中间体Br-HCA、吗啉和有机溶剂的摩尔比为1:(2~10):(60~1000)。
6.根据权利要求2所述的基于查尔酮的溶酶体荧光探针的制备方法,其特征在于,步骤1)中,所述碱性条件使用无机碱为氢氧化钠、氢氧化钾、碳酸钠、碳酸钾、碳酸铯或氢化钠,且反应原料HCA、1,3-二溴丙烷和碱的摩尔比为1:(2~10):(1~10)。
7.根据权利要求2所述的基于查尔酮的溶酶体荧光探针的制备方法,其特征在于,步骤2)中,所述碱性条件使用无机碱为氢氧化钠、氢氧化钾、碳酸钠、碳酸钾、碳酸铯或氢化钠,且反应原料Br-HCA、吗啉和碱的摩尔比为1:(1~5):(1~10)。
8. 根据权利要求2所述的基于查尔酮的溶酶体荧光探针的制备方法,其特征在于,步骤1)和步骤2)中,反应温度为60-120℃,反应时间为4~24 h。
9.权利要求1所述的基于查尔酮的溶酶体荧光探针在细胞溶酶体靶向荧光成像中的应用,所述应用为非疾病诊断目的的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210259082.6A CN114478435B (zh) | 2022-03-16 | 2022-03-16 | 一种基于查尔酮的溶酶体荧光探针及其制备方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210259082.6A CN114478435B (zh) | 2022-03-16 | 2022-03-16 | 一种基于查尔酮的溶酶体荧光探针及其制备方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114478435A CN114478435A (zh) | 2022-05-13 |
CN114478435B true CN114478435B (zh) | 2024-04-02 |
Family
ID=81486556
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210259082.6A Active CN114478435B (zh) | 2022-03-16 | 2022-03-16 | 一种基于查尔酮的溶酶体荧光探针及其制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114478435B (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112939935A (zh) * | 2019-12-10 | 2021-06-11 | 中国科学院大连化学物理研究所 | 一种用于溶酶体靶向荧光探针及其合成方法与细胞成像应用 |
-
2022
- 2022-03-16 CN CN202210259082.6A patent/CN114478435B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112939935A (zh) * | 2019-12-10 | 2021-06-11 | 中国科学院大连化学物理研究所 | 一种用于溶酶体靶向荧光探针及其合成方法与细胞成像应用 |
Also Published As
Publication number | Publication date |
---|---|
CN114478435A (zh) | 2022-05-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Lu et al. | A highly selective and sensitive fluorescent turn-on sensor for Hg 2+ and its application in live cell imaging | |
CN113072937B (zh) | 一种脂滴靶向碳点、制备方法及应用 | |
CN109867611B (zh) | 一种用于红酒和活体内硫化氢检测的水溶性双光子硫化氢荧光探针及其制备方法和应用 | |
CN108997195B (zh) | 一种定位脂滴的双光子粘度探针及其制备方法和应用 | |
CN113461609B (zh) | 一种硫酸酯酶响应的aie纳米探针及其制备方法与应用 | |
CN105154065B (zh) | 一种快速专一性识别羟基自由基的荧光探针及其制备方法和应用 | |
El-Ali et al. | Solid-state emissive O-BODIPY dyes with bimodal emissions across red and near infrared region | |
CN114478435B (zh) | 一种基于查尔酮的溶酶体荧光探针及其制备方法与应用 | |
CN114276356A (zh) | 一种线粒体靶向的荧光探针及其合成方法和应用 | |
CN114736255B (zh) | 检测β-半乳糖苷酶的黄酮衍生物荧光探针及其制备方法和应用、试剂盒及其使用方法 | |
CN116283966A (zh) | 一种新型[1,8]-萘啶衍生物及其制备方法与在细胞器荧光成像和细胞器极性检测方面的应用 | |
CN110669350B (zh) | 一种哌啶基bodipy类红光荧光染料及其制备方法和应用 | |
CN110229203B (zh) | 一种氨基己糖酶荧光探针及其制备方法和应用 | |
CN113979890A (zh) | 一种席夫碱配体及其多核稀土配合物的制备方法和应用 | |
CN114436947A (zh) | 一种对粘度和硝基还原酶双响应的荧光探针及其制备方法和应用 | |
CN115073487B (zh) | 一种罗丹明衍生物及其制备方法和应用 | |
CN112110887A (zh) | 一种3位甲酰基取代的2h-色烯衍生物的合成方法及其应用 | |
CN112945912B (zh) | 一类高亮度、大斯托克斯位移免洗脂滴荧光探针及其合成方法与应用 | |
CN113461706A (zh) | 一种快速识别羟基自由基的荧光探针及其制备方法和应用 | |
CN114702507B (zh) | 一种检测脂滴和内质网的荧光探针 | |
CN114656476B (zh) | 溶酶体靶向性罗丹明b酰肼类荧光探针及制备方法和应用 | |
CN111333565A (zh) | 一种近红外线粒体荧光探针及其合成方法 | |
CN112079860B (zh) | 氟硼二吡咯荧光探针及制备方法与其在粘度检测中的应用 | |
CN116675666B (zh) | 基于罗丹明近红外染料的pH荧光探针制备方法及应用 | |
EP4382526A1 (en) | Fluorescent dye, and preparation method therefor and use thereof |
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 |