CN116283870A - Coumarin-based chemiluminescent probe and preparation method and application thereof - Google Patents
Coumarin-based chemiluminescent probe and preparation method and application thereof Download PDFInfo
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- CN116283870A CN116283870A CN202310267896.9A CN202310267896A CN116283870A CN 116283870 A CN116283870 A CN 116283870A CN 202310267896 A CN202310267896 A CN 202310267896A CN 116283870 A CN116283870 A CN 116283870A
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- coumarin
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- chemiluminescent probe
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- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 239000000523 sample Substances 0.000 title claims abstract description 48
- 229960000956 coumarin Drugs 0.000 title claims abstract description 24
- 235000001671 coumarin Nutrition 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title abstract description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000013067 intermediate product Substances 0.000 claims abstract description 16
- 238000003756 stirring Methods 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 239000003960 organic solvent Substances 0.000 claims abstract description 10
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims abstract description 10
- AXKGIPZJYUNAIW-UHFFFAOYSA-N (4-aminophenyl)methanol Chemical compound NC1=CC=C(CO)C=C1 AXKGIPZJYUNAIW-UHFFFAOYSA-N 0.000 claims abstract description 9
- RREPYIWLDJQENS-UHFFFAOYSA-N 4-nitrophenylglyoxylic acid Chemical compound OC(=O)C(=O)C1=CC=C([N+]([O-])=O)C=C1 RREPYIWLDJQENS-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 8
- 239000000126 substance Substances 0.000 claims abstract description 8
- PFJPBLFPJISXMF-UHFFFAOYSA-N 7-bromochromen-2-one Chemical compound C1=CC(=O)OC2=CC(Br)=CC=C21 PFJPBLFPJISXMF-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910000027 potassium carbonate Inorganic materials 0.000 claims abstract description 5
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 14
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 238000010898 silica gel chromatography Methods 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 6
- 241001274216 Naso Species 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- 239000008096 xylene Substances 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 1
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 claims 1
- 239000007821 HATU Substances 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000006862 quantum yield reaction Methods 0.000 abstract description 3
- 239000002253 acid Substances 0.000 abstract description 2
- 150000003839 salts Chemical class 0.000 abstract description 2
- CMFNMSMUKZHDEY-UHFFFAOYSA-M peroxynitrite Chemical compound [O-]ON=O CMFNMSMUKZHDEY-UHFFFAOYSA-M 0.000 description 23
- 239000000243 solution Substances 0.000 description 16
- 239000007864 aqueous solution Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000007792 addition Methods 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000004907 flux Effects 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 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 2
- 239000008187 granular material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 description 1
- 102000004452 Arginase Human genes 0.000 description 1
- 108700024123 Arginases Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 238000004435 EPR spectroscopy Methods 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229960002749 aminolevulinic acid Drugs 0.000 description 1
- 150000001449 anionic compounds Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 235000003969 glutathione Nutrition 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000013930 proline Nutrition 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 235000004400 serine Nutrition 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 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
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
Abstract
The invention discloses a coumarin-based chemiluminescent probe and a preparation method and application thereof. The probe has the following molecular formula, and the preparation method comprises the following steps: s1, dissolving 2- (4-nitrophenyl) -2-oxo acetic acid and HATU in an organic solvent A, then adding N, N-diisopropylethylamine, stirring at room temperature for 10-30min, finally adding 4-amino benzyl alcohol, and stirring at room temperature for reacting for 12-24h to obtain an intermediate product; s2, dissolving potassium carbonate in the organic solvent B, adding the intermediate product and 7-bromo-2H-benzopyran-2-one, and stirring for reacting for 10-36 hours to obtain the chemical fluorescent probe. The chemiluminescent probe has the advantages of good light stability, large Stokes shift and high quantum yield, and is not easy to be subjected to H 2 O 2 、ClO ‑ Interference, peroxynitriteThe acid salt detection accuracy is high.
Description
Technical Field
The invention relates to a fluorescent probe, in particular to a coumarin-based chemiluminescent probe, and a preparation method and application thereof.
Background
Redox reactions and oxidative stress are involved in the etiology of many diseases and in the aging process, peroxynitrite (ONOO) - ) Is one of the most important endogenous Reactive Oxygen Species (ROS) in the life system and plays a vital role in various physiological and pathological processes such as signal transduction and antibacterial activity. But it is also due to the chemical nature of peroxynitrite that it is a central biological causative agent of various diseases, such as cardiovascular, neurodegenerative and inflammatory diseases, etc. Thus, for ONOO - The detection and the biological imaging research of the formula (I) have important significance for diagnosis and treatment of a plurality of diseases.
To date, several methods for detecting peroxynitrite have been developed, including ultraviolet/visible spectroscopy, electrochemical analysis, electron spin resonance, and immunohistochemistry. However, the exact pathogenic role of peroxynitrite in biological systems is still not clear due to its short life span in vivo, high activity, low concentration, elusive nature. In contrast, the fluorescent probe method for peroxynitrite detection has been developed in the past decades because of its advantages of high sensitivity, high selectivity, fast response speed, simple operation, and the like. However, existing fluorescent probes have slow response times, low quantum yields, small Stokes shifts, and are susceptible to H 2 O 2 、ClO - The interference causes the problems of poor selectivity, etc., so that the design and development of the quick, sensitive and high-selectivity peroxynitriteThe acid salt fluorescent probe has important significance.
Disclosure of Invention
In order to solve the technical problems, the invention firstly provides a coumarin-based chemiluminescent probe. The invention adopts coumarin as a luminous group to improve the optical performance of the probe, and researches show that the probe has good light stability, large Stokes displacement and difficult H-sensitivity when being used for detecting peroxynitrite 2 O 2 、ClO - Interference and the like, so that the detection accuracy is high.
Based on the second aspect of the invention, a preparation method of the coumarin-based chemiluminescent probe is also provided, and a simple and feasible process route is provided for industrial application of products.
Based on the third aspect of the invention, the application of the coumarin-based chemiluminescent probe in detecting endogenous peroxynitrite is also provided. The chemiluminescent probe can specifically react with peroxynitrite and generate a change of a fluorescent signal of turn on, and the content of peroxynitrite is detected by detecting the change of the fluorescent signal.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a coumarin-based chemiluminescent probe having the following molecular structural expression:
a method for preparing a coumarin-based chemiluminescent probe, comprising the following steps:
s1, dissolving 2- (4-nitrophenyl) -2-oxo acetic acid and 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate in an organic solvent A, then adding N, N-diisopropylethylamine, stirring at room temperature for 10-30min, finally adding 4-amino benzyl alcohol, and stirring at room temperature for reacting for 12-24h to obtain an intermediate product;
s2, dissolving potassium carbonate in the organic solvent B, adding the intermediate product and 7-bromo-2H-benzopyran-2-one, and stirring for reacting for 10-36 hours to obtain the chemical fluorescent probe.
As a preferred embodiment of the present invention, in step S1, the molar ratio of 2- (4-nitrophenyl) -2-oxoacetic acid, 4-aminobenzyl alcohol, 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine is 1 (1-1.5): (1-2): (20-50), for example, when the molar equivalent of 2- (4-nitrophenyl) -2-oxoacetic acid is 1, the molar equivalent of 4-aminobenzyl alcohol may be 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc., the molar equivalent of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate may be 1, 1.2, 1.4, 1.6, 1.8, etc., and the molar equivalent of N, N-diisopropylethylamine may be 20, 25, 30, 35, 40, 45, 50, etc.
In step S1, as a preferred embodiment of the present invention, the organic solvent a is selected from one or more of anhydrous dichloromethane, chloroform, tetrahydrofuran, acetonitrile.
As a preferred embodiment of the present invention, after the completion of the reaction in step S1, methylene chloride is added to dilute the reaction solution, and the solution is washed several times with saturated brine, anhydrous NaSO 4 And (5) performing silica gel column chromatography after drying, and purifying to obtain an intermediate product.
As a preferred embodiment of the present invention, in step S2, the molar ratio of intermediate product to 7-bromo-2H-benzopyran-2-one is 1 (1-1.5), for example 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, etc.
As a preferred embodiment of the present invention, in step S2, potassium carbonate is used in an amount of 10 to 20 times, for example, 10 times, 12 times, 14 times, 16 times, 18 times, 20 times, etc., the molar amount of the intermediate product.
As a preferred embodiment of the present invention, in step S2, the organic solvent B is selected from one or more of N, N-dimethylformamide, dimethyl sulfoxide, toluene, and xylene.
As a preferred embodiment of the present invention, after the reaction of step S2 is completed, the chemical fluorescent probe is obtained by purifying by silica gel column chromatography.
Use of a coumarin-based chemiluminescent probe as described herein and a coumarin-based chemiluminescent probe prepared by a method as described herein in the field of detection of endogenous peroxynitrite.
The chemiluminescent probe uses coumarin as a luminescent group, is applied to detection of peroxynitrite, has the advantages of good light stability, large Stokes shift and high quantum yield, and is not easy to be subjected to H 2 O 2 、ClO - Interference and high detection accuracy.
Detailed Description
The invention will now be further illustrated by means of specific examples which are given solely by way of illustration of the invention and do not limit the scope thereof.
Unless otherwise specified, the starting materials and reagents in the examples of the present invention are all commercially available.
[ example 1 ]
The chemiluminescent probe is prepared according to the following steps, and the reaction process expression is as follows:
(1) 1mmol of 2- (4-nitrophenyl) -2-oxoacetic acid and 1.2mmol of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) were dissolved in 15ml of anhydrous dichloromethane, 36.3mmol of N, N-diisopropylethylamine was then added, stirring was carried out at room temperature for 20min, and finally 1.1mmol of 4-aminobenzyl alcohol was added, and the reaction was carried out at room temperature with stirring for 12h. After the completion of the reaction, 10ml of methylene chloride was added to dilute the reaction mixture, which was washed 3 times with saturated brine, and anhydrous NaSO 4 And (5) performing silica gel column chromatography after drying, and purifying to obtain an intermediate product.
1H NMR(500MHz,Chloroform-d):δ9.25(s,1H),8.45–8.36(m,2H),8.02–7.96(m,2H),7.91–7.83(m,2H),7.49–7.38(m,2H),4.46(s,2H),1.43(s,1H).
(2) Will 3.6mmol K 2 CO 3 Dissolving in 10ml DMF, adding 0.3mmol intermediate product and 0.4mmol 7-bromo-2H-benzopyran-2-ketone, stirring for reaction for 12H, and purifying by silica gel column chromatography to obtain the chemofluorescent probe.
1H NMR(500MHz,Chloroform-d):7.49(d,J＝8.4Hz,1H),7.35(dt,J＝7.8,1.0Hz,2H),6.91(dd,J＝8.4,2.4Hz,1H),6.82(d,J＝2.4Hz,1H),6.27(d,J＝9.5Hz,1H),4.99(t,J＝1.0Hz,2H).
[ example 2 ]
The chemiluminescent probe was prepared according to the following steps:
(1) 1mmol of 2- (4-nitrophenyl) -2-oxoacetic acid and 1.8mmol of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) were dissolved in 20ml of anhydrous dichloromethane, 45mmol of N, N-diisopropylethylamine was then added, stirring was carried out at room temperature for 20min, and finally 1.5mmol of 4-aminobenzyl alcohol was added, and stirring was carried out at room temperature for 18h. After the completion of the reaction, 10ml of methylene chloride was added to dilute the reaction mixture, which was washed 3 times with saturated brine, and anhydrous NaSO 4 And (5) performing silica gel column chromatography after drying, and purifying to obtain an intermediate product.
(2) Will 5.5mmol K 2 CO 3 Dissolving in 10ml DMF, adding 0.3mmol intermediate product and 0.4mmol 7-bromo-2H-benzopyran-2-ketone, stirring for reaction for 30H, and purifying by silica gel column chromatography to obtain the chemofluorescent probe.
[ example 3 ]
The chemiluminescent probe was prepared according to the following steps:
(1) 1mmol of 2- (4-nitrophenyl) -2-oxoacetic acid and 1.6mmol of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) were dissolved in 20ml of anhydrous dichloromethane, 24mmol of N, N-diisopropylethylamine was added thereto, and the mixture was stirred at room temperature for 20 minutes, and finally 1.3mmol of 4-aminobenzyl alcohol was added thereto, and the mixture was stirred at room temperature for 22 hours. After the completion of the reaction, 10ml of methylene chloride was added to dilute the reaction mixture, which was washed 3 times with saturated brine, and anhydrous NaSO 4 And (5) performing silica gel column chromatography after drying, and purifying to obtain an intermediate product.
(2) Will be 4.5mmol K 2 CO 3 Dissolving in 10ml DMF, adding 0.3mmol intermediate product and 0.3mmol 7-bromo-2H-benzopyran-2-ketone, stirring for reaction for 25H, and purifying by silica gel column chromatography to obtain the chemofluorescent probe.
[ application example ]
(1) Sensitive detection of peroxynitrite
Firstly preparing 1mmol/L of the chemiluminescent probe aqueous solution prepared in the example 1, and then reacting sodium nitrite and hydrogen peroxide in a dilute hydrochloric acid medium to obtain a solution containing 1 mu mol/L of ONOO – Peroxynitrite solution of free radical, peroxynitrite solution is added into probe solution, and the maximum absorption wavelength, the maximum emission wavelength and Stokes shift before and after the addition are detected, and the test results are shown in table 1:
TABLE 1 test results
As can be seen from the above test results, the addition of peroxynitrite to the aqueous solution of chemiluminescent probe prepared in example 1 resulted in a significant change in the maximum absorption wavelength, maximum emission wavelength, and Stokes shift of the solution, wherein the maximum emission wavelength was changed from 527nm (green) to 654nm (red), and the chemiluminescent probe was seen to be active on ONOO – The response is sensitive, and the imaging detection of the peroxynitrite in the line granule body can be realized.
Meanwhile, the change of fluorescence flux before and after the peroxynitrite solution is added is recorded, wherein the initial fluorescence flux phi=0.04 of the solution, and after the peroxynitrite solution is added, the fluorescence flux of the solution is increased by 10-30 times within 5-30min, which indicates that the fluorescence detection intensity of the chemical fluorescence probe provided by the invention on peroxynitrite is high.
(2) Relationship between substance concentration and fluorescence intensity
Aqueous solutions of chemiluminescent probes were prepared at different concentrations according to the data in Table 2, and peroxynitrite solutions containing 1. Mu. Mol/L ONOO-radicals were added, respectively, and the fluorescence intensity was measured under 365nm ultraviolet light and the standard intensity (F/F) was recorded 0 ) Wherein F 0 The fluorescence intensity before the peroxynitrite is added, and F is the fluorescence intensity 30min after the peroxynitrite is added. The results are shown in Table 2:
TABLE 2 relationship between substance concentration and fluorescence intensity
| Concentration (mu mol/L) | 0 | 5 | 10 | 15 | 20 | 25 | 30 |
| Standard intensity (F/F) 0 ) | 0.87 | 1.35 | 1.86 | 2.51 | 3.04 | 3.37 | 4.03 |
It can be seen that the fluorescence intensity of the chemiluminescent probe aqueous solution in a certain concentration has a linear positive correlation with the probe concentration.
(3) Detection sensitivity test:
the chemiluminescent probe solutions (final concentration: 1. Mu. Mol/L) were added to peroxynitrite solutions (final concentrations: 5, 10, 25, 50, 100, 250 nmol/L) of different ONO-concentrations, respectively, to react, and the fluorescence spectra of the reacted solutions were measured. The results show that the chemiluminescent probes generate a change of a turn on type fluorescent signal during the ONO-reaction (within the range of 5-250 nmol/L) with different concentrations, which shows that the fluorescent detection sensitivity is high and the application range is wide.
(4) Fluorescence selectivity test:
adding the following anionic compounds which are common in nature or human body and comprise H into 1mmol/L chemiluminescent probe aqueous solution respectively 2 S、KCl、NaI、MgSO 4 、KNO 3 、NaNO 2 、Na 2 CO 3 、NaHCO 3 、NaOAc、H 2 O 2 NaClO and common active small molecules, including glutathione, cysteine, glutamic acid, proline, serine, 5-aminolevulinic acid, arginase (1. Mu. Mol/L), found that the fluorescence emission intensity of these solution samples was not significantly changed compared to that of the fluorescence probe alone (phi=0.03), indicating that the fluorescence probe was highly responsive and selective only to ONOO-and not susceptible to H 2 O 2 、ClO - The interference has the advantage of high accuracy when being applied to the fluorescent detection of the peroxynitrite in the linear granule.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (10)
1. A coumarin-based chemiluminescent probe, characterized by the following molecular structural expression:
2. a method for preparing a coumarin-based chemiluminescent probe, comprising the steps of:
s1, dissolving 2- (4-nitrophenyl) -2-oxo acetic acid and 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate in an organic solvent A, then adding N, N-diisopropylethylamine, stirring at room temperature for 10-30min, finally adding 4-amino benzyl alcohol, and stirring at room temperature for reacting for 12-24h to obtain an intermediate product;
s2, dissolving potassium carbonate in the organic solvent B, adding the intermediate product and 7-bromo-2H-benzopyran-2-one, and stirring for reacting for 10-36 hours to obtain the chemical fluorescent probe.
3. The method for preparing a coumarin-based chemiluminescent probe of claim 2 wherein in step S1, the molar ratio of 2- (4-nitrophenyl) -2-oxoacetic acid, 4-aminobenzyl alcohol, 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine is 1 (1-1.5): 1-2): 20-50.
4. A method for preparing a coumarin-based chemiluminescent probe according to claim 3 wherein in step S1, the organic solvent a is selected from one or more of anhydrous dichloromethane, chloroform, tetrahydrofuran, acetonitrile.
5. The method for preparing a coumarin-based chemiluminescent probe of any one of claims 2-4 wherein after the completion of the reaction in step S1, dichloromethane is added to dilute the reaction solution, and the solution is washed several times with saturated saline solution, anhydrous NaSO 4 And (5) performing silica gel column chromatography after drying, and purifying to obtain an intermediate product.
6. The method for preparing a coumarin-based chemiluminescent probe according to any one of claims 2-4 wherein the molar ratio of the intermediate to 7-bromo-2H-benzopyran-2-one in step S2 is 1 (1-1.5).
7. The method for preparing a coumarin-based chemiluminescent probe of claim 6 wherein in step S2 the amount of potassium carbonate is 10-20 times the molar amount of the intermediate.
8. The method for preparing a coumarin-based chemiluminescent probe of claim 6 wherein in step S2, the organic solvent B is selected from one or more of N, N-dimethylformamide, dimethylsulfoxide, toluene, and xylene.
9. The method for preparing a coumarin-based chemiluminescent probe of claim 8 wherein the step S2 is completed and the chemiluminescent probe is obtained by silica gel column chromatography.
10. Use of the coumarin-based chemiluminescent probe of claim 1 and the coumarin-based chemiluminescent probe prepared by the method of any one of claims 2-9 in the field of detection of endogenous peroxynitrite.
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| US20140248218A1 (en) * | 2013-02-08 | 2014-09-04 | The Regents Of The University Of California | Ros-sensitive fluorescent probes |
| CN106543125A (en) * | 2016-10-25 | 2017-03-29 | 中国工程物理研究院化工材料研究所 | Based on coumarin to NO2The fluorescent probe of enhancement effect of fluorescence and its preparation method and application |
| CN111848661A (en) * | 2020-08-03 | 2020-10-30 | 南通大学 | Coumarin fluorescent probe and preparation method and application thereof |
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| US20140248218A1 (en) * | 2013-02-08 | 2014-09-04 | The Regents Of The University Of California | Ros-sensitive fluorescent probes |
| CN106543125A (en) * | 2016-10-25 | 2017-03-29 | 中国工程物理研究院化工材料研究所 | Based on coumarin to NO2The fluorescent probe of enhancement effect of fluorescence and its preparation method and application |
| CN111848661A (en) * | 2020-08-03 | 2020-10-30 | 南通大学 | Coumarin fluorescent probe and preparation method and application thereof |
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| Title |
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| ALEKSANDRA GRZELAKOWSKA ET AL: "Water-soluble cationic boronate probe based on coumarin imidazolium scaffold: Synthesis, characterization, and application to cellular peroxynitrite detection", FREE RADICAL BIOLOGY AND MEDICINE, vol. 179, pages 34, XP086922400, DOI: 10.1016/j.freeradbiomed.2021.12.260 * |
| LULING WU ET AL: "Coumarin-based fl uorescent probe for the rapid detection of peroxynitrite‘ AND’ biological thiols", RSC ADV., vol. 10, pages 13496 - 13499 * |
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