CN113501818B - Fluorescent probe molecule and preparation method and application thereof - Google Patents
Fluorescent probe molecule and preparation method and application thereof Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 71
- 238000002360 preparation method Methods 0.000 title abstract description 8
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims abstract description 46
- 238000001514 detection method Methods 0.000 claims abstract description 15
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical group NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims abstract description 11
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960000907 methylthioninium chloride Drugs 0.000 claims abstract description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 135
- 239000000243 solution Substances 0.000 claims description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 17
- 230000002829 reductive effect Effects 0.000 claims description 16
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 15
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
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- 229940126214 compound 3 Drugs 0.000 claims description 13
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- CYPYTURSJDMMMP-WVCUSYJESA-N (1e,4e)-1,5-diphenylpenta-1,4-dien-3-one;palladium Chemical compound [Pd].[Pd].C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1 CYPYTURSJDMMMP-WVCUSYJESA-N 0.000 claims description 7
- DTUOTSLAFJCQHN-UHFFFAOYSA-N 4-bromo-1,8-naphthalic anhydride Chemical compound O=C1OC(=O)C2=CC=CC3=C2C1=CC=C3Br DTUOTSLAFJCQHN-UHFFFAOYSA-N 0.000 claims description 7
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 7
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- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 7
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 claims description 7
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- UXRZLDREKITWRO-UHFFFAOYSA-N P(c1ccccc1)c1ccccc1.CC1(C)c2ccccc2Oc2ccccc12 Chemical compound P(c1ccccc1)c1ccccc1.CC1(C)c2ccccc2Oc2ccccc12 UXRZLDREKITWRO-UHFFFAOYSA-N 0.000 claims description 4
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- 238000010438 heat treatment Methods 0.000 claims description 4
- MTVNAPYHLASOSX-UHFFFAOYSA-N 9,9-dimethylxanthene Chemical compound C1=CC=C2C(C)(C)C3=CC=CC=C3OC2=C1 MTVNAPYHLASOSX-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
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- 238000004440 column chromatography Methods 0.000 claims description 2
- 239000008247 solid mixture Substances 0.000 claims description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 abstract description 19
- 125000003368 amide group Chemical group 0.000 abstract description 3
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 6
- 230000008859 change Effects 0.000 description 6
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- 238000010586 diagram Methods 0.000 description 5
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- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- SSMIFVHARFVINF-UHFFFAOYSA-N 4-amino-1,8-naphthalimide Chemical class O=C1NC(=O)C2=CC=CC3=C2C1=CC=C3N SSMIFVHARFVINF-UHFFFAOYSA-N 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
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- 201000006306 Cor pulmonale Diseases 0.000 description 1
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- 230000000259 anti-tumor effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical group C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 description 1
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- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- QBWCMBCROVPCKQ-UHFFFAOYSA-N chlorous acid Chemical compound OCl=O QBWCMBCROVPCKQ-UHFFFAOYSA-N 0.000 description 1
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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- 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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention provides a fluorescent probe molecule, relates to the technical field of organic functional materials, and is used for simultaneously monitoring hypochlorous acid and pH; a fluorescent probe molecule is methylene blue-4-aminonaphthalimide derivative, which has an amide group reacting with hypochlorous acid and an o-phenylenediamine group responding to pH value; HClO has stronger oxidability, can break amide bonds in fluorescent probe molecules to generate an unstable intermediate, finally releases a methylene blue fluorophore with positive charges, and generates fluorescence (690nm) in a near-infrared region; when the proton is combined with 4-site o-phenylenediamine, the PET effect is inhibited, so that yellow green fluorescence (525nm) appears, and the selective recognition of double recognition sites and the simultaneous detection of hypochlorous acid and pH value are realized; the invention also provides a preparation method and application of the fluorescent probe molecule; the fluorescent probe molecule is used for detecting hypochlorous acid and pH in the multicellular system.
Description
Technical Field
The invention relates to the technical field of organic functional materials, in particular to a fluorescent probe molecule and a preparation method and application thereof.
Background
The optical probe molecular imaging technology has good application prospect in the fields of surgical operations and biomedicine. In recent years, fluorescence imaging technology has been rapidly developed, and has the characteristics of convenience, no damage, real-time in-situ dynamic visual monitoring, high sensitivity and high spatial-temporal resolution on biological samples, so that the fluorescence imaging technology gradually becomes an important tool in the field of research of biological fluorescent probes.
Fluorescent probes and biological living body fluorescence imaging studies aiming at detection of bioactive small molecules (reactive oxygen species (ROS), Reactive Nitrogen Species (RNS), Reactive Sulfur Species (RSS), and the like) are currently hot research spots. Compared with a single detection fluorescent probe, the design and synthesis of a double-site cooperative response fluorescent probe for simultaneously detecting two related bioactive small molecules and the fluorescent imaging research are more difficult and development trends at present. Fluorescent probes based on dual recognition sites for detection of different or identical active species have been reported, however, the reported dual recognition site fluorescent probes have limited detection of active species, mainly focusing on selective recognition of biological thiols (Cys, Hcy, GSH) and detection of few reactive oxygen and reactive sulfur species.
For example, Chinese patent "a fluorescent probe for simultaneously or separately detecting hydrogen sulfide and hypochlorous acid in a cell lysosome and a preparation method and application thereof" (application No. 201610263094.0, published: 2016.07.20) discloses a probe, which realizes that a single probe can simultaneously or separately detect multiple target molecules (H)2S, HClO and H2S/HClO) simultaneously or separately.
Also, for example, the fluorescent probe molecules disclosed in "design, synthesis and biological application of prodrug for antitumor therapy and fluorescent probe with double recognition sites" (Chenyu, university of south China, 2020.6.3) can be used for simultaneously detecting hypochlorous acid and hydrogen sulfide.
However, the fluorescent probe can detect hypochlorous acid and hydrogen sulfide simultaneously, but the detection of other important related substances is relatively lacked, such as hypochlorous acid and pH.
Hypochlorous acid, an important defense line against the human immune system, is produced by the reaction of chloride ions and hydrogen peroxide catalyzed by Myeloperoxidase (MPO). The hypochlorous acid level abnormality is closely related to cardiovascular disease, nervous weakening, lung injury, even cancer and the like. Organelles such as mitochondria, lysosomes, endoplasmic reticulum, etc. are maintained at a specific pH so that they maintain their respective functions in biological processes. Abnormal change of intracellular pH can induce diseases such as cardiopulmonary disease and neurodegenerative disease, so that a fluorescence research for simultaneously monitoring HClO and pH is designed and synthesized, and the research on the relation between HClO and pH in physiological and pathological processes has important significance for prevention and diagnosis of diseases.
Disclosure of Invention
The present invention is directed to overcoming at least one of the above-mentioned disadvantages of the prior art and providing a fluorescent probe molecule that can simultaneously monitor hypochlorous acid and pH.
Another objective of the present invention is to provide a method for preparing a fluorescent probe molecule.
It is a further object of the present invention to provide the use of fluorescent probe molecules.
The technical scheme adopted by the invention is that the fluorescent probe molecule has the following structure:
in the invention, the fluorescent probe molecule is a methylene blue-4-aminonaphthalimide derivative which has clear structure and stable optical property and simultaneously responds to hypochlorous acid and pH; has amide groups which react with hypochlorous acid, and o-phenylenediamine groups which respond to pH. HClO has stronger oxidability, can break amide bonds in fluorescent probe molecules to generate an unstable intermediate, finally releases a methylene blue fluorophore with positive charges, and generates fluorescence (690nm) in a near-infrared region; when the proton is combined with 4-site o-phenylenediamine, the PET effect is inhibited, so that yellow green fluorescence (525nm) appears, and the selective recognition of double recognition sites and the simultaneous detection of hypochlorous acid and pH value are realized. The probe molecule is originally designed according to the idea that the probe molecule firstly has an amide group which reacts with hypochlorous acid, and secondly the 4-position of naphthalimide has a phthalic diamine group which reacts with nitric oxide, but the phthalic diamine group of the probe molecule is not responded to the nitric oxide through spectral research, and the group is unexpectedly found to respond to the pH value.
A method for preparing the fluorescent probe molecule comprises the following steps:
s1: dissolving methylene blue and sodium carbonate in distilled water and dichloromethane, stirring, slowly adding a sodium thiosulfate aqueous solution, continuously stirring until the solution turns yellow, cooling the solution, slowly dropwise adding a dichloromethane solution of bis (trichloromethyl) carbonate, continuously stirring, then pouring into ice water, extracting with dichloromethane, drying an organic phase, filtering, distilling under reduced pressure, collecting, separating and purifying by a silica gel chromatographic column to obtain a compound 4;
s2: dissolving the compound 4 obtained in the step S1 in dichloromethane, slowly dripping dichloromethane solution of ethylenediamine into the dichloromethane solution, continuously stirring, pouring the mixture into water, extracting, drying, distilling under reduced pressure, collecting, performing silica gel chromatography, eluting, separating and purifying to obtain a compound 3;
s3: dissolving the compound 3 obtained in the step S2 and 4-bromo-1, 8-naphthalic anhydride in absolute ethyl alcohol, and refluxing and stirring under the protection of nitrogen; evaporating, separating, eluting, separating and purifying to obtain the compound 2.
S4: the compound 2 obtained in S3 and o-phenylenediamine were dissolved in anhydrous toluene, and chloroform adduct of tris (dibenzylideneacetone) dipalladium, 4, 5-bis diphenylphosphine 9, 9-dimethylxanthene and cesium carbonate were sequentially added. Heating and refluxing, evaporating, separating, and purifying under the protection of nitrogen to obtain the fluorescent probe molecule as claimed in claim 1.
Further, in S1, the specific steps are: dissolving methylene blue and sodium carbonate in distilled water and dichloromethane, stirring at normal temperature for 35 minutes, then slowly adding a sodium thiosulfate aqueous solution, continuously stirring until the solution turns yellow, cooling the solution by using an ice water bath, slowly dropwise adding a dichloromethane solution of bis (trichloromethyl) carbonate, continuously stirring for 1 hour, then pouring into ice water, extracting by using dichloromethane, drying an organic phase, filtering, distilling under reduced pressure, collecting, performing silica gel chromatography, eluting, separating and purifying to obtain a compound 4;
or in S2, the specific steps are: dissolving the compound 4 obtained in the step S1 in dichloromethane, slowly dripping a dichloromethane solution of ethylenediamine into the dichloromethane solution, continuously stirring the mixture at room temperature for 2 hours, pouring the mixture into water, extracting, drying and distilling the mixture under reduced pressure, collecting a solid mixture, and performing silica gel chromatography, elution, separation and purification to obtain a compound 3;
or in S3, the specific steps are: dissolving the compound 3 obtained in S2 and 4-bromo-1, 8-naphthalic anhydride in absolute ethyl alcohol, refluxing and stirring for 3 hours under the protection of nitrogen, evaporating the solvent under reduced pressure, and passing the crude product through a silica gel chromatographic column for elution, separation and purification to obtain a compound 2;
or in S4, the specific steps are: dissolving the compound 2 obtained in S3 and o-phenylenediamine in anhydrous toluene, sequentially adding chloroform adduct of tris (dibenzylideneacetone) dipalladium, 4, 5-bis (diphenylphosphine) 9, 9-dimethylxanthene and cesium carbonate, heating and refluxing for 10 hours under the protection of nitrogen, evaporating the solvent under reduced pressure, and passing the crude product through a silica gel chromatographic column, eluting, separating and purifying to obtain the fluorescent probe molecule as claimed in claim 1.
Further, in step S1, the mass ratio of sodium thiosulfate, methylene blue, sodium carbonate, bis (trichloromethyl) carbonate is 6.66:1.72:6.66: 1; the eluent is a mixture of n-hexane and dichloromethane, and the volume ratio of the eluent to the dichloromethane is 3: 1.
Further, in step S2, the mass ratio of the compound 4 to the ethylenediamine is 1:2.5, and the eluent is a mixture of dichloromethane and methanol with a volume ratio of 10: 1.
Further, in step S3, the mass ratio of the 4-bromo-1, 8-naphthalic anhydride to the compound 3 is 228.0mg:296.0mg, the volume of the absolute ethanol is 8.00mL, and the eluent is a mixture of dichloromethane and methanol, and the volume ratio of the mixture is 100: 1.
Further, in step S4, the mass ratio of the compound 2 to the o-phenylenediamine, the chloroform adduct of tris (dibenzylideneacetone) dipalladium, the 4, 5-bis-diphenylphosphine 9, 9-dimethylxanthene and cesium carbonate is 308mg:158.8mg:21.0mg:12mg:483.0mg, and the volume of the anhydrous toluene is 5.00 mL; the column chromatography eluent is a mixture of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 50: 1.
A fluorescent probe molecule as described above is used to detect hypochlorous acid and pH simultaneously.
Further, the fluorescent probe molecule is used for detecting hypochlorous acid and pH in the multicellular.
Further, the multicellular is one of RAW264.7, HIBEC, CT26, QBC939, HuCCT 1.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention provides a novel fluorescent probe molecule which can monitor hypochlorous acid and pH simultaneously, and has high sensitivity and good selectivity;
(2) the method for synthesizing the fluorescent probe molecule has simple steps and high yield;
(3) the fluorescent probe molecule can be used for selectively identifying hypochlorous acid and pH very well, and when HClO and other active oxygen are added into the probe, the fluorescence of HClO is only obviously enhanced at 690nm by comparing bar graphs of fluorescence intensity values of various active species at 690nm (shown in figure 5), so that the fluorescent probe molecule shows high fluorescence selectivity and has good anti-interference performance. When the pH value of the buffer solution is 7.4, the probe molecule has a smaller fluorescence emission peak at 475 nm; when the pH of the buffer solution was 7.0, the fluorescence emission peak at 475nm disappeared; as the pH of the buffer solution continued to decrease, a new fluorescence emission peak appeared at 525nm, and the fluorescence intensity increased with decreasing pH (as shown in FIG. 8); the fluorescence intensity of the probe molecule at 525nm changes with the pH cycle (as shown in FIG. 9), and the fluorescence intensity of the probe molecule hardly changes when the pH is 5.5 or 7.0, which indicates that the fluorescent probe molecule has good pH adaptability and can be used for cycle detection of pH change in internal environment.
(4) The fluorescence probe molecule of the invention has good stability of physical and chemical properties.
(5) The fluorescent probe molecule of the invention has good permeability for penetrating cells and can be used for multi-cell fluorescence imaging.
Drawings
FIG. 1 shows a synthetic route of the fluorescent probe molecule of the present invention.
FIG. 2 shows a fluorescent probe molecule of the present invention1H-NMR spectrum.
FIG. 3 shows a fluorescent probe molecule of the present invention13C-NMR spectrum.
FIG. 4 is a HRMS (ESI) spectrum of a fluorescent probe molecule of the invention.
FIG. 5 is a fluorescence spectrum variation diagram of the response of the fluorescent probe molecule of the invention to hypochlorous acid in an ethanol aqueous solution system and a fluorescence intensity bar chart of different interfering ions at 690 nm.
FIG. 6 is a graph showing the change of fluorescence spectra of the fluorescent probe molecules of the present invention against hypochlorous acid of different concentrations.
FIG. 7 is a graph of the fluorescence intensity at 690nm of the fluorescent probe molecule of the present invention fitted to the corresponding hypochlorous acid concentration.
FIG. 8 is a diagram of the change of the fluorescent probe molecule of the present invention in the detection of pH recognition site fluorescence.
FIG. 9 is a graph showing the fluorescence intensity at 525nm of the fluorescent probe molecule of the present invention as a function of pH cycle.
FIG. 10 is a graph of the fluorescence intensity at 525nm of fluorescent probe molecules of the present invention fitted to corresponding different pH values.
FIG. 11 is a diagram of the fluorescent imaging of the fluorescent probe molecules of the present invention in multicellular bodies. Wherein a1-e1 is a green fluorescence channel map; a2-e2 is a red fluorescence channel map; a3-e3 is a bright field diagram; a1-a3 is RAW264.7 cells; b1-b3 is HIBEC; FIGS. c1-c3 are CT 26; FIGS. d1-d3 are QBC 939; FIGS. e1-e3 are HuCCT 1.
Detailed Description
The examples of the present invention are provided for illustrative purposes only and are not to be construed as limiting the invention.
Example 1
Synthesis of fluorescent Probe molecule based on methylene blue-4-aminonaphthalimide derivative (the synthetic route is shown in figure 1)
(1) Preparation of compound 4: methylene blue (5.0g,15.66mmol) and sodium carbonate (6.64g,62.52mmol) were dissolved in distilled water (30mL) and dichloromethane (20mL) and stirred at ambient temperature for 35 minutes, then a solution of sodium thiosulfate (10.98g,62.52mmol) in water (40mL) was slowly added thereto and stirred until the solution turned yellow, after the above solution was cooled in an ice water bath, a solution of bis (trichloromethyl) carbonate (2.78g,9.38mmol) in dichloromethane (13mL) was slowly added dropwise thereto and stirred for 1 hour, then poured into ice water and extracted with dichloromethane, the organic phase was dried, filtered, distilled under reduced pressure and collected, and the crude product was chromatographed on a silica gel column using n-hexane: dichloromethane ═ 3:1 elution gave 2.7g of compound 4 as a white solid in 50% yield.
(2) Preparation of compound 3: 284mg (0.76mmol) of compound 4 was dissolved in 8mL of dichloromethane, a solution of ethylenediamine (228mg,1.9mmol) in dichloromethane (5mL) was slowly added dropwise thereto, stirring was continued at room temperature for 2 hours, poured into water, extracted, dried, distilled under reduced pressure, collected, and the resulting crude product was subjected to silica gel chromatography, purified with dichloromethane: elution with methanol 10:1 gave 196mg of solid compound 3 in 70% yield.
(3) Preparation of compound 2: 4-bromo-1, 8-naphthalic anhydride (228.0mg,0.82mmol), compound 3(296.0mg, 0.8mmol) were dissolved in anhydrous ethanol (8mL), stirred under reflux for 3 hours under nitrogen protection, and purified by evaporation of the solvent under reduced pressure (dichloromethane/methanol 100:1, v/v) to give compound 2 as a red solid (189.3mg, 75% yield).1H NMR(500MHz,CDCl3)δ8.66(dd,J＝7.3,0.9Hz,1H),8.60(dd,J＝8.5,0.9Hz,1H),8.41(d,J＝7.8Hz,1H),8.05(t,J＝7.0Hz,1H),7.87(dd,J＝8.4,7.4Hz,1H),7.20(d,J＝8.8Hz,2H),6.63(d,J＝2.4Hz,2H),6.54(dd,J＝8.8,2.6Hz,2H),5.39(t,J＝5.4Hz,1H),4.42–4.33(m,2H),3.66(dd,J＝11.3,5.6Hz,2H),2.92(s,12H).13C NMR(125MHz,CDCl3)δ163.87,163.83,156.10,148.86,134.07,133.34,132.21,131.40,131.07,130.61,130.33,129.12,128.35,128.06,127.25,122.98,122.12,111.29,110.86,40.77,39.81,39.64.
(4) Synthesizing a double-recognition-site fluorescent probe molecule 1 based on a methylene blue-4-aminonaphthalimide derivative: compound 2(308mg,0.49mmol), o-phenylenediamine (158.8mg,1.47mmol) were dissolved in anhydrous toluene (5mL), and chloroform adduct of tris (dibenzylideneacetone) dipalladium (21.0mg,0.02mmol),4, 5-bis-diphenylphosphine 9, 9-dimethylxanthene (12mg,0.02 mmol) were added in that ordermmol), cesium carbonate (483.0mg,1.35mmol), heated at reflux under nitrogen for 10 hours. The solvent was evaporated under reduced pressure and purified by elution (dichloromethane/methanol-50: 1, v/v) to give 210mg of compound 1 as a yellowish green solid in 50% yield.1H NMR(500MHz,DMSO-d6)δ9.01(s,1H),8.91(d,J＝8.5Hz,1H),8.52(d,J＝7.1Hz,1H),8.25(d,J＝8.5Hz,1H),7.79(t,J＝7.8Hz,1H),7.76–7.71(m,2H),7.69(dd,J＝5.7,3.1Hz,2H),7.13(d,J＝8.9Hz,1H),7.10–7.07(m,1H),6.88(d,J＝7.8Hz,1H),6.67(t,J＝7.5Hz,1H),6.63(d,J＝2.5Hz,1H),6.45(dd,J＝8.0,4.6Hz,2H),6.18(t,J＝5.5Hz,1H),5.05(s,2H),4.23(t,J＝6.5Hz,1H),4.18(d,J＝5.2Hz,1H),4.02(d,J＝6.5Hz,2H),2.84(s,12H).13C NMR(125MHz,DMSO-d6)δ166.97(s),164.16(s),163.36(s),155.02(s),149.70(s),148.31(s),144.99(s),133.85(s),133.00(s),130.75(s),129.77(s),129.22(s),128.06(s),127.76(s),127.28(s),124.46(s),123.38(s),122.24(s),120.65(s),116.52(s),115.69(s),111.04(s),110.19(s),109.53(s),65.06(s),40.23(s),38.64(s).HRMS:Calcd for C37H35N7O3S[M+H]+658.2595, m/z found,658.2597, wherein, CDCl3Is deuterated trichloromethane; DMSO-d6Is deuterated dimethyl sulfoxide. The related spectrogram is shown in figures 2-4.
Example 2
This example examines the selectivity of fluorescence detection of hypochlorous acid recognition sites based on methylene blue-4-aminonaphthalimide derivatives.
DMF is adopted: the experimental conditions were controlled with PBS (0.01mol/L, pH 7.4) solution 9:1(v: v).
Methylene blue-4-aminonaphthalimide derivative is added into DMF solvent to prepare solution with the concentration of fluorescent probe molecule being 20 mu mol/L.
Dividing the sample bottles into 14 groups, respectively adding 20 mu mol/L of methylene blue-4-aminonaphthalimide derivative-based fluorescent probe molecule solution into each group of sample bottles, taking the first bottle of solution as a blank group, and respectively adding 1200 mu mol/L of HClO, NO and HSO into the other 13 groups3 -、SO3 2-、H2S、H2O2、NO3 -、ONOO-、·OH、1O2Cys, GSH and Hcy solution. After the solutions to be tested were prepared at room temperature, each test working solution was transferred to a standard quartz cuvette of 1cm × 1cm, and the fluorescence spectrum thereof was measured. The excitation wavelength was 620nm and the emission wavelength was 690 nm. The selective detection of hypochlorous acid fluorescence based on methylene blue-4-aminonaphthalimide fluorescent probe molecules is shown in figure 5 a. It can be seen that the methylene blue-4-aminonaphthalimide derivative-based fluorescent probe molecule only has an obvious fluorescence enhancement phenomenon on HClO at 690nm, the fluorescence intensity values of all ions at 690nm are selected to make a bar graph (as shown in FIG. 5 b), and FIG. 5b can intuitively show that the fluorescence selectivity of the probe on hypochlorous acid is very good.
Example 3
This example examines the quantitative fluorescent detection of hypochlorous acid based on methylene blue-4-aminonaphthalimide derivative fluorescent probe molecules.
Ethanol is adopted: the experimental conditions were controlled with PBS (0.01mol/L, pH 7.4) solution 9:1(v: v).
The methylene blue-4-aminonaphthalimide derivative-based fluorescent probe molecule is prepared into a solution with the concentration of 20 mu mol/L. HClO solutions of different solubilities were added to the solution. After the solutions to be tested were prepared at room temperature, each test working solution was transferred to a standard quartz cuvette of 1cm × 1cm, and the fluorescence spectrum thereof was measured. The size of the gap of the fluorescence test grating is 5nm multiplied by 5 nm. FIG. 6 is a fluorescence spectrum of methylene blue-4-aminonaphthalimide derivative-based fluorescent probe molecules according to the present invention in an aqueous system as a function of hypochlorous acid concentration. Fitting the fluorescence intensity at 690nm of the fluorescence spectrum with the corresponding hypochlorous acid concentration to obtain a fitting curve (shown in FIG. 7) within the range of the hypochlorous acid concentration of 0-1200. mu. mol/L, which shows that the methylene blue-naphthalimide-based fluorescent probe molecule of the invention can quantitatively detect the hypochlorous acid concentration.
Example 4
This example examines the fluorescence detection of the pH recognition site based on methylene blue-4-aminonaphthalimide derivatives.
DMF is adopted: the experimental conditions were controlled with PBS (0.01M, pH X) solution 9:1(v: v).
A solution having a concentration of 20. mu. mol/L was prepared by adding a methylene blue-based 4-aminonaphthalimide derivative to a DMF solvent. The sample bottles were divided into 13 groups, the first bottle solution pH was 7.4, and the other 12 groups had pH of 7.0,6.5,6.0,5.5,5.0,4.5,4.0,3.5,3.0,2.5,2.0,1.5, respectively. After the solutions to be tested were prepared at room temperature, each test working solution was transferred to a standard quartz cuvette of 1cm × 1cm, and the fluorescence spectrum thereof was measured. The change of the fluorescent probe molecule based on methylene blue-4-aminonaphthalimide on the pH recognition site in the fluorescence detection is shown in FIG. 8. It can be seen that when the pH of the buffer solution is 7.4, there is a small fluorescence emission peak at 475 nm; when the pH of the buffer solution was 7.0, the fluorescence emission peak at 475nm disappeared; as the pH of the buffer solution continued to decrease, a new fluorescence emission peak appeared at 525nm, and the fluorescence intensity increased with decreasing pH. FIG. 9 shows that the fluorescence intensity of the probe molecule at 525nm changes with pH cycle, and the fluorescence intensity of the probe molecule hardly changes at pH 5.5 or 7.0, indicating that the fluorescent probe molecule has good pH adaptability and can be used for detecting pH change in internal environment in a cycle manner. The fluorescence intensity at 525nm of the fluorescence spectrum is fitted with different pH values, and a fitting curve (shown in figure 10) is obtained in the range of pH 4.0,4.5,5.0,5.5,6.0 and 6.5, which shows that the methylene blue-4-aminonaphthalimide-based fluorescent probe molecule has a good linear relation with pH value in the range of pH 4.0-6.5.
The results show that the methylene blue-4-aminonaphthalimide derivative-based fluorescent probe molecule can well identify the acidity degree of the internal environment.
Example 5
This example examines multi-cellular fluorescence imaging based on methylene blue-4-aminonaphthalimide derivative fluorescent probe molecules.
Fluorescence imaging studies were performed in this experiment using RAW264.7 (mouse macrophages), HIBEC (normal bile duct cells), CT26 (colon cancer cells), QBC939 (bile duct cancer cells), HuCCT1 (bile duct cancer cells) from the central laboratory of the southwest medical university. Shooting conditions are as follows: green Channel: λ ex is 405nm (410nm), λ em is 485 and 565nm (525nm), and the laser output power is 5%; red Channel: λ ex 638nm (620nm), λ em 650-730nm (690nm), laser output power 5%. Eyepiece magnification 10X, objective magnification 20X. The operation is as follows: the culture was first removed with a pipette and washed with PBS buffer solution, and the above procedure was repeated three times. The probe molecules were then incubated for 24h at a concentration of 20. mu. mol/L. Finally, the probe solution was removed with a pipette gun, washed with PBS buffer solution, and the above procedure was repeated three times. And (3) carrying out fluorescence imaging on the incubated cells under a confocal laser scanning microscope. The results are shown in FIG. 11. a1-e1 is a green fluorescence channel map; a2-e2 is a red fluorescence channel map; a3-e3 is a bright field diagram; a1-a3 is RAW264.7 cells; b1-b3 is HIBEC; FIGS. c1-c3 are CT 26; FIGS. d1-d3 are QBC 939; FIGS. e1-e3 are HuCCT 1. The result shows that in QBC939 and HuCCT1 cells, the green fluorescence channel and the red fluorescence channel both have obvious fluorescence enhancement, which indicates that QBC939 and HuCCT1 have stronger acidity and higher content of HClO in the intracellular environment. The experiment shows that the methylene blue-4-aminonaphthalimide derivative-based fluorescent probe molecule can identify and image HClO2 and pH in QBC939 and HuCCT1 cells.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Claims (10)
1. A fluorescent probe molecule characterized by the structure:
2. a method of preparing a fluorescent probe molecule according to claim 1, comprising the steps of:
s1: dissolving methylene blue and sodium carbonate in distilled water and dichloromethane, stirring, slowly adding a sodium thiosulfate aqueous solution, continuously stirring until the solution turns yellow, cooling the solution, slowly dropwise adding a dichloromethane solution of bis (trichloromethyl) carbonate, continuously stirring, then pouring into ice water, extracting with dichloromethane, drying an organic phase, filtering, distilling under reduced pressure, collecting, separating and purifying by a silica gel chromatographic column to obtain a compound 4;
s2: dissolving the compound 4 obtained in the step S1 in dichloromethane, slowly dripping dichloromethane solution of ethylenediamine into the dichloromethane solution, continuously stirring, pouring the mixture into water, extracting, drying, distilling under reduced pressure, collecting, performing silica gel chromatography, eluting, separating and purifying to obtain a compound 3;
s3: dissolving the compound 3 obtained in the step S2 and 4-bromo-1, 8-naphthalic anhydride in absolute ethyl alcohol, and refluxing and stirring under the protection of nitrogen; evaporating, separating by silica gel chromatography column, eluting, separating, and purifying to obtain compound 2;
s4: dissolving the compound 2 obtained in S3 and o-phenylenediamine in anhydrous toluene, sequentially adding chloroform adduct of tris (dibenzylideneacetone) dipalladium, 4, 5-bis (diphenylphosphine) 9, 9-dimethylxanthene and cesium carbonate, heating and refluxing under the protection of nitrogen, evaporating, performing silica gel chromatography, eluting, separating and purifying to obtain the fluorescent probe molecule as claimed in claim 1.
3. The method for preparing a fluorescent probe molecule according to claim 2, wherein in S1, the steps are as follows: dissolving methylene blue and sodium carbonate in distilled water and dichloromethane, stirring at normal temperature for 35 minutes, then slowly adding a sodium thiosulfate aqueous solution, continuously stirring until the solution turns yellow, cooling the solution by using an ice water bath, slowly dropwise adding a dichloromethane solution of bis (trichloromethyl) carbonate, continuously stirring for 1 hour, then pouring into ice water, extracting by using dichloromethane, drying an organic phase, filtering, distilling under reduced pressure, collecting, performing silica gel chromatography, eluting, separating and purifying to obtain a compound 4;
or in S2, the specific steps are: dissolving the compound 4 obtained in the step S1 in dichloromethane, slowly dripping a dichloromethane solution of ethylenediamine into the dichloromethane solution, continuously stirring the mixture at room temperature for 2 hours, pouring the mixture into water, extracting, drying and distilling the mixture under reduced pressure, collecting a solid mixture, and performing silica gel chromatography, elution, separation and purification to obtain a compound 3;
or in S3, the specific steps are: dissolving the compound 3 obtained in the step S2 and 4-bromo-1, 8-naphthalic anhydride in absolute ethyl alcohol, refluxing and stirring for 3 hours under the protection of nitrogen, evaporating the solvent under reduced pressure, and passing the crude product through a silica gel chromatographic column, eluting, separating and purifying to obtain a compound 2;
or in S4, the specific steps are: dissolving the compound 2 obtained in S3 and o-phenylenediamine in anhydrous toluene, sequentially adding chloroform adduct of tris (dibenzylideneacetone) dipalladium, 4, 5-bis (diphenylphosphine) 9, 9-dimethylxanthene and cesium carbonate, heating and refluxing for 10 hours under the protection of nitrogen, evaporating the solvent under reduced pressure, and passing the crude product through a silica gel chromatographic column, eluting, separating and purifying to obtain the fluorescent probe molecule as claimed in claim 1.
4. The method of claim 2 or 3, wherein in step S1, the mass ratio of sodium thiosulfate to methylene blue to sodium carbonate to bis (trichloromethyl) carbonate is 6.66:1.72:6.66: 1; the eluent is a mixture of n-hexane and dichloromethane, and the volume ratio of the eluent to the dichloromethane is 3: 1.
5. The method of claim 2 or 3, wherein in step S2, the mass ratio of compound 4 to ethylenediamine is 1:2.5, and the eluent is a mixture of dichloromethane and methanol at a volume ratio of 10: 1.
6. The method of claim 2 or 3, wherein in step S3, the mass ratio of 4-bromo-1, 8-naphthalic anhydride to compound 3 is 228.0mg:296.0mg, the volume of absolute ethanol is 8.00mL, and the eluent is a mixture of dichloromethane and methanol at a volume ratio of 100: 1.
7. The method of claim 2 or 3, wherein in step S4, the mass ratio of compound 2 to o-phenylenediamine, chloroform adduct of tris (dibenzylideneacetone) dipalladium, 4, 5-bis-diphenylphosphine 9, 9-dimethyl xanthene and cesium carbonate is 308mg:158.8mg:21.0mg:12mg:483.0mg, and the volume of the anhydrous toluene is 5.00 mL; the column chromatography eluent is a mixture of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 50: 1.
8. A fluorescent probe molecule as claimed in any one of claims 1 to 7 for use in the simultaneous detection of hypochlorous acid and pH.
9. The fluorescent probe molecule for simultaneous detection of hypochlorous acid and pH as claimed in claim 8, wherein said fluorescent probe molecule is used for detecting hypochlorous acid and pH in multiple cells.
10. The fluorescent probe molecule for simultaneous detection of hypochlorous acid and pH according to claim 9, wherein the multicellular is one of RAW264.7, HIBEC, CT26, QBC939, HuCCT 1.
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