CN113968881A - Mitochondrion targeting light-activated fluorescence imaging compound and preparation method and application thereof - Google Patents
Mitochondrion targeting light-activated fluorescence imaging compound and preparation method and application thereof Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 96
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000000799 fluorescence microscopy Methods 0.000 title abstract description 22
- 210000003470 mitochondria Anatomy 0.000 title abstract description 19
- 230000008685 targeting Effects 0.000 title description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 14
- 239000001257 hydrogen Substances 0.000 claims abstract description 14
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims abstract description 10
- 125000002947 alkylene group Chemical group 0.000 claims abstract description 9
- 125000003118 aryl group Chemical group 0.000 claims abstract description 7
- 125000001072 heteroaryl group Chemical group 0.000 claims abstract description 7
- 229910052736 halogen Inorganic materials 0.000 claims abstract description 5
- 150000002367 halogens Chemical class 0.000 claims abstract description 5
- 239000002904 solvent Substances 0.000 claims description 12
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 claims description 9
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 claims description 8
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 8
- -1 alkali metal salt Chemical class 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 5
- 229910052783 alkali metal Inorganic materials 0.000 claims description 4
- 125000004432 carbon atom Chemical group C* 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 125000002950 monocyclic group Chemical group 0.000 claims description 4
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 125000005842 heteroatom Chemical group 0.000 claims description 2
- UCFFGYASXIPWPD-UHFFFAOYSA-N methyl hypochlorite Chemical compound COCl UCFFGYASXIPWPD-UHFFFAOYSA-N 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 125000004817 pentamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 2
- 229910052717 sulfur Inorganic materials 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 abstract description 19
- 238000005286 illumination Methods 0.000 abstract description 16
- 230000002438 mitochondrial effect Effects 0.000 abstract description 16
- 210000004027 cell Anatomy 0.000 abstract description 12
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000012984 biological imaging Methods 0.000 abstract description 3
- 230000004913 activation Effects 0.000 abstract description 2
- 239000012567 medical material Substances 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 19
- 238000005481 NMR spectroscopy Methods 0.000 description 16
- 238000001228 spectrum Methods 0.000 description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 12
- 229910052799 carbon Inorganic materials 0.000 description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 238000001819 mass spectrum Methods 0.000 description 6
- IKEOZQLIVHGQLJ-UHFFFAOYSA-M mitoTracker Red Chemical compound [Cl-].C1=CC(CCl)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 IKEOZQLIVHGQLJ-UHFFFAOYSA-M 0.000 description 5
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 5
- 239000000975 dye Substances 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical group [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical group C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000002390 rotary evaporation Methods 0.000 description 3
- 238000010008 shearing Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000012757 fluorescence staining Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000002186 photoactivation Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 125000005428 anthryl group Chemical group [H]C1=C([H])C([H])=C2C([H])=C3C(*)=C([H])C([H])=C([H])C3=C([H])C2=C1[H] 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical group [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001725 pyrenyl group Chemical group 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C07F9/28—Phosphorus compounds with one or more P—C bonds
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Abstract
The invention belongs to the technical field of medical materials, and discloses a mitochondrion targeted light-activated fluorescence imaging compound and a preparation method and application thereof. The structural formula of the compound is shown as a formula I:
wherein R represents C1‑20An alkylene group; r1、R2Each independently represents hydrogen or C1‑30Alkyl radical, C1‑30Alkylene, aryl, heteroaryl, methoxy, halogen. The compound can realize mitochondrion targeted specific light-activated fluorescence imaging in living cells under the condition of illumination, and has lightHigh activation efficiency, high signal-to-noise ratio, strong cell entering ability and the like. Has the advantages of easy regulation and high space-time resolution in mitochondrial imaging or biological imaging.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a mitochondrion targeted light-activated fluorescence imaging compound, and a preparation method and application thereof.
Background
Mitochondria are "kinetic factories" of cells and have important physiological functions. The light-activated fluorescence imaging has the advantage of high space-time resolution, is beneficial to the research on the physiological function of mitochondria and is further beneficial to the research on cytopathic aspect. In the prior art, the molecular research aiming at the light activation imaging of mitochondria is little, or the imaging effect is poor, or the imaging is uncontrollable.
Therefore, it is highly desirable to provide a substance with mitochondrial targeting light-activated fluorescence imaging, which has good effect or controllable imaging on mitochondria, and thus is helpful for developing application research in the biomedical field.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the compound shows a fluorescence lighting imaging function after illumination, has a good imaging effect and is controllable in imaging.
A first aspect of the invention provides compounds for mitochondrial-targeted light-activated fluorescence imaging.
Specifically, the structural formula of the compound for mitochondrion targeted light-activated fluorescence imaging is shown as the formula I:
wherein R represents C1-20An alkylene group;
wherein R is1、R2Each independently represents hydrogen or C1-30Alkyl radical, C1-30Alkylene, aryl, heteroaryl, methoxy, halogen.
Preferably, said C1-30Alkyl is straight or branched chain alkyl; further preferably, C is1-30The alkyl group is selected from any one of methyl, ethyl, propyl, butyl, pentyl and hexyl.
Preferably, said C1-20Alkylene or C1-30Alkylene is straight or branched chain alkylene; further preferably, C is1-20Alkylene or C1-30The alkylene is at least one selected from methylene, ethylene, propylene, butylene, pentylene, hexylene, isopropyl, isobutyl and isopentyl.
Preferably, R represents C1-10An alkylene group.
Preferably, said aryl group represents a monocyclic or polycyclic aromatic group having 6 to 20 carbon atoms; further preferably, the aryl group includes at least one of phenyl, naphthyl, anthryl and pyrenyl.
Preferably, the heteroaryl group represents a monocyclic or polycyclic heteroaromatic group having 1 to 20 carbon atoms, 1 to 4 heteroatoms selected from N, S, O; further preferably, the heteroaryl group includes at least one of pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, thiazolyl, indolyl, azanaphthyl, azaanthracenyl and azapyrenyl.
Preferably, the halogen is selected from chlorine or bromine.
Preferably, said R is pentylene; r1、R2Each independently represents hydrogen or C1-30Alkyl, methoxy, chlorine.
Preferably, R is attached to a benzene ring1、R2Each independently represent 2 methoxy groups, i.e. when R is1When the methoxy group is a methoxy group, 2 methoxy groups are simultaneously connected with a benzene ring; when R is2When methoxy, it means that 2 methoxy groups are simultaneously bonded to the benzene ring.
Preferably, the structural formula of the mitochondrion-targeted light-activated fluorescence imaging compound is any one of the following formulas:
ph represents a phenyl group, and Me represents a methyl group.
The second aspect of the invention provides a preparation method of the compound for mitochondrion targeting light-activated fluorescence imaging.
Specifically, the first aspect of the present invention provides a method for preparing a compound for mitochondrial targeting light-activated fluorescence imaging, comprising the following steps:
(1) preparation of compound IV:
mixing a compound V, a compound VI, an alkali metal salt and a solvent, and reacting to obtain a compound IV, wherein the structural formula of the compound V is shown in the specification
The structural formula of the compound VI is
The structural formula of the compound IV is
(2) Preparation of compound III: dissolving compound IV and triphenylphosphine in solvent, and reactingPreparing a compound III, wherein the structural formula of the compound III is shown in the specification
(3) And mixing the compound III, hydrazine hydrate and an alcohol solvent, and reacting to obtain the mitochondrion targeted light-activated fluorescence imaging compound.
Preferably, in step (1), the alkali metal salt is potassium salt or sodium salt; further preferably, the alkali metal salt is potassium carbonate.
Preferably, in the step (1), the solvent is an organic solvent; further preferably, the solvent is acetone.
Preferably, in the step (1), the molar ratio of the compound V to the compound VI is 1: (0.5-1.5); further preferred molar ratios are 1: (0.8-1.2).
Preferably, in the step (1), the reaction temperature is 40-70 ℃, and the reaction time is 10-12 hours.
Preferably, in the step (1), after the reaction is finished, a separation and purification process is further included.
Preferably, in the step (2), the molar ratio of the compound IV to the triphenylphosphine is 0.5: (0.3-1.0); further preferred molar ratios are 1: (0.4-0.6).
Preferably, in the step (2), the solvent is an organic solvent; further preferably, the solvent is chloroform.
Preferably, in the step (2), the reaction temperature is 50-80 ℃, and the reaction time is 10-12 hours.
Preferably, in the step (2), after the reaction is finished, a separation and purification process is further included.
Preferably, in the step (3), the reaction temperature is 25-80 ℃, and the reaction time is 2-5 hours.
Preferably, in the step (3), after the reaction is finished, a separation and purification process is further included.
The third aspect of the invention provides some intermediates involved in the preparation of compounds for mitochondrial-targeted light-activated fluorescence imaging.
Specifically, the intermediate for preparing the compound has the following structural formula:
the product of the compound after the illumination reaction has the following structural formula:
wherein R represents C1-20An alkylene group;
wherein R is1Represents hydrogen, C1-30Alkyl radical, C1-30Alkylene, aryl, heteroaryl, methoxy, halogen.
Preferably, the illumination is ultraviolet light or near infrared two-photon, and the illumination intensity is 1-150mW/cm2(ii) a Further preferably, the intensity of illumination is 1-100mW/cm2。
After the compound (the compound shown in the formula I) for mitochondrial targeted light-activated fluorescence imaging is used as a light-activated mitochondrial dye to dye mitochondria, the compound shown in the formula II with fluorescence property is generated under the irradiation of light, so that the mitochondria are subjected to fluorescence imaging. The co-dyeing experiment of the compound shown in the formula I and a commercial mitochondrial dye MitoTracker Red after illumination proves that the compound shown in the formula I has a good effect of photo-activated staining mitochondrial imaging.
It should be emphasized that in the prior art, there is no document reporting that the compound of formula I of the present invention can realize mitochondrial fluorescence imaging (abbreviated as "photoshear-activated fluorescence") through photoshear reaction.
In the invention, the 'optical shearing activated fluorescence' refers to a kind of optical response molecules, which generates optical shearing reaction under illumination to generate molecules with fluorescence emission capability, and has the advantages of easy regulation and control, high space-time resolution and the like in mitochondrial imaging or biological imaging.
A third aspect of the invention provides the use of a compound for mitochondrial-targeted light-activated fluorescence imaging.
A detection reagent comprising the above compound.
Preferably, the detection reagent is used for mitochondrial imaging.
Compared with the prior art, the invention has the following beneficial effects:
the compound can realize mitochondrion targeted specific light-activated fluorescence imaging in living cells under the condition of illumination, and has the advantages of high light-activated efficiency, high signal-to-noise ratio, strong cell entering capability and the like. Has the advantages of easy regulation and high space-time resolution in mitochondrial imaging or biological imaging.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of compound IV-1;
FIG. 2 is a NMR carbon spectrum of Compound IV-1;
FIG. 3 is a high resolution mass spectrum of compound IV-1;
FIG. 4 is a NMR chart of Compound III-1;
FIG. 5 is a NMR carbon spectrum of Compound III-1;
FIG. 6 is a high resolution mass spectrum of compound III-1;
FIG. 7 is a NMR spectrum of Compound I-1;
FIG. 8 is a NMR carbon spectrum of Compound I-1;
FIG. 9 is a graph showing the change of fluorescence spectrum of Compound I-1 under UV irradiation with the lapse of time;
FIG. 10 is a schematic diagram of the photocleavage reaction of compound I-1 to compound II-1 under UV irradiation;
FIG. 11 is a co-staining pattern of Compound I-1 in HeLa cells after 10min of illumination at 365nm with MitoTracker Red.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
Example 1: preparation of compound for mitochondrion targeted light-activated fluorescence imaging and preparation of intermediate
(1) Preparation of Compound IV-1:
dissolving the compound V-1(276mg, 1.0mmol) and the compound VI-1(301mg, 1.0mmol) in acetone (i.e. acetone) (10mL), adding potassium carbonate (137mg, 1.0mmol), mixing to form a reaction solution, reacting the reaction solution under reflux for 12 hours, cooling to room temperature (for example, 20 ℃) after the reaction is finished, removing the acetone by rotary evaporation, extracting the residue by chloroform and water, and further recrystallizing in n-hexane and ethyl acetate to obtain the compound IV-1(460mg, 93%) after the reaction is finished;
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of compound IV-1; FIG. 2 is a NMR carbon spectrum of Compound IV-1; FIG. 3 is a high-resolution mass spectrum of Compound IV-1 (the abscissa "m/z" in FIG. 3 represents the mass-to-charge ratio and the ordinate "Intens" represents the relative intensity). The abscissa of the hydrogen and carbon nuclear magnetic resonance spectrum represents the chemical shift in ppm.
The characterization results of the nuclear magnetic resonance hydrogen spectrum, the nuclear magnetic resonance carbon spectrum and the mass spectrum of the compound IV-1 are as follows:1H NMR(CDCl3,400MHz):δ10.2(s,1H),7.77–7.74(m,3H),6.61–6.58(m,2H),5.52(s,2H),4.07(s,3H),4.04(t,J＝6.0Hz,2H),3.97(s,3H),3.42(t,J＝6.8Hz,2H),1.90–1.83(m,4H),1.52–1.50(m,4H)；13CNMR(CDCl3,100MHz):188.0,165.7,161.0,154.3,147.9,138.6,134.1,128.4,119.1,109.7,107.9,106.7,99.8,68.3,67.3,56.7,56.4,33.6,32.5,28.8,27.8,25.2；HRMS(ESI):m/z[M+H]+calcd for C22H27BrNO7:496.0971，found:496.0972。
(2) preparation of Compound III-1:
compound IV-1(248mg,0.5mmol), triphenylphosphine (i.e. PPh)3131mg,0.5mmol) in chloroform (6mL), followed by reaction at reflux for 12 hours, further cooling to room temperature, rotary evaporation to remove the solvent, and separation of the residue by column chromatography to give compound III-1(273mg, yield 72%);
FIG. 4 is a NMR chart of Compound III-1; FIG. 5 is a NMR carbon spectrum of Compound III-1; FIG. 6 is a high-resolution mass spectrum of Compound III-1 (the abscissa "m/z" in FIG. 6 represents the mass-to-charge ratio and the ordinate "Intens" represents the relative intensity). The abscissa of the hydrogen and carbon nuclear magnetic resonance spectrum represents the chemical shift in ppm.
The characterization results of the nuclear magnetic resonance hydrogen spectrum, the nuclear magnetic resonance carbon spectrum and the mass spectrum of the compound III-1 are as follows:1H NMR(CDCl3,400MHz):δ10.13(s,1H),7.82–7.66(m,16H),7.19–7.10(m,2H),6.66–6.53(m,2H),5.46(s,2H),4.03(s,3H),3.98(t,J＝6.0Hz,2H),3.92(s,3H),3.73(t,J＝6.4Hz,2H),1.73–1.47(m,8H)；13CNMR(CDCl3,100MHz):188.0,165.6,160.8,154.2,147.8,138.6,134.9,133.6,133.5,130.4,130.3,128.2,118.9,118.6,117.7,110.1,107.8,106.7,99.8,68.3,67.3,56.7,56.3,29.9,29.8,28.5,25.4,22.5；HRMS(ESI):m/z[M+H]+calcd for C40H42BrNO7P:758.1882；found:758.1711；
(3) preparation of mitochondrion-targeted photo-activated fluorescence imaging compound:
compound III-1(189mg,0.25mmol) was dissolved in anhydrous ethanol (10mL), followed by addition of hydrazine hydrate (12.5mg,0.125mmol), reaction solution was refluxed for 4 hours, further cooled to room temperature, and then solvent was removed by rotary evaporation, and further isolated by column chromatography to give compound I-1(170mg, 90% yield).
FIG. 7 is a NMR spectrum of Compound I-1; FIG. 8 is a NMR carbon spectrum of Compound I-1. The abscissa of the hydrogen and carbon nuclear magnetic resonance spectrum represents the chemical shift in ppm.
The nuclear magnetic resonance hydrogen spectrum and the nuclear magnetic resonance carbon spectrum of the compound I-1 have the following characteristic results:1H NMR(CDCl3,400MHz):δ9.10(s,2H),7.99–7.38(m,32H),7.38(s,2H),6.54-6.49(m,4H),5.50(s,4H),3.99–3.79(m,20H),1.73–1.48(m,16H)；13C NMR(CDCl3,100MHz):δ162.8,158.6,156.1,153.9,147.8,138.8,134.9,133.5,133.4,130.4,130.3,128.4,118.5,117.7,115.8,109.7,107.9,107.1,100.0,67.9,67.4,56.4,56.3,30.0,29.8,28.6,25.4,22.4。
example 2
The compound I-1 prepared in example 1 was dropped on a wet filter paper, and then the change of the fluorescence spectrum was measured as the illumination time increased under the irradiation of ultraviolet light (365nm), and the result of the fluorescence spectrum is shown in FIG. 9. FIG. 9 is a graph showing the change of fluorescence spectrum of compound I-1 under ultraviolet illumination with the increase of illumination time, and it can be seen from FIG. 9 that the fluorescence intensity is also enhanced with the increase of illumination time, which indicates that compound I-1 can effectively complete the photo-shearing reaction within 7 minutes, and compared with the prior art, the fluorescence intensity is obviously increased, the photo-activation efficiency is high, and the signal-to-noise ratio is high, thereby laying the foundation for the high signal-to-noise ratio photo-activation imaging of cells. FIG. 10 is a schematic diagram of the photocleavage reaction of compound I-1 to compound II-1 under UV irradiation.
Example 3
The compound I-1 prepared in example 1 was added to HeLa cell culture broth (3. mu. mol/L) and then co-stained with a commercial mitochondrial dye MitoTracker Red by UV light (10 min at 365 nm). fig. 11 is a co-staining graph of the compound I-1 in HeLa cells after 10min at 365nm and the MitoTracker Red, (a) in fig. 11 shows a HeLa cell field map, (b) shows a cell fluorescence staining graph of the compound I-1 after 10min at 365nm, (c) shows a cell fluorescence staining graph of the MitoTracker Red, (d) shows a cell imaging overlay, and it was concluded from fig. 11 that the compound I-1 has excellent mitochondrial specificity and high signal-to-noise ratio imaging ability after illumination.
It should be noted that, within the scope of the claimed invention, for example, 9 specific structural formulas of the compound for mitochondrial targeting light-activated fluorescence imaging are listed in the summary of the invention, and the specific embodiment only lists the imaging effect of one of the structural formulas, but the imaging effects corresponding to the remaining 8 specific structural formulas are also similar (i.e. the mitochondrial imaging effects of the structural formulas of the 9 compounds are similar). Alternatively, when R, R in formula I1、R2The groups were selected within the scope defined in the present invention, and the mitochondrial imaging effect of the corresponding specific compounds was similar to that of compound I-1 described above.
The invention establishes a simple, convenient and efficient preparation method of the mitochondrion targeting compound. Under illumination, the mitochondrion targeting compound can perform photoshearing reaction to further lighten fluorescence and can be used as a mitochondrion targeting light-activated fluorescent probe.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A compound having the structural formula I:
wherein R represents C1-20An alkylene group;
R1、R2each independently represents hydrogen or C1-30Alkyl radical, C1-30Alkylene, aryl, heteroaryl, methoxy, halogen.
2. The compound of claim 1, wherein C is1-30Alkyl is straight or branched chain alkyl.
3. The compound of claim 1, wherein C is1-20Alkylene or C1-30The alkylene group is a linear or branched alkylene group.
4. A compound according to claim 1, characterized in that said aryl group represents a monocyclic or polycyclic aromatic group having 6 to 20 carbon atoms; the heteroaryl group represents a monocyclic or polycyclic heteroaromatic group having 1 to 20 carbon atoms, 1 to 4 heteroatoms selected from N, S, O.
5. The compound of claim 1, wherein R is pentylene; r1、R2Each independently represents hydrogen or C1-30Alkyl, methoxy, chlorine.
6. The compound of any one of claims 1-5, wherein the compound has the structural formula of any one of:
7. a process for the preparation of a compound according to any one of claims 1 to 6, comprising the steps of:
(1) preparation of compound IV:
mixing a compound V, a compound VI, an alkali metal salt and a solvent, and reacting to obtain a compound IV, wherein the structural formula of the compound V is shown in the specification
The structural formula of the compound VI is
The structural formula of the compound IV is
(2) Preparation of compound III: dissolving a compound IV and triphenylphosphine in a solvent, and reacting to obtain a compound III, wherein the structural formula of the compound III is shown in the specification
(3) And mixing the compound III, hydrazine hydrate and alcohol solvent, and reacting to obtain the compound.
8. An intermediate for the preparation of a compound according to any one of claims 1 to 6, wherein the intermediate has the formula:
9. the product of the photoreactive compound of any of claims 1-6, wherein the product has the formula II:
10. a detection reagent comprising a compound according to any one of claims 1 to 6.
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