CN115010691B - 一种荧光探针及其制备方法和应用 - Google Patents
一种荧光探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN115010691B CN115010691B CN202210588887.5A CN202210588887A CN115010691B CN 115010691 B CN115010691 B CN 115010691B CN 202210588887 A CN202210588887 A CN 202210588887A CN 115010691 B CN115010691 B CN 115010691B
- Authority
- CN
- China
- Prior art keywords
- reaction
- solution
- probe
- application according
- fluorescent probe
- 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 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 239000000523 sample Substances 0.000 claims abstract description 40
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 5
- 235000018417 cysteine Nutrition 0.000 claims abstract description 5
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims abstract 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 10
- 238000004440 column chromatography Methods 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- WJKHJLXJJJATHN-UHFFFAOYSA-N triflic anhydride Chemical compound FC(F)(F)S(=O)(=O)OS(=O)(=O)C(F)(F)F WJKHJLXJJJATHN-UHFFFAOYSA-N 0.000 claims description 8
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- VZXOZSQDJJNBRC-UHFFFAOYSA-N 4-chlorobenzenethiol Chemical compound SC1=CC=C(Cl)C=C1 VZXOZSQDJJNBRC-UHFFFAOYSA-N 0.000 claims description 6
- 239000012065 filter cake Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000000975 dye Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 4
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000003834 intracellular effect Effects 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims 10
- 235000008160 pyridoxine Nutrition 0.000 claims 5
- 239000011677 pyridoxine Substances 0.000 claims 5
- 229940011671 vitamin b6 Drugs 0.000 claims 5
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 claims 4
- 239000007788 liquid Substances 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 238000006462 rearrangement reaction Methods 0.000 abstract description 3
- 230000007488 abnormal function Effects 0.000 abstract 1
- 230000003647 oxidation Effects 0.000 abstract 1
- 238000007254 oxidation reaction Methods 0.000 abstract 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 10
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 9
- 238000002189 fluorescence spectrum Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 229940125782 compound 2 Drugs 0.000 description 5
- 229960003180 glutathione Drugs 0.000 description 5
- 239000003642 reactive oxygen metabolite Substances 0.000 description 5
- 238000001816 cooling Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 1
- 206010048962 Brain oedema Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- -1 F - Chemical class 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 102000003896 Myeloperoxidases Human genes 0.000 description 1
- 108090000235 Myeloperoxidases Proteins 0.000 description 1
- 229910019093 NaOCl Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 208000032140 Sleepiness Diseases 0.000 description 1
- 206010041349 Somnolence Diseases 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 208000006752 brain edema Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- CUILPNURFADTPE-UHFFFAOYSA-N hypobromous acid Chemical compound BrO CUILPNURFADTPE-UHFFFAOYSA-N 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 230000006540 mitochondrial respiration Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000035806 respiratory chain Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
Classifications
-
- 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
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
- C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
-
- 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
-
- 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
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Materials Engineering (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及荧光探针领域，具体涉及一种荧光探针及其制备方法和应用。正常情况下，细胞内的氧化系统和还原系统处于有机平衡状态，细胞内的氧化还原环境一旦失衡，将会表现出应激和功能异常，进而引发一系列疾病。本发明开发了一种荧光探针，可以同时传感半胱氨酸(Cys)和超氧自由基(O2 ·‑)，该荧光探针可以与Cys发生“取代‑重排”反应生成具有绿色荧光的物质，与O2 ·‑发生“氧化‑水解”反应生成具有蓝色荧光的物质，因此，探针可以分别从绿色和蓝色两个通道实现对Cys和O2 ·‑的同时传感。
Description
技术领域
本发明属于荧光探针领域，具体涉及一种荧光探针及其制备方法和应用。
背景技术
线粒体呼吸作用除了为细胞的生存与发展提供能量之外，也会因为呼吸链的电子泄露诱发大量的活性氧(ROS)。O2 ·-是呼吸链电子泄露所产生的第一个ROS，产生的O2 ·-在超氧歧化酶的催化下转化为H2O2，H2O2不仅能在髓过氧化物酶的催化下与细胞内Cl-和Br-反应生成HClO和HBrO，而且能在Fe2+离子的催化下发生Fenton反应产生HO·。在细胞氧化还原平衡失衡的条件下，过度产生的ROS能够氧化细胞内各种生物大分子，造成细胞正常功能的损伤，最终引发各种各样的疾病。其中，O2 ·-能引发体内脂质过氧化，加快从皮肤到内部器官整个肌体的衰老过程，并可诱发皮肤病变、心血管疾病、癌症等，严重危害人体健康。实际上，为了维持氧化还原平衡，细胞天然表达了各种各样的抗氧系统(如小分子生物硫醇、大分子抗氧化酶类、维生素C等)，在维持细胞内氧化还原平衡方面扮演了关键的角色。在小分子生物硫醇中，半胱氨酸(Cysteine，Cys)除了与同型半胱氨酸(Hcy)、谷胱甘肽(GSH)共同维持着生物体内氧化-还原平衡外，在蛋白质合成、解毒和新陈代谢方面等方面也扮演着重要的角色。Cys浓度的异常也会引发一系列疾病，高浓度的Cys与神经系统疾病密切相关，Cys缺失可以导致生长缓慢、脑水肿、嗜睡、肝损伤等疾病。鉴于此，开发一种可以同时传感Cys和O2 ·-的荧光探针，不仅对于Cys和O2 ·-的生理病理疾病的相关性研究，而且对于相关治疗药物的开发均意义重大。
发明内容
针对上述问题，本发明提供了一种可以同时传感Cys和O2 ·-的荧光探针及其制备和应用。该探针可与Cys发生“取代-重排”反应，生成一个绿荧光的“胺基-吡啰红”染料；与GSH发生仅发生“取代”反应，生成一个红色荧光的“硫代吡啰红”染料；Hcy和Cys在结构上仅仅是一个亚甲基的差异，具有相似的反应性能，但考虑到Hcy在细胞内极低的浓度，由Hcy所造成的干扰通常可忽略。此外，探针可以与O2 ·-发生“氧化-水解”反应，生成一个蓝荧光的“吡啰红酮”染料。总的来说，探针可以从绿色和蓝色两个通道实现对Cys和O2 ·-的同时传感。
为了达到上述目的，本发明采用了下列技术方案：
一种荧光探针，所述探针可以同时传感半胱氨酸和超氧自由基，探针为PyClTP，其结构式为：
一种所述荧光探针的制备方法，包括以下步骤：
(1)将吡啰红B(化合物1)和氰化钾加入水中，回流后，冷却至室温，过滤，洗涤，滤饼溶解在盐酸水溶液中，并逐滴加入到六水氯化铁的盐酸水溶液中，反应液反应后冷却至室温，过滤，洗涤，滤饼溶解在饱和的碳酸氢钠溶液中回流，冷却至室温有固体析出，过滤得粗品，粗产品经柱色谱分离得亮黄色固体吡啰红酮(化合物2)；
(2)在氮气保护下，将吡啰红酮(化合物2)溶于溶剂中，搅拌得到吡啰红酮溶液，然后向上述吡啰红酮溶液中逐渐滴加三氟甲基磺酸酐，反应液反应后加入4-氯苯硫酚，混合物在室温下反应过夜，旋干溶剂，粗产品经柱色谱分离得到“对氯苯硫酚功能化”的吡啰红染料，即所述探针。
进一步，所述步骤(1)中吡啰红B、氰化钾与六水氯化铁的摩尔比为1：3：3.5。
进一步，所述步骤(1)中盐酸水溶液的浓度为2mol/L。
进一步，所述步骤(1)中吡啰红B和氰化钾加入水中回流的时间为18h；所述反应液的反应温度为90℃，反应时间为12h；所述在饱和的碳酸氢钠溶液中回流3h。
进一步，所述步骤(1)中洗涤用水洗涤，所述柱色谱展开剂CH2Cl2/MeOH的体积比为50：1。
进一步，所述步骤(2)中吡啰红酮、三氟甲基磺酸酐与4-氯苯硫酚的摩尔比为1：4：10。
进一步，所述步骤(2)中溶剂为乙腈，搅拌的温度为0℃，时间为10min，反应液反应的时间为30min。
进一步，所述步骤(2)中柱色谱分离展开剂CH2Cl2：CH3OH的体积比为20：1。
一种所述荧光探针的应用，在制备同时检测细胞内半胱氨酸和超氧自由基试剂中的应用。
与现有技术相比本发明具有以下优点：
本发明提供的探针能与Cys发生“取代-重排”反应生成绿色的荧光物质，能与O2 ·-发生“氧化-水解”反应生成蓝色的荧光物质。因此本发明提供的探针能分别从绿色和蓝色两个通道实现对Cys和O2 ·-的同时传感，为研究Cys和O2 ·-的相关性提供了一个潜在的工具。
附图说明
图1为化合物2的1H NMR图(CDCl3,600MHz)；
图2为化合物2的13C NMR图(CDCl3,150MHz)；
图3为化合物2的HRMS图；
图4为探针PyClTP的1H NMR图(CDCl3,600MHz)；
图5为探针PyClTP的13C NMR图(CDCl3,150MHz)；
图6为探针PyClTP的HRMS图；
图7为探针在PBS中荧光光谱随时间(0-20min)的变化图；
图8为探针PyClTP与Cys反应的荧光光谱图，(图8A)探针与Cys反应的荧光强度随时间的变化图；(图8B)探针对Cys的荧光滴定图；(图8C)探针分别与Cys、Hcy和GSH反应的荧光光谱图；(图8D)探针分别与各种氨基酸(包括Ala、Arg、Asp、Gln、Glu、His、Ile、Leu、Lys、Nac、Phe、Pro、Ser、Thr、Try、Tyr和Val)反应的荧光光谱图；
图9为探针PyClTP与O2 ·-反应的荧光光谱图，(图9A)探针与O2 ·-反应的荧光强度随时间的变化图；(图9B)探针对O2 ·-的荧光滴定图；(图9C)探针分别与各种阴离子(包括F-、Cl-、Br-、I-、CO3 2-、PO3 2-、HPO3 -、SO3 2-、SCN-)反应的荧光光谱图；(图9D)探针分别与各种ROS(包括BO3 -、NO、NO2 -、S2-、H2O2、NaOCl、HO·、KO2)反应的荧光光谱图；
图10为本发明探针PyClTP与Cys和O2 ·-的传感机理示意图。
具体实施方式
实施例1
一种荧光探针，可以同时传感Cys和O2 ·-，其结构式为：
一种可以同时传感Cys和O2 ·-的荧光探针的制备方法，包括以下步骤：
(1)将吡啰红B(0.358g，1.0mmol)和KCN(0.195g，3.0mmol)加入10mL水中，回流18小时后，冷却至室温，过滤，用水洗涤。滤饼溶解在2.0M的HCl中(100mL)，逐滴加入到10mL的FeCl3.6H2O(0.947g，3.5mmol)的2.0M的HCl溶液中，反应液在90℃时反应12h，然后冷却至室温，过滤，用水洗涤。滤饼溶解在50mL饱和的NaHCO3中，回流3天，冷却至室温有固体析出，过滤的粗品。粗产品经柱色谱(CH2Cl2/MeOH,50：1v/v)分离得亮黄色固体(0.134g，产率39.7％)。
1H NMR(600Hz,CDCl3)8.10(d,J＝9.0Hz,2H),6.65(dd,J1＝1.8Hz,J2＝9.0Hz,2H),6.69(s,2H),3.46(q,J＝7.2Hz,8H),1.25(t,J＝7.2Hz,12H)；
13C NMR(150MHz,CDCl3)δ177.099,161.402,154.921,130.754,114.531,111.513,99.266,47.652,15.459；ESI-MS:[M+H]+calcd for 339.2066,Found 339.2067.
(2)在N2保护下，将吡啰红酮(0.1g，0.3mmol)的CH3CN溶液在0℃搅拌10分钟，然后向上述溶液中逐渐滴加三氟甲基磺酸酐(200μL，1.2mmol)，反应液反应30分钟后加入4-氯苯硫酚(0.42g，3mmol)，混合物在室温下反应过夜。旋干溶剂，粗产品经柱色谱(CH2Cl2/MeOH＝20/1v/v)分离得到所述探针(65mg，产率44.3％)。
1H NMR(600Hz,CDCl3)δ8.07(d,J＝13.8Hz,2H),7.29(m,4H),6.91(m,4H),3.63(q,J＝9.0Hz,8H),1.34(t,J＝9.6Hz,12H).
13C NMR(150MHz,CDCl3)δ157.2,155.7,153.1,134.5,132.7,131.1,130.3,115.6,114.5,96.9,46.3,12.7.ESI-MS:[M]+calcd for465.1762,Found 465.1761.
实施例2
1.测试溶液配制
将探针用乙腈配成2mM的储存液，随后用20mM的PBS(pH 7.4)稀释至测试浓度。
2.探针稳定性实验
为了确保探针在PBS体系中的稳定性，首先将探针(1μM)置于比色皿中并在荧光光谱仪上连续扫描20分钟。如图7所示，探针在PBS中的发射波长为620nm，连续扫描20分钟后，荧光光谱未发生明显变化。上述结果表明，阳离子特性赋予了探针优良的水溶性且可以在PBS中稳定存在，该特性对于生物应用至关重要。
3.探针与Cys的反应性能研究
在PBS体系中，选取450nm作为激发光时，PyClTP(1μM)本身在620nm处具有微弱的红色荧光；当加入100μM的Cys后，探针在620nm处的发射峰消失，在534nm处出现了一个新的发射峰，荧光强度约在15分钟时达到饱和，随后的实验均选取15分钟作为反应时间(图8A)。进一步的荧光滴定实验表明，随着Cys浓度的增加，534nm处的荧光强度逐渐升高，当Cys的浓度达到100μM时，荧光强度趋于稳定且增加92倍(图8B)。选择性实验表明，当向PyClTP(1μM)中分别加入100μM的Hcy、GSH以及其余常见氨基酸时，如我们预期的那样，仅有Hcy可以引起534nm处荧光强度的增加(图8C)，其余氨基酸(包括GSH)均不会引发534nm处荧光强度的改变(图8D)。考虑到Hcy在细胞内的浓度仅有12μM左右，其造成的影响可以忽略不计。因此，探针PyClTP可以从绿色通道高选择性的传感Cys。
4.探针与O2 ·-的反应性能研究
在PBS体系中，选取400nm作为激发光时，PyClTP(1μM)本身在620nm处具有微弱的红色荧光；当加入300μM的O2 ·-后，探针在620nm处的发射峰消失，在450nm处出现了一个新的发射峰，荧光强度约在2分钟时达到饱和，随后的实验均选取2分钟作为反应时间(图9A)。进一步的荧光滴定实验表明，随着O2 ·-浓度的增加，450nm处的荧光强度逐渐升高，当O2 ·-的浓度达到200μM时，荧光强度趋于稳定且增加6.4倍(图9B)。选择性实验表明，当向PyClTP(1μM)中分别加入200μM的各种阴离子以及活性氧化物时，上述化合物均不会引发450nm处荧光强度的改变(图9C和图9D)。因此，探针PyClTP可以从蓝色通道高选择性的传感O2 ·-。
综上所述，探针PyClTP本身的发射峰在620nm处，当向探针的PBS溶液中分别加入Cys和O2 ·-后，二者可以与探针分别发生“取代-重排”以及“氧化-水解”反应，生成了光物理性质完全不同的两个荧光物质，探针最终实现了对Cys和O2 ·-从绿色和蓝色两个通道的同时传感(图9)。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，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)
1.一种荧光探针在制备同时检测细胞内半胱氨酸和超氧自由基试剂中的应用，其特征在于，所述荧光探针的结构式为：
。
2.根据权利要求1所述的应用，其特征在于，所述荧光探针的制备方法包括以下步骤：
（1）将吡啰红B和氰化钾加入水中，回流后，冷却至室温，过滤，洗涤，滤饼溶解在盐酸水溶液中，并逐滴加入到六水氯化铁的盐酸水溶液中，反应液反应后冷却至室温，过滤，洗涤，滤饼溶解在饱和的碳酸氢钠溶液中回流，冷却至室温有固体析出，过滤得粗品，粗产品经柱色谱分离得亮黄色固体吡啰红酮；
（2）在氮气保护下，将吡啰红酮溶于溶剂中，搅拌得到吡啰红酮溶液，然后向上述吡啰红酮溶液中逐渐滴加三氟甲基磺酸酐，反应液反应后加入4-氯苯硫酚，混合物在室温下反应过夜，旋干溶剂，粗产品经柱色谱分离得到“对氯苯硫酚功能化”的吡啰红染料，即所述探针。
3.根据权利要求2所述的应用，其特征在于，所述步骤（1）中吡啰红B、氰化钾与六水氯化铁的摩尔比为1：3：3.5。
4.根据权利要求2所述的应用，其特征在于，所述步骤（1）中盐酸水溶液的浓度为2mol/L。
5.根据权利要求2所述的应用，其特征在于，所述步骤（1）中吡啰红B和氰化钾加入水中回流的时间为18h；所述反应液的反应温度为90℃，反应时间为12h；所述在饱和的碳酸氢钠溶液中回流3h。
6.根据权利要求2所述的应用，其特征在于，所述步骤（1）中洗涤用水洗涤，所述柱色谱展开剂CH2Cl2/MeOH的体积比为 50：1。
7.根据权利要求2所述的应用，其特征在于，所述步骤（2）中吡啰红酮、三氟甲基磺酸酐与4-氯苯硫酚的摩尔比为1：4：10。
8.根据权利要求2所述的应用，其特征在于，所述步骤（2）中溶剂为乙腈，搅拌的温度为0℃，时间为10min，反应液反应的时间为30min。
9.根据权利要求2所述的应用，其特征在于，所述步骤（2）中柱色谱分离展开剂CH2Cl2：CH3OH的体积比为20：1。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210588887.5A CN115010691B (zh) | 2022-05-26 | 2022-05-26 | 一种荧光探针及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210588887.5A CN115010691B (zh) | 2022-05-26 | 2022-05-26 | 一种荧光探针及其制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115010691A CN115010691A (zh) | 2022-09-06 |
| CN115010691B true CN115010691B (zh) | 2023-12-26 |
Family
ID=83070686
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210588887.5A Active CN115010691B (zh) | 2022-05-26 | 2022-05-26 | 一种荧光探针及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115010691B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2006317506A1 (en) * | 2005-11-22 | 2007-05-31 | Two-Tag Holdings Pty Ltd | Methods for determining the redox status of proteins |
| CN109666012A (zh) * | 2018-12-24 | 2019-04-23 | 云南大学 | 基于苯并吡喃腈检测心肌黄酶的荧光探针分子、制备及用途 |
| CN111518066A (zh) * | 2020-05-25 | 2020-08-11 | 河南省农业科学院农业质量标准与检测技术研究所 | 用于识别次氯酸根和亚硫酸氢根的双功能荧光探针及其制备方法和应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160151469A1 (en) * | 2014-11-30 | 2016-06-02 | Regen BioPharma, Inc. | Immune modulation by tlr activation for treatment of filovirus infections including ebola |
-
2022
- 2022-05-26 CN CN202210588887.5A patent/CN115010691B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2006317506A1 (en) * | 2005-11-22 | 2007-05-31 | Two-Tag Holdings Pty Ltd | Methods for determining the redox status of proteins |
| CN109666012A (zh) * | 2018-12-24 | 2019-04-23 | 云南大学 | 基于苯并吡喃腈检测心肌黄酶的荧光探针分子、制备及用途 |
| CN111518066A (zh) * | 2020-05-25 | 2020-08-11 | 河南省农业科学院农业质量标准与检测技术研究所 | 用于识别次氯酸根和亚硫酸氢根的双功能荧光探针及其制备方法和应用 |
Non-Patent Citations (6)
Also Published As
| Publication number | Publication date |
|---|---|
| CN115010691A (zh) | 2022-09-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sun et al. | Mitochondria targetable time-gated luminescence probe for singlet oxygen based on a β-diketonate–europium complex | |
| CN110128435B (zh) | 一种过氧化亚硝基比率型荧光探针的制备和应用 | |
| Carneiro et al. | Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex—A system capable of producing nitric oxide and singlet oxygen | |
| CN108030921B (zh) | 一种白蛋白负载金属卟啉配合物纳米颗粒的制备方法及其应用 | |
| CN115385861B (zh) | 一种荧光探针及其制备方法和应用 | |
| CN108658995B (zh) | 一种二硫联吡啶修饰锌酞菁及其制备方法和应用 | |
| Al-Raqa et al. | A novel water soluble axially substituted silicon (IV) phthalocyanine bearing quaternized 4-(4-pyridinyl) phenol groups: Synthesis, characterization, photophysicochemical properties and BSA/DNA binding behavior | |
| CN106929003A (zh) | 一种多功能近红外荧光探针及其制备方法和应用 | |
| Pinto et al. | Therapeutic potential of two visible light responsive luminescent photoCORMs: Enhanced cellular internalization driven by lipophilicity | |
| CN104230944B (zh) | 二酞菁锌配合物及其制备方法和应用 | |
| Yao et al. | Optimization of axial ligands to promote the photoactivation of BODIPY-conjugated platinum (IV) anticancer prodrugs | |
| CN111662333A (zh) | 一类双三联吡啶铱(ⅲ)配合物及其合成方法 | |
| Ahmadi et al. | A cystine-based dual chemosensor for fluorescent-colorimetric detection of CN− and fluorescent detection of Fe3+ in aqueous media: Synthesis, spectroscopic, and DFT studies | |
| CN117701025B (zh) | 尼罗蓝-parp抑制剂染料及其合成方法与应用 | |
| CN118515681B (zh) | 一种近红外荧光探针及其制备方法和应用 | |
| Shen et al. | A efficient light-controlled nitric oxide releaser in aqueous solution and its red fluorescence imaging in lysosome | |
| Tian et al. | Synthesis and antitumor study of novel porphyrin–coumarin derivatives for chemotherapy and photodynamic therapy | |
| Güleç et al. | Peripheral (E)‐2‐[(4‐hydroxybenzylidene)‐3, 4‐dihydronaphthalen‐1 (2H)‐one)]‐coordinated phthalocyanines with improved enzyme inhibition properties and photophysicochemical behaviors | |
| EP0350948A2 (en) | Porphyrin derivatives | |
| CN115010691B (zh) | 一种荧光探针及其制备方法和应用 | |
| CN112409365B (zh) | 3-磺酸基丙烷巯基修饰酞菁及其制备方法与在制药领域的应用 | |
| CN110903325B (zh) | 一种环金属铱配合物探针及其制备方法和应用 | |
| CN113735890B (zh) | 可避免细胞内GSH干扰的Cys荧光探针及制备和应用 | |
| CN118812600A (zh) | 具有线粒体靶向且光动力活性的bodipy纳米光敏剂及其制备方法与应用 | |
| Saha et al. | Reaction of a nitrosyl complex of cobalt porphyrin with hydrogen peroxide: putative formation of peroxynitrite intermediate |
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 |