CN116675623A - Fluorescent probe for detecting peroxynitrite and preparation method and use method thereof - Google Patents
Fluorescent probe for detecting peroxynitrite and preparation method and use method thereof Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 17
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 title claims abstract 20
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 239000000523 sample Substances 0.000 claims abstract description 30
- -1 3-formyl-4-hydroxystyryl Chemical group 0.000 claims abstract description 20
- CUONGYYJJVDODC-UHFFFAOYSA-N malononitrile Chemical compound N#CCC#N CUONGYYJJVDODC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims description 26
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 16
- 239000012086 standard solution Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 229960000583 acetic acid Drugs 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 239000012362 glacial acetic acid Substances 0.000 claims description 9
- 230000005284 excitation Effects 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- DPZSNGJNFHWQDC-ARJAWSKDSA-N (z)-2,3-diaminobut-2-enedinitrile Chemical compound N#CC(/N)=C(/N)C#N DPZSNGJNFHWQDC-ARJAWSKDSA-N 0.000 claims description 6
- KYPOHTVBFVELTG-UPHRSURJSA-N (z)-but-2-enedinitrile Chemical compound N#C\C=C/C#N KYPOHTVBFVELTG-UPHRSURJSA-N 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 238000002189 fluorescence spectrum Methods 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 238000000295 emission spectrum Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 13
- 230000004044 response Effects 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 7
- 238000006400 oxidative hydrolysis reaction Methods 0.000 abstract description 3
- SQEQNZJGQVHATH-UHFFFAOYSA-N 2,4,4-trimethyl-6-oxocyclohexene-1,3-dicarbonitrile Chemical compound CC1=C(C#N)C(=O)CC(C)(C)C1C#N SQEQNZJGQVHATH-UHFFFAOYSA-N 0.000 abstract description 2
- 125000003172 aldehyde group Chemical group 0.000 abstract description 2
- 239000000975 dye Substances 0.000 abstract description 2
- 238000012546 transfer Methods 0.000 abstract description 2
- 125000000879 imine group Chemical group 0.000 abstract 1
- CMFNMSMUKZHDEY-UHFFFAOYSA-M peroxynitrite Chemical compound [O-]ON=O CMFNMSMUKZHDEY-UHFFFAOYSA-M 0.000 description 55
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- 239000010413 mother solution Substances 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 4
- 229920002554 vinyl polymer Polymers 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 150000007857 hydrazones Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 150000002466 imines Chemical group 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 244000005700 microbiome Species 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 238000006396 nitration reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000007248 oxidative elimination reaction Methods 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- HXITXNWTGFUOAU-UHFFFAOYSA-N phenylboronic acid Chemical class OB(O)C1=CC=CC=C1 HXITXNWTGFUOAU-UHFFFAOYSA-N 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000003244 pro-oxidative effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000000954 titration curve Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C255/00—Carboxylic acid nitriles
- C07C255/61—Carboxylic acid nitriles containing cyano groups and nitrogen atoms being part of imino groups bound to the same carbon skeleton
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C253/00—Preparation of carboxylic acid nitriles
- C07C253/30—Preparation of carboxylic acid nitriles by reactions not involving the formation of cyano groups
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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"
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- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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Abstract
The invention discloses a fluorescent probe for detecting peroxynitrite, and a preparation method and a use method thereof. The invention utilizes (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile to construct a classical Intramolecular Charge Transfer (ICT) system. The probe has good ICT effect and almost no fluorescence, but under the condition of peroxynitrite, peroxynitrite and imine part on the probe molecule are subjected to oxidative hydrolysis reaction to be broken to generate aldehyde group, so that the ICT effect of the molecule is blocked, and the probe molecule emits strong red fluorescence. The open type peroxynitrite probe of the dicyanoisophorone dye provided by the invention has good response to peroxynitrite solution, can realize sensitive and quantitative detection of trace peroxynitrite in a sample, and has the advantages of simplicity and convenience in operation, low cost, sensitive response, easiness in popularization and application and the like.
Description
Technical Field
The invention belongs to the field of organic micromolecule fluorescent probes, and particularly relates to 2-amino-3- (((E) -5- ((E) -2- (3- (dicyanomethylene) -5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) maleonitrile used as a peroxynitrite fluorescent probe, and a preparation method and a use method thereof.
Background
Peroxynitrite (ONOO) - ) Is typically endogenous active nitrogen, usually composed of the highly active free radical Nitric Oxide (NO) and superoxide (O) 2 ·- ) Is generated in situ in organism, and has high oxidation and high nitration activity. Based on these characteristics, it exhibits a bi-directional effect (i) positive: peroxynitrite is an indispensable physiological activator and signal molecule in organisms. ONOO under normal physiological conditions - As a signaling molecule, modulates a range of physiological homeostasis. ONOO (oxide-nitride-oxide) - Helps to regulate the dynamic redox homeostasis of organisms and to resist invasion by exogenous microorganisms. It can also regulate redox-sensitive transcription factors in cells and affect the expression of subsequent genes. (ii) negative: when ONOO is formed - If the level exceeds a reasonable value, the living system may fail to react and utilize the excess ONOO - Causing the redox state of the cell to change to a pro-oxidative state. This state induces oxidation of biomolecules, resulting in damage to organelles and subcellular organelles ultimately inducing severe inflammation and diseases such as cardiovascular disease, neurodegenerative disease, alzheimer's disease, and even cancer. Thus, a rapid, sensitive and selective detection of ONOO was developed - Is helpful for better research of ONOO - The dynamic action in biochemistry is also helpful for early diagnosis and treatment of diseases, and has important significance.
Fluorescent detection methods are widely focused by researchers because of their excellent detection sensitivity and selectivity and the ability to perform real-time, on-line detection of samples to be detected.
The small molecule fluorescent probe for detecting peroxynitrite which has been developed at present is designed mainly by utilizing the selective oxidation reaction, deprotection reaction, oxidative cleavage reaction and the like of peroxynitrite as the basis. Under the condition that peroxynitrite exists, the detection group in the probe molecule reacts with peroxynitrite specifically, so that the original PET or ICT effect of the molecule is changed, the fluorescence property of the probe molecule is changed, and the specific recognition of peroxynitrite is realized.
However, based on reactive peroxynitrite fluorescent probes (see review, Q.Ma, S.Xu, Z.Zhai, K.Wang, X.Liu, H.Xiao, S.Zhuo, Y.Liu, chem.Eur.J.2022,28, e 202200828.) including the oxidation of phenylboronic acids or esters to phenolic compounds, selective oxidative hydrolysis of oximes or hydrazones, etc. have become the common selective recognition mechanism for peroxynitrite fluorescent probes. However, these reported probes still have the defects of poor stability of fluorophores, difficult synthesis, short detection wavelength, inability to be used for visualizing fluctuation of peroxynitrite in a real-time system, and the like, which are unfavorable for accurate evaluation of dynamic level of peroxynitrite in a complex system.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a fluorescent open type fluorescent probe (DCI-OH-DAMN) for quantitatively detecting peroxynitrite. The invention can quantitatively detect the trace peroxynitrite in the sample.
The core of the invention is to construct a classical Intramolecular Charge Transfer (ICT) system by using (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile. The probe has good ICT effect and almost no fluorescence, but under the condition of peroxynitrite, peroxynitrite and imine part on the probe molecule are subjected to oxidative hydrolysis reaction to generate aldehyde group, thereby blocking ICT effect of the molecule, and the probe molecule emits strong red fluorescence.
The peroxynitrite fluorescent probe is named DCI-OH-DAMN, and the molecular formula is C 24 H 20 N 6 O has a structural formula shown in formula (I):
the preparation method of the fluorescent probe comprises the following steps: an amount of (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-en-1-ylidene malononitrile, diaminomaleonitrile and glacial acetic acid are dissolved in absolute ethanol and reacted for a certain time, after which the compound 2-amino-3- (((E) -5- ((E) -2- (3- (dicyano-methylene) -5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) maleonitrile is obtained after filtration, namely: DCI-OH-DAMN.
The preparation reaction formula of the probe is as follows:
preferably, the molar ratio of the (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile, the diaminomaleonitrile and the glacial acetic acid is 1:1-2:3-5; (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile and absolute ethyl alcohol in a mass volume ratio of 1:10-200; the reaction temperature is 0-50 ℃ and the reaction time is 1-24 hours.
Preferably, the molar ratio of the (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile, the diaminomaleonitrile and the glacial acetic acid is 1:1:3.
Preferably, the mass volume ratio of the (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile to the absolute ethyl alcohol is 100.
Preferably, the reaction temperature is 50 ℃.
Preferably, the reaction time is 24 hours.
The use method of the peroxynitrite fluorescent probe comprises the following steps:
step 1: adding the compound shown in the formula (I) in the same concentration into phosphate buffer solutions (10 mM, pH=7.4) with different peroxynitrite concentrations, and preparing standard solutions containing the compound shown in the formula (I) with at least 5 different peroxynitrite contents;
the concentration of the compound shown in the formula (I) in the standard solution is 1 nM-10 mu M;
the content of peroxynitrite in the standard solution is 0.1 nM-1 mM;
step 2: respectively measuring fluorescence emission spectra of the standard solutions, wherein the excitation wavelength is 532nm, the concentration of peroxynitrite is taken as an abscissa, and I is taken as 653 Establishing a standard curve for the ordinate;
I 653 representing the fluorescence emission peak intensity value of the standard solution at the wavelength of 653 nm;
step 3: adding a compound shown in a formula (I) into a sample to be detected, and controlling the concentration of the compound to be detected to be equal to that of the compound shown in the formula (I) in the standard solution; and measuring the fluorescence emission spectrum of the sample under excitation light with excitation wavelength of 532nm, namely calculating the peroxynitrite content of the sample to be measured according to a standard curve.
The invention has the following characteristics:
1) The fluorescent probe provided by the invention is brown solid powder and has a stable structure.
2) According to the fluorescent probe provided by the invention, the solution is sensitive to the concentration of peroxynitrite, and along with the increase of the concentration of peroxynitrite, the fluorescence of the aqueous solution is observed to be changed from weak fluorescence to bright red fluorescence under an ultraviolet lamp.
3) The fluorescent probe provided by the invention has the emission wavelength of 653nm, is a fluorescent 'on' type response, has larger change (about 30 times) of fluorescent intensity before and after reaction, can greatly eliminate the influence of detection condition difference on the result during detection, and improves the detection sensitivity.
4) The fluorescent probe provided by the invention has a linear relation to the concentration of peroxynitrite, and can be used for accurately measuring the concentration of peroxynitrite.
The 'open' -type peroxynitrite probe based on the dicyanoisophorone dye provided by the invention has good response to peroxynitrite solution, can realize sensitive quantitative detection of peroxynitrite in a sample, and has the advantages of simplicity and convenience in operation, low cost, sensitive response, easiness in popularization and application and the like.
Drawings
Fig. 1: nuclear magnetic resonance hydrogen spectrum of fluorescent probe DCI-OH-DAMN.
Fig. 2: color response plot of fluorescent probe DCI-OH-DAMN to peroxynitrite phosphate buffer solution.
Fig. 3: fluorescent response plot of fluorescent probe DCI-OH-DAMN to peroxynitrite phosphate buffer solution.
Fig. 4: ultraviolet absorption spectrum of fluorescent probe DCI-OH-DAMN before and after reaction with peroxynitrite in phosphate buffer solution, wherein the probe concentration is 10.0 mu M, and the peroxynitrite concentration is 20.0 mu M.
Fig. 5: fluorescence titration curve of peroxynitrite in phosphate buffer solution of fluorescent probe DCI-OH-DAMN, wherein excitation wavelength is 532nm, and probe concentration is 10.0 mu M.
Fig. 6: fluorescent response diagram of fluorescent probe DCI-OH-DAMN to common active oxygen small molecule, halogen ion, anion and metal ion, wherein excitation wavelength is 532nm, probe concentration is 10.0 mu M, and analyte concentration is 100.0 mu M.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below were obtained commercially unless otherwise specified.
The numbers of the compounds in the examples correspond to the numbers in the above-mentioned compounds.
Example 1 synthesis of the Compound DCI-OH-DAMN.
Synthesis of the Compound 2-amino-3- (((E) -5- ((E) -2- (3- (dicyandiamide-5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) maleonitrile.
5.0g of (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-ylidene malononitrile (1) (15.70 mmol), 1.7g of diamino Ma Laijing (2) (15.70 mmol) were dissolved in 500mL of absolute ethanol and 2.70mL of glacial acetic acid (3) (47.11 mmol) were added and reacted at 50℃for 24 hours, after filtration 3.1g of the compound 2-amino-3- (((E) -5- ((E) -2- (3- (dicyandiamide) -5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) Ma Laijing (4) was obtained in 48% yield.
1 H NMR(400MHz,DMSO-d 6 ) δ10.96 (s, 1H), 8.58 (s, 1H), 8.43 (d, j=2.2 hz, 1H), 8.01 (s, 2H), 7.69 (dd, j=8.6, 2.3hz, 1H), 7.33 (d, j=16.1 hz, 1H), 7.23 (d, j=16.1 hz, 1H), 6.99 (d, j=8.6 hz, 1H), 6.85 (s, 1H), 2.63 (s, 2H), 2.55 (s, 2H), 1.03 (s, 6H); as shown in fig. 1;
example 2, color response of the compound DCI-OH-DAMN to peroxynitrite.
Preparing a dimethyl sulfoxide mother liquor solution of the fluorescent probe DCI-OH-DAMN for detecting peroxynitrite, which is prepared by the method, with the concentration of 0.1 mM. 1mL of the mother solution is measured, 8.9mL of phosphate buffer solution is used for dilution, then peroxynitrite solution with a certain concentration is added, finally, the phosphate buffer solution is used for fixing the volume to 10mL, so that the concentration of a probe in the test solution is 10.0 mu M, and the concentration of peroxynitrite is 10.0 mu M for color response test. As shown in fig. 2 and 3, after the peroxynitrite solution is added, the color of the solution is visually observed to be changed from pale yellow to light red, and meanwhile, the fluorescence of the solution is also changed from weak fluorescence to bright red fluorescence, so that the probe DCI-OH-DAMN has visual color development response to the peroxynitrite.
Example 3 uv titration detection of compound DCI-OH-DAMN with different concentrations of peroxynitrite.
Preparing a dimethyl sulfoxide mother liquor solution of the fluorescent probe DCI-OH-DAMN for detecting peroxynitrite, which is prepared by the method, with the concentration of 0.1 mM. 1mL of the mother solution is measured, 8.9mL of phosphoric acid buffer solution is used for dilution, then peroxynitrite solution with a certain concentration is added, finally the volume is fixed to 10mL by the phosphoric acid buffer solution, so that the concentration of a probe in the test solution is 10.0 mu M, and the concentration of peroxynitrite is 0 and 20.0 mu M respectively for absorption spectrum test. As shown in FIG. 4, after adding a certain amount of peroxynitrite, the ultraviolet absorption peak was red-shifted from 413nm to 505nm.
Example 4 fluorescence titration detection of different concentrations of peroxynitrite on the compound DCI-OH-DAMN.
Preparing a dimethyl sulfoxide mother liquor solution of the fluorescent probe DCI-OH-DAMN for detecting peroxynitrite, which is prepared by the method, with the concentration of 0.1 mM. 1mL of the mother solution is measured, diluted by 8.9mL of phosphoric acid buffer solution, then peroxynitrite solution with a certain concentration is added, finally, the volume is fixed to 10mL by the phosphoric acid buffer solution, so that the concentration of a probe in the test solution is 10.0 mu M, and the concentration of peroxynitrite is 0-30.0 mu M for fluorescence detection (λex=532 nm). Obtaining the fluorescence intensity in each system, and establishing a standard curve of the fluorescence intensity and the peroxynitrite concentration. As shown in FIG. 5, as the peroxynitrite concentration increases, the fluorescence intensity at 653nm of the system gradually increases, and when the peroxynitrite concentration reaches 20.0. Mu.M, the fluorescence intensity of the reaction system reaches a maximum.
Example 5, selectivity of the compound DCI-OH-DAMN for different common reactive oxygen species, halogen ions, anions and metal ions.
Preparing a dimethyl sulfoxide mother liquor solution of the fluorescent probe DCI-OH-DAMN for detecting peroxynitrite, which is prepared by the method, with the concentration of 0.1 mM. Solutions of various small molecules to be tested were prepared at a concentration of 10mM for use. 1mL of the mother solution is measured, diluted by 8.9mL of phosphoric acid buffer solution, then a solution of an object to be detected with a certain concentration is added, finally the volume is fixed to 10mL by the phosphoric acid buffer solution, so that the concentration of a probe in the test solution is 10.0 mu M, and the concentration of a small molecule to be detected is 20.0 mu M for fluorescence detection (λex=532 nm). The fluorescence spectrum of each system is shown in fig. 6, and the fluorescence of the probe DCI-OH-DAMN is hardly influenced by common small molecules to be detected.
Example 6 synthesis of the compound 2-amino-3- (((E) -5- ((E) -2- (3- (dicyandiamide-5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) maleonitrile.
5.0g of (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-ylidene malononitrile (15.70 mmol), 2.55g of diamino Ma Laijing (23.55 mmol) were dissolved in 100mL of absolute ethanol, 3.60mL of glacial acetic acid (62.80 mmol) were added and reacted at 50℃for 24 hours, and after filtration the residue was washed with absolute ethanol to give 2.7g of the compound 2-amino-3- (((E) -5- ((E) -2- (3- (dicyanomethylene) -5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) maleimide in 42% yield.
Example 7 synthesis of the compound 2-amino-3- (((E) -5- ((E) -2- (3- (dicyandiamide-5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) maleonitrile.
5.0g of (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-ylidene malononitrile (15.70 mmol), 3.4g of diamino Ma Laijing (31.40 mmol) were dissolved in 800mL of absolute ethanol, 4.50mL of glacial acetic acid (78.50 mmol) were added and reacted at 50℃for 24 hours, and after filtration the residue was washed with absolute ethanol to give 2.9g of the compound 2-amino-3- (((E) -5- ((E) -2- (3- (dicyanomethylene) -5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) Ma Laijing in 45% yield.
Claims (8)
1. A fluorescent probe for detecting peroxynitrite, characterized in that: its molecular formula is C 24 H 20 N 6 O, DCI-OH-DAMN for short, has a structural formula (I);
2. the method for preparing a fluorescent probe for detecting peroxynitrite according to claim 1, wherein the synthesis steps are as follows:
dissolving (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-en-1-ylidene malononitrile, diaminomaleonitrile and glacial acetic acid in absolute ethanol, and reacting for a period of time to obtain 2-amino-3- (((E) -5- ((E) -2- (3- (dicyanomethylene) -5, 5-dimethylcyclohexenyl) vinyl) -2-hydroxybenzylidene) amino) maleonitrile, namely: DCI-OH-DAMN.
3. The method for preparing the fluorescent probe for detecting peroxynitrite according to claim 2, which is characterized in that:
the molar ratio of the (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile, diaminomaleonitrile and glacial acetic acid is 1:1-2:1-5; (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile and absolute ethyl alcohol in a mass volume ratio of 1:20-200; the reaction temperature is 0-50 ℃ and the reaction time is 1-24 hours.
4. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, wherein: the molar ratio of the (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile, diaminomaleonitrile and glacial acetic acid is 1:1:3.
5. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, wherein: the mass volume ratio of the (E) -2- (3-formyl-4-hydroxystyryl) -5, 5-dimethylcyclohexyl-2-alkene-1-subunit malononitrile to the absolute ethyl alcohol is 1:100.
6. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, wherein: the reaction temperature was 50 ℃.
7. A method for preparing a fluorescent probe for detecting peroxynitrite according to claim 2 or 3, wherein: the reaction time was 24 hours.
8. A method of using a fluorescent probe for detecting peroxynitrite according to claim 1; the method is characterized in that:
1) Adding the compound shown in the formula (I) with the same concentration into phosphate buffer solutions with different concentrations of peroxynitrite, and preparing at least 5 standard solutions with different peroxynitrite contents and containing the compound shown in the formula (I);
the concentration of the compound shown in the formula (I) in the standard solution is 1 nM-10 mu M;
the content of peroxynitrite in the standard solution is 0.1 nM-1 mM;
2) Determination of the fluorescence emission of the standard solutions, respectivelyThe emission spectrum, the excitation wavelength is 532nm, the concentration of peroxynitrite is taken as the abscissa, I 653 Establishing a standard curve for the ordinate;
I 653 representing the fluorescence emission peak intensity value of the standard solution at the wavelength of 653 nm;
3) Adding a compound shown in a formula (I) into a sample to be detected, and controlling the concentration of the compound to be detected to be equal to that of the compound shown in the formula (I) in the standard solution; and measuring the fluorescence emission spectrum of the sample under excitation light with excitation wavelength of 532nm, namely calculating the peroxynitrite content of the sample to be measured according to a standard curve.
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