CN114195811B - Synthesis of difunctional fluorescent probe and application of difunctional fluorescent probe in simultaneous distinguishing detection of hydrogen peroxide and HClO - Google Patents
Synthesis of difunctional fluorescent probe and application of difunctional fluorescent probe in simultaneous distinguishing detection of hydrogen peroxide and HClO Download PDFInfo
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- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 27
- 230000015572 biosynthetic process Effects 0.000 title claims description 11
- 238000003786 synthesis reaction Methods 0.000 title claims description 11
- 238000001514 detection method Methods 0.000 title abstract description 22
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 66
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 15
- -1 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione Chemical compound 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 12
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 12
- 239000003068 molecular probe Substances 0.000 claims description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000005977 Ethylene Substances 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 6
- 125000004527 pyrimidin-4-yl group Chemical group N1=CN=C(C=C1)* 0.000 claims description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000002194 synthesizing effect Effects 0.000 claims description 5
- 230000001588 bifunctional effect Effects 0.000 claims description 4
- SHHGMESNVFQZIV-UHFFFAOYSA-N 2-pyrimidin-4-ylacetonitrile Chemical compound N#CCC1=CC=NC=N1 SHHGMESNVFQZIV-UHFFFAOYSA-N 0.000 claims description 3
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 125000004076 pyridyl group Chemical group 0.000 claims description 3
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 claims description 2
- 238000010992 reflux Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 21
- 230000005284 excitation Effects 0.000 abstract description 10
- 239000003086 colorant Substances 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 238000000799 fluorescence microscopy Methods 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000008346 aqueous phase Substances 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 102000003896 Myeloperoxidases Human genes 0.000 description 2
- 108090000235 Myeloperoxidases Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- RFKJHQXSLBUONF-UHFFFAOYSA-N methyl blue free acid Chemical compound C1=CC(S(=O)(=O)O)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=NC=2C=CC(=CC=2)S(O)(=O)=O)C=2C=CC(NC=3C=CC(=CC=3)S(O)(=O)=O)=CC=2)C=C1 RFKJHQXSLBUONF-UHFFFAOYSA-N 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- IVDFJHOHABJVEH-UHFFFAOYSA-N pinacol Chemical compound CC(C)(O)C(C)(C)O IVDFJHOHABJVEH-UHFFFAOYSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- CBUOGMOTDGNEAW-UHFFFAOYSA-N 2-[4-(bromomethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane Chemical compound O1C(C)(C)C(C)(C)OB1C1=CC=C(CBr)C=C1 CBUOGMOTDGNEAW-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000002781 deodorant agent Substances 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000003891 environmental analysis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 238000002407 reforming Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
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Abstract
The invention discloses aThe species simultaneously distinguish hydrogen peroxide (H) by two different fluorescence emission signals, red and green 2 O 2 ) And hypochlorous acid (HClO), the fluorescent probe having the chemical structural formula:
. The fluorescent probe can utilize the probe and H under the same detection condition 2 O 2 Different chemical reactions with HClO are carried out to generate different fluorescent substances, thereby emitting fluorescence of red and green colors under specific excitation wavelength, and achieving the purpose of simultaneously distinguishing and detecting H 2 O 2 And HClO. Probe and H 2 O 2 Red light of 648nm was emitted at 560nm excitation wavelength after reaction, and green light of 548nm was emitted at 427nm excitation wavelength after reaction with HClO. The probe detects H 2 O 2 And HClO has the advantages of high sensitivity, good selectivity, large distinguishing degree and the like, and has great application prospect in the technical fields of analytical chemistry, life science, environmental science and the like.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and in particular relates to a dual-channel method for simultaneously distinguishing hydrogen peroxide (H) 2 O 2 ) And hypochlorous acid (HClO), and the simultaneous quantitative detection of H in the environment 2 O 2 And HClO, two-channel fluorescence imaging cell endogenous H 2 O 2 And HClO applications.
Background
Reactive oxygen species (ROS, including H 2 O 2 ，O 2 - And 1 O 2 ) Maintaining a range of concentrations under normal physiological conditions plays an important role in maintaining cell morphology and essential function (anal. Chem.,2014,86,9970-9976). Active oxygen is considered as an index of oxidative stress as an endogenous metabolite. Wherein hydrogen peroxide (H) 2 O 2 ) Is one of the most important ROS. When H is 2 O 2 At a low concentration of<0.7 μm) as a second messenger within the cell may activate signaling pathways to stimulate cell proliferation, differentiation and migration. Unlike other messengers, hydrogen peroxide (H 2 O 2 ) As an oxidizing agent, hydrogen peroxide is abnormally produced under stress or under stimulation by exogenous chemical substances, resulting in oxidative stress, and the ROS produced may attack cellular structures or biomolecules such as proteins, liposomes and DNA, which are associated with aging, alzheimer's disease and cancer (Analyst, 2017,142,4522-4528). It is believed that HClO can catalyze the production of hydrogen peroxide and chloride ions in an organism by Myeloperoxidase (MPO) (anal. Chem.,2014,86,671-677). As active oxygenHClO plays an irreplaceable role in daily life and in vivo. In one aspect, HClO is used as a bleaching agent, disinfectant, deodorant, etc. On the other hand, excessive HClO causes various diseases such as cardiovascular diseases, arthritis, arteriosclerosis and cancer. Therefore, the method has important significance for accurate detection of in-vivo HClO. Currently, fluorescent probes for detecting the two have been reported, but only one of the fluorescent probes or the fluorescent probes has a small degree of distinction. In the invention, a method for simultaneously distinguishing H is developed by modifying and reforming coumarin fluorophores 2 O 2 And HClO, has the advantages of high detection sensitivity, good selectivity, large discrimination and the like, and has good application prospects in environmental analysis and biological imaging.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a method for simultaneously differentiating H 2 O 2 And HClO fluorescent molecular probes, and provide some analysis and detection methods and ideas for the fields of analysis and detection, environmental science and the like.
The invention also aims to provide a synthesis and application method of the difunctional fluorescent molecular probe, which is simple in preparation method.
The invention adopts the specific technical scheme that the synthesis of the difunctional fluorescent probe and the simultaneous distinguishing detection of H 2 O 2 And HClO, the chemical structural formula of the probe is as follows:
at the same time distinguish H 2 O 2 And a HClO bifunctional fluorescent probe, wherein the preparation method of the fluorescent molecular probe comprises the following steps:
step 1, synthesizing 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione;
a. under the protection of nitrogen, adding 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridyl [3,2,1-ij ] quinolin-11-one and a proper amount of Lawson reagent into a proper amount of dry and re-steamed toluene, stirring at 110 ℃ for 12 hours to obtain a red solution,
b. c, spin-drying the red reaction liquid in the step a, and recrystallizing ethyl acetate to obtain a red solid, namely 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione;
a. Stirring 2- (pyrimidine-4-yl) acetonitrile and 1.2 times equivalent of NaH in anhydrous acetonitrile for 30 minutes at normal temperature under the protection of argon and in a dark condition, adding an acetonitrile solution of 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione, stirring for 2 hours at normal temperature, and finally adding silver nitrate to react for 90 minutes to terminate the reaction;
b. purifying the reaction liquid by column chromatography to obtain a purple solid, namely (E) -2- (pyrimidine-4-yl) -2- (9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridine [3,2,1-ij ] quinoline-11-ethylene) acetonitrile;
step 3, synthesizing the fluorescent probe
Refluxing 1:1 equivalent of (E) -2- (pyrimidin-4-yl) -2- (9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridine [3,2,1-ij ] quinoline-11-ethylene) acetonitrile and 4-bromomethyl phenylboronic acid pinacol ester synthesized in the step 2 in acetonitrile solvent at 80 ℃ for 12 hours, separating out solid, filtering, washing and drying to obtain ink blue solid, namely the fluorescent probe.
The difunctional fluorescent molecular probe simultaneously distinguishes H 2 O 2 And HClO usage method: unless otherwise specified, probe molecules are typically dissolved for analytical detection at room temperature in an environment of 5:5 volume ratio of organic phase to aqueous phase, the organic phase being Acetonitrile (ACN), the aqueous phase being Phosphate Buffered Saline (PBS) at ph=7.4 and an aqueous solution of the analyte.
The dual-function fluorescent probe of the invention detects H 2 O 2 And HClO are specifically characterized as follows: dimethyl for molecular fluorescent probeDissolution of the base sulfoxide (DMSO), dissolution of the probe molecule in organic and aqueous (5:5, v/v) solutions, and H 2 O 2 After 30 minutes of reaction at 37 ℃, strong red fluorescence of 648nm is emitted at an excitation wavelength of 560 nm; after 15 minutes reaction with HClO at room temperature, strong green fluorescence at 548nm was emitted at 427nm excitation wavelength. The probe itself does not fluoresce significantly. Thus specific excitation and fluorescence emission signals are achieved for detection of specific analytes, and when both active oxygen species are present, the two can be well distinguished by using different excitation and fluorescence emission signals. The fluorescent molecular probe realizes the simultaneous differentiated detection of H under the same detection condition 2 O 2 And HClO for NAC, gly, ala, his, met, thr, lys, asp, glu, pro, ser, O 2 - , 1 O 2 ,ONOO - The amino acid, sulfur-containing derivatives and other active oxygen substances such as TBHP, OH, NO, n-butyl amine, aniline and the like have NO obvious response to H 2 O 2 And HClO as low as 0.136 μm and 0.28 μm, respectively. Therefore, the dual-function fluorescent molecular probe disclosed by the invention can realize high-sensitivity distinguishing detection of the two.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a bifunctional fluorescent probe of the present invention.
FIG. 2 shows the endogenous H of RAW 264.7 (macrophage) cells imaged by the dual-function fluorescent probe of the invention through fluorescence of red and green colors 2 O 2 And fluorescence imaging of HClO.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The synthetic route of the fluorescent molecular probe is shown as the following formula:
EXAMPLE 1 Synthesis of 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione
I. 2.00g (6.47 mmol) of 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridinyl [3,2,1-ij ] quinolin-11-one and 3.92g (9.72 mmol) of Lawson reagent, 1:1.5 equivalents, are added to 20mL of dry redistilled toluene under nitrogen and stirred at 110℃for 12 hours to give a red solution;
and II, spin-drying the red reaction liquid in the step a, and recrystallizing ethyl acetate to obtain red solid, namely 1.4g of 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione, wherein the yield is 66.54%.
EXAMPLE 2 Synthesis of (E) -2- (pyrimidin-4-yl) -2- (9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridin [3,2,1-ij ] quinolin-11-ethylene) acetonitrile
i. Under the protection of argon and in the dark, 0.47g (4.0 mmol) of 2- (pyrimidin-4-yl) acetonitrile and an equivalent amount of NaH (0.16 g,4.00 mmol) are stirred in anhydrous acetonitrile for 30 minutes at normal temperature, then 1.00g (3.07 mmol) of acetonitrile solution of 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione is added, and the mixture is stirred for 2 hours at room temperature, and finally 0.68g (4.0 mmol) of silver nitrate is added to react for 90 minutes to terminate the reaction;
purifying the reaction liquid by column chromatography to obtain a purple solid, namely (E) -2- (pyrimidin-4-yl) -2- (9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridine [3,2,1-ij ] quinoline-11-ethylene) acetonitrile; 1.00g, yield 79.28%.
Example 3 Synthesis of the fluorescent probes
1.00g (2.44 mmol) of (E) -2- (pyrimidin-4-yl) -2- (9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridin [3,2,1-ij ] quinolin-11-ethylene) acetonitrile and 0.72g (2.44 mmol) of pinacol 4-bromomethylbenzoborate 1:1 equivalent were refluxed in acetonitrile solvent at 80℃for 12 hours to precipitate a solid, which was filtered, washed and dried to give an ink blue solid, namely 0.70g of the fluorescent probe in 45.9% yield.
Example 4 differential detection of H by fluorescent molecular probes in an in vitro Environment 2 O 2 And the use of HClO
The invention relates to a spectrum property experiment of a difunctional fluorescent molecular probe: dissolving the probe in dimethyl sulfoxide (DMSO)Placing into 1mM probe solution, and respectively preparing 1mM HClO and H 2 O 2 An aqueous solution. The specific test mode is as follows: mu.L of 1mM probe solution, 980. Mu.L of analytically pure acetonitrile, the required amount of PBS buffer in water and the required 1mM HClO/H were taken 2 O 2 Aqueous solution in a 2mL sample tube, all tests maintained a volume ratio of organic phase to aqueous phase of 5:5 (total volume of each test sample of 2 mL), for example when test H was required 2 O 2 Probe and H at a concentration of 10. Mu.M 2 O 2 The fluorescent intensity after reaction, the sample preparation conditions are: mu.L of 1mM probe solution, 980. Mu.L of analytically pure acetonitrile, 20. Mu.L of 1mM H were taken 2 O 2 The fluorescence emission intensity of the aqueous solution and 980 mu L of PBS buffer solution in a 2mL sample tube can be measured by using 560nm excitation wavelength after shaking uniformly for 30 minutes at 37 ℃, and other testing operations are similar to the above steps. The probe molecule realizes the differentiated detection of H by using different excitation wavelengths and fluorescence emission signals 2 O 2 And HClO, has high sensitivity to H 2 O 2 The detection limits of both HClO and HClO are as low as 0.136 mu M and 0.28 mu M respectively, which is very suitable for endogenous H of living cells 2 O 2 And imaging and analysis of HClO.
EXAMPLE 5 RAW 264.7 (macrophage) endogenous H 2 O 2 And HClO dual-channel fluorescence imaging analysis RAW 264.7 cells were passaged into confocal dish cell culture medium, cultured for 24 hours under standard growth conditions, then added with appropriate amount of probe (5. Mu.M) to continue culturing for 30 minutes under standard growth conditions, then photographed under confocal fluorescence microscope, and fluorescence imaged with red and green fluorescence channels, respectively, RAW 264.7 cells endogenous H 2 O 2 And HClO, as can be seen from FIG. 2, the dual detection fluorescent probe of the present invention successfully realizes endogenous and exogenous H in cells 2 O 2 And HClO double-channel fluorescence imaging analysis, and has great application value in the fields of biochemistry, analysis detection and the like.
The synthesis of the dual-function fluorescent probe and the simultaneous differentiation of H 2 O 2 And HClO application, develop a high-efficiency simple dual-functionFluorescent probe for detecting active oxygen substances and capable of being used for simultaneously distinguishing and detecting H 2 O 2 And HClO based on the same probe and under the same detection conditions using the probe and H 2 O 2 Different chemical reactions with HClO are carried out to generate different fluorescent substances, and then fluorescence of red and green colors is emitted under specific excitation wavelength, thus achieving the purpose of simultaneous differential detection and successfully realizing the endogenous H in the double-channel simultaneous fluorescence imaging cell 2 O 2 And HClO. It is hoped to provide some ideas for the development of the two-channel and multi-channel active oxygen fluorescent probes in future. While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Thus, synthesis of bifunctional fluorescent probes having the features described herein and simultaneous differential detection of H 2 O 2 And the use of HClO, all fall within the scope of protection of this patent.
Claims (3)
1. At the same time distinguish H 2 O 2 And a bifunctional fluorescent probe for HClO, wherein the fluorescent molecular probe has a chemical structural formula shown in (1):
2. the method for synthesizing the fluorescent probe according to claim 1, wherein the preparation method of the fluorescent molecular probe comprises the following steps:
step 1, synthesizing 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione;
a. under the protection of nitrogen, adding 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridyl [3,2,1-ij ] quinolin-11-one and Lawson reagent into dry and re-steamed toluene, stirring at 110 ℃ for 12 hours to obtain a red solution,
b. c, spin-drying the red reaction liquid in the step a, and recrystallizing ethyl acetate to obtain a red solid, namely 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione;
step 2, synthesizing (E) -2- (pyrimidine-4-yl) -2- (9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridine [3,2,1-ij ] quinoline-11-ethylene) acetonitrile;
a. stirring 2- (pyrimidine-4-yl) acetonitrile and 1.2 times equivalent of NaH in anhydrous acetonitrile for 30 minutes at normal temperature under the protection of argon and in a dark condition, adding an acetonitrile solution of 9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyrido [3,2,1-ij ] quinoline-11-thione, stirring for 2 hours at normal temperature, and finally adding silver nitrate to react for 90 minutes to terminate the reaction;
b. purifying the reaction liquid by column chromatography to obtain a purple solid, namely (E) -2- (pyrimidine-4-yl) -2- (9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridine [3,2,1-ij ] quinoline-11-ethylene) acetonitrile;
step 3. Synthesis of the fluorescent Probe according to claim 1
Refluxing 1:1 equivalent of (E) -2- (pyrimidin-4-yl) -2- (9- (trifluoromethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-F ] pyridine [3,2,1-ij ] quinoline-11-ethylene) acetonitrile and 4-bromomethylbenzofuran pinacol ester synthesized in the step 2 for 12 hours at 80 ℃ in acetonitrile solvent, separating out solid, filtering, washing and drying to obtain black blue solid, namely the fluorescent probe of claim 1.
3. The use of a fluorescent probe according to claim 1 for the preparation of a device capable of detecting H in an environment 2 O 2 And HClO, and simultaneously differentiating imaging H within cells 2 O 2 And HClO.
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