WO2016070854A1 - Photoactivatable bioprobes: design, method of preparation and applications - Google Patents
Photoactivatable bioprobes: design, method of preparation and applications Download PDFInfo
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- WO2016070854A1 WO2016070854A1 PCT/CN2015/099420 CN2015099420W WO2016070854A1 WO 2016070854 A1 WO2016070854 A1 WO 2016070854A1 CN 2015099420 W CN2015099420 W CN 2015099420W WO 2016070854 A1 WO2016070854 A1 WO 2016070854A1
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- tpp3m
- photoactivatable
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- probes
- product
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- 239000007789 gas Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 239000012216 imaging agent Substances 0.000 description 1
- 125000002632 imidazolidinyl group Chemical group 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 125000003392 indanyl group Chemical group C1(CCC2=CC=CC=C12)* 0.000 description 1
- 125000003453 indazolyl group Chemical group N1N=C(C2=C1C=CC=C2)* 0.000 description 1
- 125000003454 indenyl group Chemical group C1(C=CC2=CC=CC=C12)* 0.000 description 1
- 125000003406 indolizinyl group Chemical group C=1(C=CN2C=CC=CC12)* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 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
- 125000000904 isoindolyl group Chemical group C=1(NC=C2C=CC=CC12)* 0.000 description 1
- 125000000555 isopropenyl group Chemical group [H]\C([H])=C(\*)C([H])([H])[H] 0.000 description 1
- 125000002183 isoquinolinyl group Chemical group C1(=NC=CC2=CC=CC=C12)* 0.000 description 1
- 125000003965 isoxazolidinyl group Chemical group 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- PGXWDLGWMQIXDT-UHFFFAOYSA-N methylsulfinylmethane;hydrate Chemical compound O.CS(C)=O PGXWDLGWMQIXDT-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000004776 molecular orbital Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000002757 morpholinyl group Chemical group 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000000160 oxazolidinyl group Chemical group 0.000 description 1
- 125000003585 oxepinyl group Chemical group 0.000 description 1
- 125000003566 oxetanyl group Chemical group 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 125000001791 phenazinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3N=C12)* 0.000 description 1
- 125000001484 phenothiazinyl group Chemical group C1(=CC=CC=2SC3=CC=CC=C3NC12)* 0.000 description 1
- 125000001644 phenoxazinyl group Chemical group C1(=CC=CC=2OC3=CC=CC=C3NC12)* 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 125000004592 phthalazinyl group Chemical group C1(=NN=CC2=CC=CC=C12)* 0.000 description 1
- 125000004193 piperazinyl group Chemical group 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 125000001042 pteridinyl group Chemical group N1=C(N=CC2=NC=CN=C12)* 0.000 description 1
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 description 1
- 125000003072 pyrazolidinyl group Chemical group 0.000 description 1
- 125000005551 pyridylene group Chemical group 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
- 125000001422 pyrrolinyl group Chemical group 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 1
- 125000001567 quinoxalinyl group Chemical group N1=C(C=NC2=CC=CC=C12)* 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003660 reticulum Anatomy 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229960001866 silicon dioxide Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 125000005329 tetralinyl group Chemical group C1(CCCC2=CC=CC=C12)* 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 125000005556 thienylene group Chemical group 0.000 description 1
- 125000004568 thiomorpholinyl group Chemical group 0.000 description 1
- XJDNKRIXUMDJCW-UHFFFAOYSA-J titanium tetrachloride Chemical compound Cl[Ti](Cl)(Cl)Cl XJDNKRIXUMDJCW-UHFFFAOYSA-J 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 125000001834 xanthenyl group Chemical group C1=CC=CC=2OC3=CC=CC=C3C(C12)* 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0023—Di-or triarylmethane dye
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
- C07D213/30—Oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/10—Aza-phenanthrenes
Definitions
- the present invention relates to the synthesis and applications of new photoactivatable probes with taking advantage of RIR, TICT, and Photo-oxidative Dehydrocyclization, in which the luminescence of the photoactivatable probes could be photoactivated by lights.
- Photoactivatable fluorophores with the conversion from weak or non-fluorescent state to fluorescent state when irradiated at the appropriate wavelengths have become a powerful tool in biochemical and biological research such as cell lineage in development, macromolecular movement tracking in living cells, and super-resolved fluorescence imaging (PALM, STORM, etc. ) .
- a good photoactivatable fluorophore should be less harmful and perturbative to biological system, rapid and efficient photoactivation, large signal to background ratio (S/B) , high photostability, pH or environmental insensitivity of fluorescence, specificity, and easily prepared.
- the traditional photoactivatable fluorophores are photoactivatable fluorescent proteins which have the advantage of being genetically targeted; however, they are less brightness, easier to photobleach, and more easily influenced by pH or environment than good small-molecule fluorophores. Therefore, small organic photoactivatable fluorophores possessing the brighter emission, less perturbation, and more readily tailored have attracted more and more attention recently.
- Caged dyes as the most concerned small synthetic photoactivatable fluorophores bearing the photo-removable protecting groups (PRPG) such as 2-nitrobenzyl, azidomethyl, diazo groups, and their derivatives, are mainly caged-fluorescein, caged-rhodamine, caged-coumarin, caged-BODIPY, and their derivatives, of which fluorescence recovered by the photocleavage of PRPG.
- PRPG photo-removable protecting groups
- AIE Aggregation-induced emission
- RIR intramolecular rotation
- AIE molecules structured with electron donor and acceptor also possess twisted intramolecular charge-transfer (TICT) effect.
- TCT intramolecular charge-transfer
- the emission of molecules owning TICT effect will be greatly red-shifted and severely quenched with increasing the solvent polarity. Therefore, a competition between AIE and TICT effects will emerge in the AIE system with D-A structure when aggregation happened in solvent with large polarity. It is not difficult to deduce that one AIE molecule structured with D-A will show very weak even almost no emission both in solution and aggregation state if the TICT effect much larger than AIE effect in polar solvent.
- FIG. 1A we developed a new approach to design photoactivatable probes based on the smart combination of RIR, TICT and Photo-oxidative Dehydrocyclization, as shown in FIG. 1A.
- o-TPP3M mitochondria specific photoactivatable probe o-TPP3M
- FIG. 1B a new mitochondria specific photoactivatable probe o-TPP3M
- the pre-irradiated state with almost non-fluorescence would be realized by taking advantaging of RIR mechanism and TICT effect not like PET or FRET reported previously, and then the post-irradiated state with strong fluorescence would be photo-induced by photo-oxidative dehydrocyclization not like the photocleavage of quenching groups.
- Such photoactivatable property of o-TPP3M enables it to be applied in spatiotemporal selective living cell imaging and the super-resolved fluorescence imaging of mitochondria due to the specific to mitochondria without any auxiliaries.
- the present invention provides photoactivatable probes with AIE characteristics, wherein the photoactivatable probes comprise a backbone structure of a formula selected from the group consisting of:
- each R 1 , R 1 ′ , R 1 ” and R 1 ”’ is independently selected from the group consisting of:
- each R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is independently selected from the group consisting of H, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, alkoxy, alkenoxy, cycloalkoxy, cycloalkenoxy, heterocycloxy, aryloxy, heteroaryloxy, alkylthio, cycloalkylthio, cycloalkenylthio, heterocyclylthio, arylthio, heteroarylthio, and heteroaryloxy, amino, each of which is optionally substituted by one or more substituents selected from halogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, alkoxy, alkenoxy, cycloalkoxy, cycloalkenoxy, heterocycloxy, aryloxy, heteroaryloxy, alky
- each A - is a monovalent counter ion.
- each R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is independently selected from the group consisting of C n H 2n+1 , C 10 H 7 , C 12 H 9 , OC 6 H 5 , OC 10 H 7 and OC 12 H 9 , C n H 2n COOH, C n H 2n NCS, C n H 2n N 3 , C n H 2n NH 2 , C n H 2n SH, C n H 2n Cl, C n H 2n Br, C n H 2n I, N (C n H m ) 2 , SC n H m , wherein the symbol “n” is an integer preferably selected from 1-10, and m is 2n+1;
- each A - is independently selected from the group consisting of I - , Cl - , Br - , PF6 - , ClO 4 - , BF 4 - , BPh 4 - , CH 3 PhSO 3 - and other monovalent counter ions.
- the photoactivatable probes comprise a backbone structure selected from the above formula 6, wherein R 1 represents Particularly, an example of the photoactivatable probes according to the invention is o-TPP3M of the following formula:
- the photoactivatable probes are mitochondria specific, even without any auxiliaries.
- the photoactivatable probes can be photoactivated by lights undergoing photo-oxidative dehydrocyclization.
- said photoactivatable probes can be photoactivated in solution or aggregate state by using UV-light or visible light.
- the present invention further provides a method of preparation of the photoactivatable probes, wherein the photoactivatable probes are constructed based on RIR, TICT and Photo-oxidative Dehydrocyclization.
- the present invention further provides use of these photoactivatable probes in spatiotemporal selective cells (e.g. HeLa cells) , organelles and macromolecules imaging and super-resolved fluorescence imaging.
- spatiotemporal selective cells e.g. HeLa cells
- these photoactivatable probes are dissolved in an organic polar solvent such as DMSO, and then added to the cell culture medium.
- the fluorescence images can be taken by fluorescent microscope and confocal laser scanning microscope, wherein the laser used can be preferably set up in two models with low and high powers, or different wavelengths.
- the present invention also provides a photoactivated product of any of the photoactivatable probes as defined above, wherein the photoactivated product is a photocyclized product.
- the photoactivated product is a photocyclized product.
- at least one single bond is newly produced between pyridinium ring and phenyl ring of the photocyclized product under photoactivation.
- the photoactivated product according to the invention is a photoactivated product of the o-TPP3M.
- photoactivated product of the o-TPP3M is c-TPP3M of the following formula:
- FIG. 1 Design of new photoactivatable fluorophores: (A) Photoactivatable probes principle; (B) Chemical structure of o-TPP3M; (C) PL spectra of o-TPP3M before (empty circles) and after (filled circles) photoactivation.
- FIG. 2 (A) PL spectra and (B) the fluorescence intensity enhancement at 595 nm of o-TPP3M with different fractions (f w ) between H 2 O and DMSO. Excitation: 435 nm.
- FIG. 3 Synthetic route to o-TPP3M.
- FIG. 4 HR-MS of compound 1.
- FIG. 5 1 H-NMR of compound 1.
- FIG. 6 13 C-NMR of compound 1.
- FIG. 7 HR-MS of the cation part of compound o-TPP3M.
- FIG. 8 HR-MS of the anion part of compound o-TPP3M.
- FIG. 9 1 H-NMR of compound o-TPP3M.
- FIG. 10 13 C-NMR of compound o-TPP3M.
- FIG. 11 (A) Photoreaction synthesis route for c-TPP3M; (B) ORTEP drawing of c-TPP3M.
- FIG. 12 HR-MS of the cation part of compound c-TPP3M.
- FIG. 13 HR-MS of the anion part of compound c-TPP3M.
- FIG. 14 1 H-NMR of compound c-TPP3M.
- FIG. 15 13 C-NMR of compound c-TPP3M.
- FIG. 16 Density functional theory (DFT) calculation: (A) Optimized molecular conformation for o-TPP3M; (B) Molecular orbital amplitude plot of HOMO and LUMO for o-TPP3M; (C) Optimized lowest energy molecular conformations of c-TPP3M and c-TPP3M (b) (simulated product with photocyclization happened between two phenyl rings) .
- DFT Density functional theory
- FIG. 17 (A) PL and (B) UV spectra of o-TPP3M in DMSO solution irradiated with 365 nm, 2.48 mW/cm 2 UV-light for different times. Concentration: 10 -5 M. Inset: a) , b) and c) , d) are the fluorescent and daylight images before and after UV-light irradiation.
- FIG. 18 (A) The structure of c-TPP3M with two typed proton peaks: H a and H b ; (B) HR-MS spectrum of o-TPP3M after irradiation for about 2 h in DMSO solution; (C) Change in 1 H NMR spectrum of o-TPP3M after 180 W high-pressure mercury vapor lamp irradiation for (a) 0 h, (b) 1 h, (c) 3 h, (d) 5 h and (e) c-TPP3M in d 6 -DMSO solution; (D) Photo-converted yield from o-TPP3M to c-TPP3M against irradiation time. Concentration: 1 ⁇ 10 -3 M.
- FIG. 19 (A) Cytotoxicity of o-TPP3M to HeLa cells determined by MTT array; (B) Bright-field and fluorescent images of living HeLa cells stained with o-TPP3M (10 ⁇ M) after UV-light irradiation for 0 s, 4 s, and 8 s; (C) Fluorescent images of living HeLa cells stained with o-TPP3M (10 ⁇ M) for 10 min and Mito-Tracker red FM (MT, 100 nM) for 15 min. Excitation wavelength: 330-385 nm (for o-TPP3M after UV-light irradiation for 8 s) and 540-580 nm (for MT) . Irradiation wavelength: 330-385 nm. Scale bar: 30 ⁇ m.
- FIG. 20 (A) Plot of fluorescence enhancement (I/I 0 ) against irradiation time with increasing the laser power: 1%, 2%and 5%in living HeLa cells. Insets: the confocal images of HeLa cells (a) before and (b) after irradiation; (B) Selective fluorescent images of living HeLa cells. Cells in the white ellipse region were selected to be irradiated for 1 s under 35%power. HeLa cells were stained with 10 ⁇ M o-TPP3M. Scale bar: 20 ⁇ m. Irradiation and Excitation: 405-nm laser. Excitation: 1.5%power.
- Photoactivatable fluorophores have provided unique tools for super-resolved fluorescence imaging and spatiotemporal tracking of living cells, organelles and macromolecules.
- novel mitochondria photoactivatable fluorophores which will be photoactivated to a strong emissive state undergoing an individual photo-oxidative dehydrocyclization reaction occurred between pyridinium and phenyl rings.
- the inventive photoactivatable probes or fluorophores were very good candidates in bioresearch with highly spatiotemporal resolution, almost no cytotoxicity, very large signal to background, efficient photoactivation, very specific to mitochondria, environmental stability, and readily preparation.
- the specificity to mitochondria without any decoration imply that PALM or STORM imaging of mitochondria with easier operation will be expected to realized based on the photoactivatable probes according to the invention.
- halogen represents fluorine, chlorine, bromine and iodine, particularly chlorine or fluorine, preferably fluorine.
- alkyl represents a linear or branched alkyl radical having the number of carbon atoms specifically indicated, e.g. C1-C10 alkyl means a linear or branched alkyl radical having one, two, three, four, five, six, seven, eight, nine or ten carbon atoms, e.g.
- alkyl represents a linear or branched alkyl radical having, as a rule, 1 to 40, particularly 1 to 30, preferably 1 to 20 carbon atoms. Particularly, the alkyl group has 1, 2, 3, 4, 5 or 6 carbon atoms ( “C1-C6-alkyl” ) , e.g.
- the alkyl group has 1, 2 or 3 carbon atoms ( “C1-C3-alkyl” ) , methyl, ethyl, n-propyl-, isopropyl, n-butyl, tert-butyl, pentyl, isopentyl, hexyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neo-pentyl, 1, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3, 3-dimethylbutyl, 2, 2-dimethylbutyl, 1, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl.
- the alkyl group has 1, 2 or 3 carbon atoms ( “C
- alkenyl is to be understood as preferably meaning a linear or branched, monovalent hydrocarbon group, which contains one double bond, and which has 2 or more carbon atoms (such as “C2-C6-alkenyl” ) .
- Said alkenyl group is, for example, a vinyl, allyl, (E) -2-methylvinyl, (Z) -2-methylvinyl or isopropenyl group.
- alkoxy- is to be understood as preferably meaning a linear or branched, saturated, monovalent, hydrocarbon group of formula -O-alkyl, in which the term “alkyl” is defined as above, e.g. a methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, tert-butoxy, sec-butoxy, pentyloxy, iso-pentyloxy, n-hexyloxy group, or an isomer thereof.
- the “alkoxy-” group is a “C1-C6-alkoxy-” , “C1-C4-alkoxy-” , a “C1-C3-alkoxy-” , a methoxy, ethoxy, or propoxy group, preferably a methoxy, ethoxy or propoxy group. Further preferred is a “C1-C2-alkoxy-” group, particularly a methoxy or ethoxy group.
- alkylthio - is to be understood as preferably meaning a linear or branched, saturated, monovalent, hydrocarbon group of formula –S-alkyl, in which the term “alkyl” is defined as above.
- cycloalkyl is to be understood as preferably meaning a saturated, monovalent, monocyclic hydrocarbon ring which contains such as 3, 4, 5, 6, 7 or 8 carbon atoms.
- C3-C8-cycloalkyl group is for example, a monocyclic hydrocarbon ring, e.g. a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl group.
- said cycloalkyl group is a C4-C6-cycloalkyl, a C5-C6-cycloalkyl or a cyclohexyl group.
- C3-C6-cycloalkyl is to be understood as preferably meaning a saturated, monovalent, monocyclic hydrocarbon ring which contains 3, 4, 5 or 6 carbon atoms.
- said C3-C6-cycloalkyl group is a monocyclic hydrocarbon ring such as a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group.
- cycloalkoxy- is to be understood as preferably meaning a group of formula -O-cycloalkyl, in which the term “cycloalkyl” is defined as above.
- cycloalkylthio- is to be understood as preferably meaning a group of formula -S-cycloalkyl, in which the term “cycloalkyl” is defined as above.
- cycloalkenyl ring is to be understood as preferably meaning a non-aromatic monocyclic hydrocarbon ring which contains one or more double bonds. such as a cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl or cycloheptenyl group, wherein the bond between said ring with the rest of the molecule may be to any carbon atom of said ring, be it saturated or unsaturated.
- alkenoxy- is to be understood as preferably meaning a group of formula -O-cycloalkyl, in which the term “cycloalkyl” is defined as above.
- heterocyclyl is to be understood as meaning a saturated or partially unsaturated, monovalent, mono-or bicyclic hydrocarbon ring which contains such as 3, 4, 5, 6, 7, 8 or 9 carbon atoms and further containing 1, 2 or 3 heteroatom-containing groups selected from oxygen, sulfur, nitrogen.
- heterocyclyl is to be understood as meaning a “4-to 10-membered heterocyclic ring” .
- the “heterocyclyl” used herein is unaromatic.
- Said heterocyclic ring is for example, a monocyclic heterocyclic ring such as an oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, 1, 3-dioxolanyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, 1, 4-dioxanyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, 1, 3-dithianyl, thiomorpholinyl, piperazinyl, or chinuclidinyl group.
- a monocyclic heterocyclic ring such as an oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, 1, 3-dioxolanyl, imidazolidinyl, pyrazolidinyl, ox
- said heterocyclic ring can contain one or more double bonds, e. g. 4H-pyranyl, 2H-pyranyl, 2, 5-dihydro-1H-pyrrolyl, 1, 3-dioxolyl, 4H-1, 3, 4-thiadiazinyl, 2, 5-dihydrofuranyl, 2, 3-dihydrofuranyl, 2, 5-dihydrothienyl, 2, 3-dihydrothienyl, 4, 5-dihydrooxazolyl, 4, 5-dihydroisoxazolyl, or 4H-1, 4-thiazinyl group, or, it may be benzo fused.
- 4H-pyranyl 2H-pyranyl, 2, 5-dihydro-1H-pyrrolyl, 1, 3-dioxolyl, 4H-1, 3, 4-thiadiazinyl, 2, 5-dihydrofuranyl, 2, 3-dihydrofuranyl, 2, 5-dihydrothienyl, 2, 3-dihydrothienyl, 4,
- heterocyclyl is to be understood as being a heterocyclic ring which contains 3, 4 or 5 carbon atoms, and 1, 2 or 3 of the above-mentioned heteroatom-containing groups (a “4-to 8-membered heterocyclic ring” ) , more particularly said ring can contain 4 or 5 carbon atoms, and 1, 2 or 3 of the above-mentioned heteroatom-containing groups (a “5-to 8-membered heterocyclic ring” ) , more particularly said heterocyclic ring is a “6-membered heterocyclic ring” , which is to be understood as containing 4 carbon atoms and 2 of the above-mentioned heteroatom-containing groups or 5 carbon atoms and one of the above-mentioned heteroatom-containing groups, preferably 4 carbon atoms and 2 of the above-mentioned heteroatom-containing groups.
- heterocycloxy- is to be understood as preferably meaning a group of formula -O-heterocyclyl, in which the term “heterocyclyl” is defined as above.
- heterocyclylthio- is to be understood as preferably meaning a group of formula -S-heterocyclyl, in which the term “heterocyclyl” is defined as above.
- aryl is to be understood as preferably meaning a monovalent, aromatic or partially aromatic, mono-, or bi-or tricyclic hydrocarbon ring having 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (a “C6-C14-aryl” group) , particularly a ring having 6 carbon atoms (a “C6-aryl” group) , e.g. a phenyl group; or a biphenyl group, or a ring having 9 carbon atoms (a “C9-aryl” group) , e.g. an indanyl or indenyl group, or a ring having 10 carbon atoms (a “C10-aryl” group) , e.g.
- heteroaryl is understood as preferably meaning a monovalent, monocyclic-, bicyclic-or tricyclic aromatic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms (a “5-to 14-membered heteroaryl” group, e.g. 6-membered heteroaryl) , particularly 5 or 6 or 9 or 10 atoms, and which contains at least one heteroatom which may be identical or different, said heteroatom being such as oxygen, nitrogen or sulfur, and in addition in each case can be benzocondensed.
- heteroaryl is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl etc., and benzo derivatives thereof, such as, for example, benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and benzo derivatives thereof, such as, for example, quinolinyl, quinazolinyl, isoquinolinyl, etc.;
- 5-membered heteroaryl is understood as preferably meaning a monovalent, aromatic ring system having 5 ring atoms and which contains at least one heteroatom which may be identical or different, said heteroatom being such as oxygen, nitrogen or sulfur.
- “5-membered heteroaryl” is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, tetrazolyl.
- 6-membered heteroaryl is understood as preferably meaning a monovalent, aromatic ring system having 6 ring atoms and which contains at least one heteroatom which may be identical or different, said heteroatom being such as oxygen, nitrogen or sulfur.
- “6-membered heteroaryl” is selected from pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl.
- the heteroarylic or heteroarylenic radicals include all the possible isomeric forms thereof, e.g. the positional isomers thereof.
- the term pyridinyl or pyridinylene includes pyridin-2-yl, pyridin-2-ylene, pyridin-3-yl, pyridin-3-ylene, pyridin-4-yl and pyridin-4-ylene; or the term thienyl or thienylene includes thien-2-yl, thien-2-ylene, thien-3-yl and thien-3-ylene.
- aryloxy- or “heteroaryloxy-” is to be understood as preferably meaning a group of formula -O-aryl or -O-heteroaryl, in which the term “aryl” and “heteroaryl” are defined as above respectively.
- arylthio- or “heteroarylthio -” is to be understood as preferably meaning a group of formula -S-aryl or -S-heteroaryl, in which the term “aryl” and “heteroaryl” are defined as above respectively.
- numeric range “1-10” is to be understood as meaning a group having a finite number of atoms, such as carbon atoms, of 1 to 10, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
- the term “one or more” e.g. in the definition of the substituents of the compounds of the general formulae of the present invention, is understood as meaning “one, two, three, four or five, particularly one, two, three or four, more particularly one, two or three, even more particularly one or two” .
- the invention also includes all suitable isotopic variations of a compound of the invention.
- An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature.
- isotopes that can be incorporated into a compound of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine and iodine.
- o-TPP3M a photoactivatable fluorophore named o-TPP3M as shown in FIG. 1B, which was structured with three 4-methoxyphenyl groups as electron donor (D) and 1-methylpyridinium cation as electron acceptor (A) .
- D 4-methoxyphenyl groups
- A 1-methylpyridinium cation
- o-TPP3M behaved AIE property with the quantum yield of 0.0018 in solution and 0.051 in solid (Table 1) .
- DMSO solution of o-TPP3M presents almost no emission because of the free rotation of four rings.
- FIG. 16A shows the optimized molecular conformation of o-TPP3M.
- HOMO and LUMO of o-TPP3M were caluculated in FIG. 16B.
- the electron density in HOMO was delocalized throughout the molecule, however that in LUMO was mainly localized on the pyridinium ring. This suggests that the pyridinium ring is more reactive compared to the other phenyl rings in the excited state, and the photocyclization will prefer to occurre between the pyridinium ring and phenyl ring.
- ⁇ F, o-TPP3M, Soln 0.0018
- ⁇ F, c-TPP3M shows a little emission with a quantum yield of 0.051 in solid state, the TICT effect will quench this weak emission to a weaker level in polar solvent as explained in the design section.
- FIG. 17 shows the PL and UV spectra to monitor the whole photoactivation process in DMSO solution in air.
- the emission color of a solution of o-TPP3M in DMSO was not observed without UV-light irradiation (FIG. 17A, inset a) .
- the fluorescence spectrum of o-TPP3M also suggested the fluorescence signal was too weak to record by PL as FIG. 17A shown, which very well consist with the quantum yield of 0.0018 in Table 1. And then, the green color emission was emerged (FIG.
- FIG. 18B shows a new signal at m/z 436.1915 comparing to m/z 438.2050 ( [M-PF 6 ] + ) of o-TPP3M for the solution of o-TPP3M after irradiation with exposure to air, which accurately corresponded to the molecular weight of c-TPP3M minus PF 6 .
- 1 H NMR signals of o-TPP3M were transformed to those of c-TPP3M gradually were recorded in FIG.
- the rate of photo-conversion was faster at the beginning and then became slower and slower until it reached to a platform. This is due to combined action involving the decrease of the amount of oxygen dissolved in solvent and the partial photodecomposition of both the reactant and product.
- the maximum yield was as high as 80%, which indicates that the photoactivation from o-TPP3M to c-TPP3M is very efficient.
- cytotoxicity of o-TPP3M to living cells was assessed using a 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) assay (see FIG. 19A) .
- MTT 5-diphenyltetrazolium bromide
- FIG. 19B monitored the whole photoactivation process in living HeLa cells by the fluorescent microscope.
- Living HeLa cells stained with o-TPP3M were shown in FIG. 19B with the bright-field image, and the fluorescent images of them were recorded after UV-light (330-385 nm) irradiation for different periods of time (0 s, 4 s, and 8 s) .
- UV-light 330-385 nm
- FIG. 19B fluorescent image at 0 s
- FIG. 20A The kinetic of the photoactivation for o-TPP3M in living HeLa cells was investigated by altering irradiation power using confocal fluorescence microscope technique.
- Insets a and b in FIG. 20A displayed the confocal images of living HeLa cells before and after irradiation of 405-nm laser.
- the green fluorescence from the in situ photo-converted c-TPP3M was obviously emerged compared to the initial state, and the monitoring of the whole fluorescence turn-on process was recorded by increasing the fluorescence intensity against irradiation time.
- the plot of the fluorescence intensity enhanced multiple (I/I 0 ) against irradiation time for 1%, 2%and 5%power used was shown in FIG. 20A.
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Abstract
Photoactivatable fluorophores have provided unique tools for supe-resolved fluorescence imaging and spatiotemporal tracking of living cells, organelles and macromolecules. Here, we report a new strategy to design a novel mitochondria specific photoactivatable fluorophore o-TPP3M which will be photoactivated to a strong emissive c-TPP3M (Φ =0.624) undergoing an individual photooxidative dehydrocyclization reaction occurred between pyridinium and phenyl rings. Highly efficient photoactivation and selective imaging of o-TPP3M in living HeLa cells suggests that this fluorophore was a very good candidate in bioresearch with highly spatiotemporal resolution, almost no cytotoxicity, very large signal to background, efficient photoactivation, ver specific to mitochondria, environmental stability, and readily preparation. Furthermore, the specificity to mitochondria without any decoration imply that PALM or STORM imaging of mitochondria with easier operation will be expected to realized based on o-TPP3M. Because of this artful molecular design, photo-oxidative dehydrocyclization based method should provide a new platform for developing photoactivatable fluorophores for various bioresearch.
Description
RELATED APPLICATION
The present application claims the benefit of priority of U.S. Provisional Application No. 62/123,012, which was filed on Nov. 5, 2014. The entire text of the aforementioned application is incorporated herein by reference.
The present invention relates to the synthesis and applications of new photoactivatable probes with taking advantage of RIR, TICT, and Photo-oxidative Dehydrocyclization, in which the luminescence of the photoactivatable probes could be photoactivated by lights.
Photoactivatable fluorophores with the conversion from weak or non-fluorescent state to fluorescent state when irradiated at the appropriate wavelengths have become a powerful tool in biochemical and biological research such as cell lineage in development, macromolecular movement tracking in living cells, and super-resolved fluorescence imaging (PALM, STORM, etc. ) . A good photoactivatable fluorophore should be less harmful and perturbative to biological system, rapid and efficient photoactivation, large signal to background ratio (S/B) , high photostability, pH or environmental insensitivity of fluorescence, specificity, and easily prepared. The traditional photoactivatable fluorophores are photoactivatable fluorescent proteins which have the advantage of being genetically targeted; however, they are less brightness, easier to photobleach, and more easily influenced by pH or environment than good small-molecule fluorophores. Therefore, small organic photoactivatable fluorophores possessing the brighter emission, less perturbation, and more readily tailored have attracted more and more attention recently. Caged dyes, as the most concerned small synthetic photoactivatable fluorophores bearing the photo-removable protecting groups (PRPG) such as 2-nitrobenzyl, azidomethyl, diazo groups, and their derivatives, are mainly caged-fluorescein, caged-rhodamine, caged-coumarin, caged-BODIPY, and their derivatives, of which fluorescence recovered by the photocleavage of PRPG. [Methods Enzymol. 1998, 291, 63–78] Another two kinds of photoactivatable dyes based on azido-DCDHF pull-push fluorogen and intramolecular tetrazole-alkene compound have been reported by researchers recently, which made use of the photo-chemical conversion from azido to amino and the photoclick cycloaddition reaction of intramolecular tetrazole-alkene to implement the fluorescence photoactivation. [J. Am. Chem. Soc. 2008, 130, 9204–9205; J. Am. Chem. Soc.
2011, 133, 11912–11915] However, there still existed some drawbacks for these aforementioned small-molecule fluorophores. For example, perturbation and harmfulness of the PRPG and high active intermediates generated (e.g., nitrene) to bio-system, not very high signal to background ratio due to insufficient quenching effect, very serious side-products produced due to the reactions occurred between active intermediates and species in local environment, and complicated and inefficient synthesis. Moreover, the specific to mitochondria for them is not very good without the help of bioconjugation. Therefore, better and easier photoactivatable systems are strongly expected to be developed for the advance of bioresearch.
Aggregation-induced emission (AIE) undergoing the mechanism of restriction of intramolecular rotation (RIR) has been investigated for about fourteen years by our group and others. [Adv. Mater. 2014, 26, 5429–5479] Molecules with AIE property present almost no emission in solution state due to the non-radiative consumption of the energy of excited state in the form of intramolecular rotation, and then be induced to emit strongly after the formation of aggregates which suppresses the intramolecular rotation, rigidifies the molecular structure, and actives the radiative decay channel. Besides the aggregation, many methods to restrict the rotation by covalent and non-covalent bonds have been developed for the study of RIR mechanism. Among of them, photo-oxidative dehydrocyclization has attracted our attention due to the participation of light with appropriate wavelengths. Some of AIE molecules structured with electron donor and acceptor (D-A) also possess twisted intramolecular charge-transfer (TICT) effect. As we known, the emission of molecules owning TICT effect will be greatly red-shifted and severely quenched with increasing the solvent polarity. Therefore, a competition between AIE and TICT effects will emerge in the AIE system with D-A structure when aggregation happened in solvent with large polarity. It is not difficult to deduce that one AIE molecule structured with D-A will show very weak even almost no emission both in solution and aggregation state if the TICT effect much larger than AIE effect in polar solvent.
Inventions of photoactivatable probes have been reported in prior art, examples of which have been reported by Stefan W. Hell (US8617827 B2) , Wen-Hong Li (US7304168 B2, US8153103 B2) , Joan C Politz (WO1998006875 A1) , and Robort J. Twieg (US8772048 B2) .
SUMMARY OF THE INVENTION
In this invention, we developed a new approach to design photoactivatable probes based on the smart combination of RIR, TICT and Photo-oxidative Dehydrocyclization, as shown in FIG. 1A. Herein, we reported a new mitochondria specific photoactivatable probe o-TPP3M (FIG. 1B) , of which the pre-irradiated state with almost non-fluorescence would be realized by taking advantaging of RIR mechanism and TICT effect not like PET or FRET reported previously, and then the post-irradiated state with strong fluorescence would be
photo-induced by photo-oxidative dehydrocyclization not like the photocleavage of quenching groups. Such photoactivatable property of o-TPP3M enables it to be applied in spatiotemporal selective living cell imaging and the super-resolved fluorescence imaging of mitochondria due to the specific to mitochondria without any auxiliaries.
In particular, the present invention provides photoactivatable probes with AIE characteristics, wherein the photoactivatable probes comprise a backbone structure of a formula selected from the group consisting of:
wherein each R1, R1′ , R1” and R1”’ is independently selected from the group consisting of:
each R2, R3, R4, R5, R6 and R7 is independently selected from the group consisting of H, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, alkoxy, alkenoxy, cycloalkoxy, cycloalkenoxy, heterocycloxy, aryloxy, heteroaryloxy, alkylthio, cycloalkylthio, cycloalkenylthio, heterocyclylthio, arylthio, heteroarylthio, and heteroaryloxy, amino, each of which is optionally substituted by one or more substituents selected from halogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, alkoxy, alkenoxy, cycloalkoxy, cycloalkenoxy, heterocycloxy, aryloxy, heteroaryloxy, alkylthio, cycloalkylthio, cycloalkenylthio, heterocyclylthio, arylthio, heteroarylthio, and heteroaryloxy, amino, azide (-N3) , OH, SH, COOH, NCS; and
each A- is a monovalent counter ion.
Preferably, each R2, R3, R4, R5, R6 and R7 is independently selected from the group consisting of CnH2n+1, C10H7, C12H9, OC6H5, OC10H7 and OC12H9, CnH2nCOOH, CnH2nNCS, CnH2nN3, CnH2nNH2, CnH2nSH, CnH2nCl, CnH2nBr, CnH2nI, N (CnHm) 2, SCnHm, wherein the symbol “n” is an integer preferably selected from 1-10, and m is 2n+1;
each A- is independently selected from the group consisting of I-, Cl-, Br-, PF6-, ClO4
-, BF4
-, BPh4
-, CH3PhSO3
-and other monovalent counter ions.
As a specific example, the photoactivatable probes comprise a backbone structure selected from the above formula 6, wherein R1 represents Particularly, an example of the photoactivatable probes according to the invention is o-TPP3M of the following formula:
According to the invention, the photoactivatable probes are mitochondria specific, even without any auxiliaries.
According to the present invention, the photoactivatable probes can be photoactivated by lights undergoing photo-oxidative dehydrocyclization. In particular, said photoactivatable probes can be photoactivated in solution or aggregate state by using UV-light or visible light. The present invention further provides a method of preparation of the photoactivatable probes, wherein the photoactivatable probes are constructed based on RIR, TICT and Photo-oxidative Dehydrocyclization.
The present invention further provides use of these photoactivatable probes in spatiotemporal selective cells (e.g. HeLa cells) , organelles and macromolecules imaging and super-resolved fluorescence imaging.
In some embodiments, these photoactivatable probes are dissolved in an organic polar solvent such as DMSO, and then added to the cell culture medium.
In some embodiments, the fluorescence images can be taken by fluorescent microscope and confocal laser scanning microscope, wherein the laser used can be preferably set up in two models with low and high powers, or different wavelengths.
The present invention also provides a photoactivated product of any of the photoactivatable probes as defined above, wherein the photoactivated product is a photocyclized product. In particular, as compared to the structure of the photoactivatable probe as defined above, at least one single bond is newly produced between pyridinium ring and phenyl ring of the photocyclized product under photoactivation.
As a specific example, the photoactivated product according to the invention is a photoactivated product of the o-TPP3M. Specifically, photoactivated product of the o-TPP3M is c-TPP3M of the following formula:
DESCRIPTION OF DRAWINGS
FIG. 1 Design of new photoactivatable fluorophores: (A) Photoactivatable probes principle; (B) Chemical structure of o-TPP3M; (C) PL spectra of o-TPP3M before (empty circles) and after (filled circles) photoactivation.
FIG. 2 (A) PL spectra and (B) the fluorescence intensity enhancement at 595 nm of o-TPP3M with different fractions (fw) between H2O and DMSO. Excitation: 435 nm.
Concentration: 10 μM.
FIG. 3 Synthetic route to o-TPP3M.
FIG. 4 HR-MS of compound 1.
FIG. 5 1H-NMR of compound 1.
FIG. 6 13C-NMR of compound 1.
FIG. 7 HR-MS of the cation part of compound o-TPP3M.
FIG. 8 HR-MS of the anion part of compound o-TPP3M.
FIG. 9 1H-NMR of compound o-TPP3M.
FIG. 10 13C-NMR of compound o-TPP3M.
FIG. 11 (A) Photoreaction synthesis route for c-TPP3M; (B) ORTEP drawing of c-TPP3M.
Solvent and anion are omitted for clarity.
FIG. 12 HR-MS of the cation part of compound c-TPP3M.
FIG. 13 HR-MS of the anion part of compound c-TPP3M.
FIG. 14 1H-NMR of compound c-TPP3M.
FIG. 15 13C-NMR of compound c-TPP3M.
FIG. 16 Density functional theory (DFT) calculation: (A) Optimized molecular conformation for o-TPP3M; (B) Molecular orbital amplitude plot of HOMO and LUMO for o-TPP3M; (C) Optimized lowest energy molecular conformations of c-TPP3M and c-TPP3M (b) (simulated product with photocyclization happened between two phenyl rings) .
FIG. 17 (A) PL and (B) UV spectra of o-TPP3M in DMSO solution irradiated with 365 nm, 2.48 mW/cm2 UV-light for different times. Concentration: 10-5 M. Inset: a) , b) and c) , d) are the fluorescent and daylight images before and after UV-light irradiation.
FIG. 18 (A) The structure of c-TPP3M with two typed proton peaks: Ha and Hb; (B) HR-MS spectrum of o-TPP3M after irradiation for about 2 h in DMSO solution; (C) Change in 1H NMR spectrum of o-TPP3M after 180 W high-pressure mercury vapor lamp irradiation for (a) 0 h, (b) 1 h, (c) 3 h, (d) 5 h and (e) c-TPP3M in d6-DMSO solution; (D) Photo-converted yield from o-TPP3M to c-TPP3M against irradiation time. Concentration: 1×10-3 M.
FIG. 19 (A) Cytotoxicity of o-TPP3M to HeLa cells determined by MTT array; (B) Bright-field and fluorescent images of living HeLa cells stained with o-TPP3M (10 μM) after UV-light irradiation for 0 s, 4 s, and 8 s; (C) Fluorescent images of living HeLa cells stained with o-TPP3M (10 μM) for 10 min and Mito-Tracker red FM (MT, 100 nM) for 15 min.
Excitation wavelength: 330-385 nm (for o-TPP3M after UV-light irradiation for 8 s) and 540-580 nm (for MT) . Irradiation wavelength: 330-385 nm. Scale bar: 30 μm.
FIG. 20 (A) Plot of fluorescence enhancement (I/I0) against irradiation time with increasing the laser power: 1%, 2%and 5%in living HeLa cells. Insets: the confocal images of HeLa cells (a) before and (b) after irradiation; (B) Selective fluorescent images of living HeLa cells. Cells in the white ellipse region were selected to be irradiated for 1 s under 35%power. HeLa cells were stained with 10 μM o-TPP3M. Scale bar: 20 μm. Irradiation and Excitation: 405-nm laser. Excitation: 1.5%power.
DETAILS DESCRIPTION OF THE INVENTION
Photoactivatable fluorophores have provided unique tools for super-resolved fluorescence imaging and spatiotemporal tracking of living cells, organelles and macromolecules. Provided herein novel mitochondria photoactivatable fluorophores which will be photoactivated to a strong emissive state undergoing an individual photo-oxidative dehydrocyclization reaction occurred between pyridinium and phenyl rings. The inventive photoactivatable probes or fluorophores were very good candidates in bioresearch with highly spatiotemporal resolution, almost no cytotoxicity, very large signal to background, efficient photoactivation, very specific to mitochondria, environmental stability, and readily preparation. Furthermore, the specificity to mitochondria without any decoration imply that PALM or STORM imaging of mitochondria with easier operation will be expected to realized based on the photoactivatable probes according to the invention.
For the purposes of the present invention, the substituents have the following meaning, unless otherwise specified:
The term “halogen” , “halogen atom” or “halo” represents fluorine, chlorine, bromine and iodine, particularly chlorine or fluorine, preferably fluorine.
The term “alkyl” represents a linear or branched alkyl radical having the number of carbon atoms specifically indicated, e.g. C1-C10 alkyl means a linear or branched alkyl radical having one, two, three, four, five, six, seven, eight, nine or ten carbon atoms, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl-, decyl-, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neo-pentyl, 1, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3, 3-dimethylbutyl, 2, 2-dimethylbutyl, 1, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl. If the number of carbon atoms is not specifically indicated the term “alkyl” represents a linear or branched alkyl radical having, as a rule, 1 to 40, particularly 1 to 30, preferably 1 to 20 carbon atoms. Particularly, the alkyl group has 1, 2, 3, 4, 5 or 6 carbon atoms ( “C1-C6-alkyl” ) , e.g. methyl, ethyl, n-propyl-, isopropyl, n-butyl, tert-butyl, pentyl, isopentyl, hexyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl,
neo-pentyl, 1, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3, 3-dimethylbutyl, 2, 2-dimethylbutyl, 1, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl. Preferably, the alkyl group has 1, 2 or 3 carbon atoms ( “C1-C3-alkyl” ) , methyl, ethyl, n-propyl or isopropyl.
The term “alkenyl” is to be understood as preferably meaning a linear or branched, monovalent hydrocarbon group, which contains one double bond, and which has 2 or more carbon atoms (such as “C2-C6-alkenyl” ) . Said alkenyl group is, for example, a vinyl, allyl, (E) -2-methylvinyl, (Z) -2-methylvinyl or isopropenyl group.
The term “alkoxy-” is to be understood as preferably meaning a linear or branched, saturated, monovalent, hydrocarbon group of formula -O-alkyl, in which the term “alkyl” is defined as above, e.g. a methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, tert-butoxy, sec-butoxy, pentyloxy, iso-pentyloxy, n-hexyloxy group, or an isomer thereof. Particularly, the “alkoxy-” group is a “C1-C6-alkoxy-” , “C1-C4-alkoxy-” , a “C1-C3-alkoxy-” , a methoxy, ethoxy, or propoxy group, preferably a methoxy, ethoxy or propoxy group. Further preferred is a “C1-C2-alkoxy-” group, particularly a methoxy or ethoxy group.
The term “alkylthio -” is to be understood as preferably meaning a linear or branched, saturated, monovalent, hydrocarbon group of formula –S-alkyl, in which the term “alkyl” is defined as above.
The term “cycloalkyl” is to be understood as preferably meaning a saturated, monovalent, monocyclic hydrocarbon ring which contains such as 3, 4, 5, 6, 7 or 8 carbon atoms. C3-C8-cycloalkyl group is for example, a monocyclic hydrocarbon ring, e.g. a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl group. Particularly, said cycloalkyl group is a C4-C6-cycloalkyl, a C5-C6-cycloalkyl or a cyclohexyl group. For instance, the term “C3-C6-cycloalkyl” is to be understood as preferably meaning a saturated, monovalent, monocyclic hydrocarbon ring which contains 3, 4, 5 or 6 carbon atoms. In particular said C3-C6-cycloalkyl group is a monocyclic hydrocarbon ring such as a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group.
The term “cycloalkoxy-” is to be understood as preferably meaning a group of formula -O-cycloalkyl, in which the term “cycloalkyl” is defined as above.
The term “cycloalkylthio-” is to be understood as preferably meaning a group of formula -S-cycloalkyl, in which the term “cycloalkyl” is defined as above.
The term “cycloalkenyl” ring is to be understood as preferably meaning a non-aromatic monocyclic hydrocarbon ring which contains one or more double bonds. such as a cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl or cycloheptenyl group, wherein the bond between said ring with the rest of the molecule may be to any carbon atom of said ring, be it saturated or unsaturated.
The term “alkenoxy-” is to be understood as preferably meaning a group of formula -O-cycloalkyl, in which the term “cycloalkyl” is defined as above.
The term “heterocyclyl” is to be understood as meaning a saturated or partially unsaturated, monovalent, mono-or bicyclic hydrocarbon ring which contains such as 3, 4, 5, 6, 7, 8 or 9 carbon atoms and further containing 1, 2 or 3 heteroatom-containing groups selected from oxygen, sulfur, nitrogen. Particularly, the term “heterocyclyl” is to be understood as meaning a “4-to 10-membered heterocyclic ring” . Preferably, the “heterocyclyl” used herein is unaromatic.
Said heterocyclic ring is for example, a monocyclic heterocyclic ring such as an oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, 1, 3-dioxolanyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, 1, 4-dioxanyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, 1, 3-dithianyl, thiomorpholinyl, piperazinyl, or chinuclidinyl group. Optionally, said heterocyclic ring can contain one or more double bonds, e. g. 4H-pyranyl, 2H-pyranyl, 2, 5-dihydro-1H-pyrrolyl, 1, 3-dioxolyl, 4H-1, 3, 4-thiadiazinyl, 2, 5-dihydrofuranyl, 2, 3-dihydrofuranyl, 2, 5-dihydrothienyl, 2, 3-dihydrothienyl, 4, 5-dihydrooxazolyl, 4, 5-dihydroisoxazolyl, or 4H-1, 4-thiazinyl group, or, it may be benzo fused.
Particularly, the term “heterocyclyl” is to be understood as being a heterocyclic ring which contains 3, 4 or 5 carbon atoms, and 1, 2 or 3 of the above-mentioned heteroatom-containing groups (a “4-to 8-membered heterocyclic ring” ) , more particularly said ring can contain 4 or 5 carbon atoms, and 1, 2 or 3 of the above-mentioned heteroatom-containing groups (a “5-to 8-membered heterocyclic ring” ) , more particularly said heterocyclic ring is a “6-membered heterocyclic ring” , which is to be understood as containing 4 carbon atoms and 2 of the above-mentioned heteroatom-containing groups or 5 carbon atoms and one of the above-mentioned heteroatom-containing groups, preferably 4 carbon atoms and 2 of the above-mentioned heteroatom-containing groups.
The term “heterocycloxy-” is to be understood as preferably meaning a group of formula -O-heterocyclyl, in which the term “heterocyclyl” is defined as above.
The term “heterocyclylthio-” is to be understood as preferably meaning a group of formula -S-heterocyclyl, in which the term “heterocyclyl” is defined as above.
The term “aryl” is to be understood as preferably meaning a monovalent, aromatic or partially aromatic, mono-, or bi-or tricyclic hydrocarbon ring having 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (a “C6-C14-aryl” group) , particularly a ring having 6 carbon atoms (a “C6-aryl” group) , e.g. a phenyl group; or a biphenyl group, or a ring having 9 carbon atoms (a “C9-aryl” group) , e.g. an indanyl or indenyl group, or a ring having 10 carbon atoms (a “C10-aryl” group) , e.g. a tetralinyl, dihydronaphthyl, or naphthyl group, or a ring having 13 carbon atoms, (a “C13-aryl” group) , e.g. a fluorenyl group, or a ring having 14 carbon atoms, (a “C14-aryl” group) , e.g. an anthranyl group.
The term “heteroaryl” is understood as preferably meaning a monovalent, monocyclic-, bicyclic-or tricyclic aromatic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms (a “5-to 14-membered heteroaryl” group, e.g. 6-membered heteroaryl) , particularly 5 or 6 or 9 or 10 atoms, and which contains at least one heteroatom which may be identical or different, said heteroatom being such as oxygen, nitrogen or sulfur, and in addition in each case can be benzocondensed. Particularly, heteroaryl is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl etc., and benzo derivatives thereof, such as, for example, benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and benzo derivatives thereof, such as, for example, quinolinyl, quinazolinyl, isoquinolinyl, etc.; or azocinyl, indolizinyl, purinyl, etc., and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthpyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, xanthenyl, or oxepinyl, etc.
The term “5-membered heteroaryl” is understood as preferably meaning a monovalent, aromatic ring system having 5 ring atoms and which contains at least one heteroatom which may be identical or different, said heteroatom being such as oxygen, nitrogen or sulfur. Particularly, “5-membered heteroaryl” is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, tetrazolyl.
The term “6-membered heteroaryl” is understood as preferably meaning a monovalent, aromatic ring system having 6 ring atoms and which contains at least one heteroatom which may be identical or different, said heteroatom being such as oxygen, nitrogen or sulfur. Particularly, “6-membered heteroaryl” is selected from pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl.
In general, and unless otherwise mentioned, the heteroarylic or heteroarylenic radicals include all the possible isomeric forms thereof, e.g. the positional isomers thereof. Thus, for some illustrative non-restricting example, the term pyridinyl or pyridinylene includes pyridin-2-yl, pyridin-2-ylene, pyridin-3-yl, pyridin-3-ylene, pyridin-4-yl and pyridin-4-ylene; or the term thienyl or thienylene includes thien-2-yl, thien-2-ylene, thien-3-yl and thien-3-ylene.
The term “aryloxy-” or “heteroaryloxy-” is to be understood as preferably meaning a group of formula -O-aryl or -O-heteroaryl, in which the term “aryl” and “heteroaryl” are defined as above respectively.
The term “arylthio-” or “heteroarylthio -” is to be understood as preferably meaning a group of formula -S-aryl or -S-heteroaryl, in which the term “aryl” and “heteroaryl” are defined as above respectively.
The numeric range “1-10” , as well as those sub-range comprised therein as used throughout this text, is to be understood as meaning a group having a finite number of atoms, such as carbon atoms, of 1 to 10, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties.
As used herein, the term “one or more” , e.g. in the definition of the substituents of the compounds of the general formulae of the present invention, is understood as meaning “one, two, three, four or five, particularly one, two, three or four, more particularly one, two or three, even more particularly one or two” .
The invention also includes all suitable isotopic variations of a compound of the invention. An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be incorporated into a compound of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine and iodine.
Examples
The foregoing and other feathers and advantages of the invention will become further apparent from the following detailed description of the presently preferred embodiment, read in conjunction with the accompanying drawings. The detailed description and drawings are nerely illustrative of the invention, rather than limiting the scope of the invention being defined by appended claims and equivalent thereof.
Following the general strategy in FIG. 1A, we developed a photoactivatable fluorophore named o-TPP3M as shown in FIG. 1B, which was structured with three 4-methoxyphenyl groups as electron donor (D) and 1-methylpyridinium cation as electron acceptor (A) . Firstly, o-TPP3M behaved AIE property with the quantum yield of 0.0018 in solution and 0.051 in solid (Table 1) . According to the RIR mechanism, DMSO solution of o-TPP3M presents almost no emission because of the free rotation of four rings. However, the aggregation of o-TPP3M also showed almost no emission due to the quenching effect of TICT is larger than the induced emission of AIE in polar solution like H2O. This uncommon phenomenon was investigated in FIG. 2. The emission of o-TPP3M in aggregates was not induced with increasing the poor polar solvent H2O in DMSO-H2O system, in contrast, decreased to weaker comparing to the initial almost-non-emissive state. Therefore, o-TPP3M exhibited no emission in polar solution whether it disperses or aggregates due to the RIR and TICT effects, which will ensure the pre-irradiated o-TPP3M in dark state with very low background. (FIG. 1C, curve with empty circles) Secondly, photo-oxidative dehydrocyclization as a new photoactivatable mechanism was introduced to restrict the intramolecular rotation to build post-irradiated state with strong fluorescence (FIG. 1C, curve with filled circles) . Thirdly, the
pyridinium cationic functional group used made o-TPP3M very specific to mitochondria without any extra modification and also improved the hydrophilicity for biocompatibility.
The synthesis of o-TPP3M through a very simple route was shown in FIG. 3. Compound 1 was synthesized starting from compounds 2 and 3. Compound 2 was prepared according to literature procedures, and compound 3 was obtained commercially. And then, o-TPP3M was obtained with a very high yield by the reaction of compound 1 with iodomethane and the ion-exchanging between iodide and hexafluorophosphate. Compared to the reported photoactivatable fluorophores with complicated and inefficient synthesis, here o-TPP3M presented much more readily to be prepared with high efficiency. All of these synthesized products were confirmed by NMR, HR-MS. (FIG. 4-10)
Synthesis of Compound 1
TiCl4 (1 mL, 9.0 mmol) was slowly added into a suspension of Zn dust (1.17 g, 18.0 mmol) in dry THF (50 mL) under -78 ℃. And then the mixture was refluxed for 2 h. Afterward, a mixture of compound 2 (0.642 g, 3 mmol) and compound 3 (1.089 g, 4.5 mmol) in dry THF (20 mL) was added to the reaction, and continued to reflux for another 5 h. THF was removed with the condensation gas. The residue was extracted by DCM and dried by anhydrous MgSO4. The crude product was purified on a silica-gel column using DCM as eluent. Compound 1 was isolated as light yellow solid in 61 %yield. 1H NMR (400 MHz, CD2Cl2) : δ 8.28 (d, J = 6.0 Hz, 2H) , 6.96-6.90 (m, 8H) , 6.69-6.65 (m, 6H) , 3.74 (m, 9H) ; 13C NMR (100 MHz, CD2Cl2) : δ 158.74, 158.48, 158.32, 152.49, 149.13, 141.97, 136.28, 135.76, 135.47, 135.31, 132.43, 132.40, 126.06, 113.28, 113.21, 113.04, 55.09, 55.04; HRMS (m/z) : [M+] calcd. for C28H25NO3, 423.1834; found, 423.1898.
Synthesis of Compound o-TPP3M
Compound 1 (0.106 mg, 0.25 mmol) was dissolved in 20 mL toluene, and 0.1 mL CH3I (large overdose) was added into. The whole reaction mixture was refluxed overnight. Afterward, the reaction mixture was cooled down. The precipitation was filtered and washed with cold toluene three times. The filter was dissolved in 20 mL acetone. KPF6 (100 mg) was added and ion-exchanging for 2 h. Removed acetone and washed with water. The pure yellow product was obtained by recrystallization using DCM and hexane (1: 5) in 95 %yield. 1H NMR (400 MHz, CD2Cl2) : δ 8.10 (d, J = 6.8 Hz, 2H) , 7.38 (d, J = 6.8 Hz, 2H) , 7.00-6.93 (m, 6H) , 6.81-6.76 (m, 4H) , 6.68 (d, J = 8.8 Hz, 2H) , 4.20 (s, 3H) , 3.80 (s, 3H) , 3.77 (s, 3H) , 3.75 (s, 3H) ; 13C NMR (100 MHz, CD2Cl2) : δ 162.95, 160.48, 159.71, 159.23, 150.32, 143.02, 134.14, 133.61, 133.37, 133.21, 133.00, 132.93, 132.79, 129.66, 114.27, 114.22, 113.20, 55.32, 55.22, 47.47; HRMS (m/z) : [M-PF6] + calcd. for [C29H28NO3] +, 438.2069; found, 438.2021.
Synthesis of Compound c-TPP3M
In order to understand how the photo-oxidative dehydrocyclization work as the new photoactivatable mechanism for o-TPP3M, photoreaction using o-TPP3M as the starting material was carried out under irradiation with a 500 W high-pressure mercury vapor lamp in degased methanol solution as shown in FIG. 11A. Iodine was used as an oxidant, and propylene epoxide (PO) was as the scavenging agent for removing the generated hydrogen iodide to avoid side-reactions between o-TPP3M and hydrogen iodide.
A solution of o-TPP3M (145.8 mg, 0.25 mmol) , I2 (69.79 mg, 0.275 mmol) and PO (50 mL) in methanol (250 ml) in the photoreaction vessel, N2 was bubbled through a stirred solution for 30 minutes before photo-irradiation, and then the N2 flow was maintained throughout irradiation with a 500 W high-pressure mercury vapor lamp placed in the immersion quartz well. The progress of the reaction was monitored by U.V. spectroscopy. After completion of the reaction, the solvent was evaporated under reduced pressure and the crude product was purified by column chromatography, and recrystallized from methanol-chloroform to give c-TPP3M in single crystal (30 mg, 21%) , 1H NMR (400 MHz, CD2Cl2) : δ 10.08 (s, 1H) , 8.25 (s, 1H) , 8.23 (d, J = 6.8 Hz, 1H) , 7.91 (d, J = 6.8 Hz, 1H) , 7.69 (d, J = 6.8 Hz, 1H) , 7.36 (d, J = 6.8 Hz, 1H) , 7.08-7.03 (m, 4H) , 6.88-6.85 (m, 4H) , 4.64 (s, 3H) , 4.12 (s, 3H) , 3.80 (s, 6H) ; 13C NMR (100 MHz, CD2Cl2) : δ 161.06, 159.13, 159.10, 150.01, 142.95, 140.15, 136.08, 132.46, 132.04, 131.20, 130.79, 130.21, 129.54, 128.08, 128.07, 125.60, 124.95, 121.22, 113.88, 113.42, 102.98, 56.16, 55.21, 48.37; HRMS (m/z) : [M-PF6] + calcd. for [C29H26NO3] +, 436.1913; found, 436.1899.
NMR and HR-MS spectra for c-TPP3M were used to confirmed its structure as shown in FIG. 12-15. Fortunately, the single crystal of c-TPP3M was obtained in mixed solution of chloroform and methanol by slow evaporation. The ORTEP drawing of molecular structure for c-TPP3M in the single crystal was depicted in FIG. 11B. There is a new single bond produced between pyridinium ring and phenyl ring, suggests that the photo-oxidative dehydrocyclization indeed happened under irradiation and the position of photocyclization was localized between pyridinium ring and phenyl ring. This result is different from the previous reports in which the photocyclization usually occurred between two phenyl rings. Therefore, a question was naturally emerged in mind that why the photocyclization occurred between the pyridinium ring and phenyl ring for o-TPP3M.
To answer this question, density functional theory (DFT) calculation was carried out in FIG. 16. The reason for the distinctive photo-oxidative dehydrocyclizaion in o-TPP3M could be explained in dynamics and thermodynamics. First, FIG. 16A shows the optimized molecular conformation of o-TPP3M. The torsion angles between pyridinium ring and double bond (C1-C2-C6-C17 = 32.52°) and between the phenyl ring and double bond (C2-C1-C3-C16 =43.14°) in the left of double bond were smaller than those between two phenyl rings and double bond (C1-C2-C5-C26 = 39.97° and C2-C1-C4-C7 = 53.17°) in the right of double
bond, respectively, which indicates that the pyridinium and phenyl rings in the left are more coplanar to double bond than the two phenyl rings in the right. Thus, the photocyclization between the pyridinium ring and phenyl ring is easier to perform due to the allowance of steric position. Second, HOMO and LUMO of o-TPP3M were caluculated in FIG. 16B. The electron density in HOMO was delocalized throughout the molecule, however that in LUMO was mainly localized on the pyridinium ring. This suggests that the pyridinium ring is more reactive compared to the other phenyl rings in the excited state, and the photocyclization will prefer to occurre between the pyridinium ring and phenyl ring. Third, if the photocyclization happened between two phenyl rings in the right of double bond in o-TPP3M following the general ways, the photocyclization product, named as c-TPP3M (b) , was simulated and optimized in FIG. 16C. By comparing the lowest energy of the most optimized comformation for c-TPP3M and c-TPP3M (b) , it was found that the energy for c-TPP3M was 21.47 KJ/mol lower than that for c-TPP3M (b) . This suggests that c-TPP3M is a more stable thermodynamic product than c-TPP3M (b) . Therefore, a very unique photocyclization for o-TPP3M to c-TPP3M was sufficiently domenstrated here not only in experiment but also in theoretical calulation, and it will be beneficial to understand the photoactivation in various bioresearch application.
Photophysical properties of o-TPP3M and c-TPP3M
The photophysical properties of o-TPP3M and c-TPP3M were summarized in Table 1. o-TPP3M exhibited the maximum wavelength of absorption at 431 nm, and extremely weak emission at 593 nm with a negligible quantum yield (ΦF, o-TPP3M, Soln = 0.0018) . In contract, its photocyclization product c-TPP3M was very emissive at 521 nm with a considerable quantum yield (ΦF, c-TPP3M, Soln = 0.624) , and still show very strong emission with a high quantum yield (ΦF, c-TPP3M, Solid = 0.201) in solid state. Although o-TPP3M shows a little emission with a quantum yield of 0.051 in solid state, the TICT effect will quench this weak emission to a weaker level in polar solvent as explained in the design section.
Table 1. Photophysical properties of o-TPP3M and c-TPP3M in solution (Soln) a and solid (solid) b states.
a. In DMSO solution with concentration is 10-3 M; b. solid state; c. maximum absorption wavelength; d. maximum emission wavelength; e. absolute quantum yield; f. average fluorescence lifetime.
Next, the photoactivation property of o-TPP3M in vitro was investigated with UV-light irradiation. FIG. 17 shows the PL and UV spectra to monitor the whole photoactivation process in DMSO solution in air. At the beginning, because of the RIR mechanism, the emission color of a solution of o-TPP3M in DMSO was not observed without UV-light irradiation (FIG. 17A, inset a) . In fact, the fluorescence spectrum of o-TPP3M also suggested the fluorescence signal was too weak to record by PL as FIG. 17A shown, which very well consist with the quantum yield of 0.0018 in Table 1. And then, the green color emission was emerged (FIG. 17A, inset b) and the fluorescence intensity at around 521 nm enhanced gradually upon UV-light (365 nm) (FIG. 17A) . Meanwhile, the absorption spectrum of o-TPP3M varied regularly with the absorption maximum wavelength transformed from 431 nm to 407 nm (FIG. 17B) . Appealingly, these new fluorescence and absorption were rather similar to those of c-TPP3M synthesized in FIG. 11A (Table 1) , which indeed suggests that o-TPP3M was photo-converted to c-TPP3M upon UV-light irradiation in the case of the presence of oxygen dissolved in solvent.
Both HR-MS and 1H NMR spectroscopic data in the intermediate states of the photoactivation process were collected to support the photocyclization of o-TPP3M to c-TPP3M. FIG. 18B shows a new signal at m/z 436.1915 comparing to m/z 438.2050 ( [M-PF6] +) of o-TPP3M for the solution of o-TPP3M after irradiation with exposure to air, which accurately corresponded to the molecular weight of c-TPP3M minus PF6. Moreover, 1H NMR signals of o-TPP3M were transformed to those of c-TPP3M gradually were recorded in FIG. 18C after irradiation for different periods of time. There were two easily identified new singlet proton peaks at 10.70 ppm and 4.57 ppm gradually appeared, which corresponding to Ha and Hb of c-TPP3M. In fact, besides these two peaks, the others after photoactivation were all consisted with those of c-TPP3M. Thus, we concluded that o-TPP3M was indeed converted into c-TPP3M after irradiation for a certain period of time. Furthermore, the photo-converted yield from o-TPP3M to c-TPP3M was calculated by 1H NMR spectra, and be plotted against irradiation time in FIG. 18D. The rate of photo-conversion was faster at the beginning and then became slower and slower until it reached to a platform. This is due to combined action involving the decrease of the amount of oxygen dissolved in solvent and the partial photodecomposition of both the reactant and product. The maximum yield was as high as 80%, which indicates that the photoactivation from o-TPP3M to c-TPP3M is very efficient.
Application of o-TPP3M
Before the application for living cell imaging, the cytotoxicity of o-TPP3M to living cells was assessed using a 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) assay (see FIG. 19A) . This MTT assay for o-TPP3M was carried out under different concentrations (0, 2.5, 5, 7.5 and 10 μM) . The cell viability was not significant altered even
when the concentration of o-TPP3M was up to 10 μM in culture medium, suggests that o-TPP3M is almost no cytotoxicity to living cells.
And then, we evaluated the photoactivation behavior of o-TPP3M in living HeLa cells. FIG. 19B monitored the whole photoactivation process in living HeLa cells by the fluorescent microscope. Living HeLa cells stained with o-TPP3M were shown in FIG. 19B with the bright-field image, and the fluorescent images of them were recorded after UV-light (330-385 nm) irradiation for different periods of time (0 s, 4 s, and 8 s) . Before UV-light irradiation, almost no green emission was observed, only existing the very weak blue auto-fluorescence of HeLa cells (FIG. 19B, fluorescent image at 0 s) . Not like other AIE fluorophores with very strong emission when staining cells reported by our group or others, the intrinsic emission of o-TPP3M was not induced after binding to the organelles in cells, which could be attributed to the fluorescence quenching effect of TICT as discussed in the design section. When using UV-light to irradiate, the gradually enhanced green fluorescence was observed with increasing the irradiation time (as the fluorescent images at 4 s and 8 s shown in FIG. 19B) . This photoactivation behavior in living HeLa cells was consisted with that in vitro. The green fluorescence was from the strong emitted c-TPP3M generated in situ in living HeLa cells by photo-oxidative dehydrocyclization. Noteworthily, the fluorescence signal was significantly bright for bio-imaging only spending 8 s compared to the background fluorescence at 0 s, which suggests that the photoactivation of o-TPP3M was very rapid and efficient in living HeLa cells.
During the living HeLa cell imaging, we found that the reticulum structures of mitochondria were clearly visible with the aid of o-TPP3M after UV-light irradiation for 8 s, as shown in FIG. 19B. In order to assess the specific targeting of o-TPP3M to mitochondria in living HeLa cells, Mito Tracker red FM (MT) , a commercially available mitochondria imaging agent, was chosen to stain the living HeLa cells together with o-TPP3M in FIG. 19C. This co-staining experiment suggests that the observed green fluorescence from o-TPP3M after UV-light irradiation for 8 s is localized on the mitochondria of the living HeLa cells (FIG. 19C) . Person’s correlation coefficient (Rr; from -1 to 1) , indicating the degree of linear dependence between two variables, is utilized to qualify the overlap degree of staining region between o-TPP3M and MT. Fluorescent signals of these two fluorophores collected from two different channels are perfectly overlapped with Rr = 0.98, which demonstrates the specific targeting of o-TPP3M on mitochondria.
The kinetic of the photoactivation for o-TPP3M in living HeLa cells was investigated by altering irradiation power using confocal fluorescence microscope technique. Insets a and b in FIG. 20A displayed the confocal images of living HeLa cells before and after irradiation of 405-nm laser. The green fluorescence from the in situ photo-converted c-TPP3M was obviously emerged compared to the initial state, and the monitoring of the whole fluorescence turn-on process was recorded by increasing the fluorescence intensity against
irradiation time. The plot of the fluorescence intensity enhanced multiple (I/I0) against irradiation time for 1%, 2%and 5%power used was shown in FIG. 20A. The faster fluorescence intensity enhancement for using 5%power, with a feature of larger slope in the curve, suggests that the faster rate and shorter time to reach the photoactivation were induced by the higher irradiated power used. In addition, the maximum value platform of I/I0 for 5%power seemed to be faster to reach but smaller than that for 2%and 1%powers. This is because not only the faster photoactivating but also the more serious photobleaching will be brought with the larger laser power used.
A spatiotemporal control over the in situ green-fluorescent c-TPP3M generation by modulating the power of 405-nm laser was allowed by the study of photoactivation kinetic for o-TPP3M in living HeLa cells, which would be successfully utilized to image the select cells in space and time. Thus, living HeLa cells stained with o-TPP3M were prepared in FIG. 20B. A select subpopulation of three living HeLa cells in the optical window (region selected in the white ellipse in FIG. 20B) was exposed to a short-time (about 1 s) 405-nm laser irradiation at 35%power while the living HeLa cells outside the selected region were not. And then, the full optical window was scanning for fluorescence imaging with 1.5%power 405-nm laser. Strongly green turn-on fluorescence was observed for cells within the white ellipse active region, in sharp contrast to the very weak background for cells outside the activated region. The fluorescence intensity of mitochondria in activated living HeLa cells was quantified in FIG. 20B and revealed a 17-fold enhancement relative to the unactivated cells. These results demonstrated that o-TPP3M was indeed a very efficient photoactivatable fluorophore, and could be applied in other various bioresearch.
Claims (10)
- Photoactivatable probes with AIE characteristics, wherein the photoactivatable probes comprise a backbone structure of a formula selected from the group consisting of:wherein each R1, R1′, R1” and R1”’ is independently selected from the group consisting of:each R2, R3, R4, R5, R6 and R7 is independently selected from the group consisting of H, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, alkoxy, alkenoxy, cycloalkoxy, cycloalkenoxy, heterocycloxy, aryloxy, heteroaryloxy, alkylthio, cycloalkylthio,cycloalkenylthio, heterocyclylthio, arylthio, heteroarylthio, and heteroaryloxy, amino, each of which is optionally substituted by one or more substituents selected from halogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, heteroaryl, alkoxy, alkenoxy, cycloalkoxy,cycloalkenoxy, heterocycloxy, aryloxy, heteroaryloxy, alkylthio, cycloalkylthio,cycloalkenylthio, heterocyclylthio, arylthio, heteroarylthio, and heteroaryloxy, amino, azide, OH, SH, COOH, NCS; andeach A- is a monovalent counter ion.
- The photoactivatable probes of claim 1, wherein each R2, R3, R4, R5, R6 and R7 is independently selected from the group consisting of CnH2n+1, C10H7, C12H9, OC6H5, OC10H7 and OC12H9, CnH2nCOOH, CnH2nNCS, CnH2nN3, CnH2nNH2, CnH2nSH, CnH2nCl, CnH2nBr, CnH2nI, N (CnHm) 2, SCnHm, wherein the symbol “n” is an integer preferably selected from 1-10, and m is 2n+1; andeach A- is independently selected from the group consisting of I-, Cl-, Br-, PF6-, ClO4 -, BF4 -,BPh4 -, CH3PhSO3 - and other monovalent counter ions.
- The photoactivatable probes of any of claims 1-4, wherein the photoactivatable probes can be photoactivated, e.g. in solution or aggregate state, by lights such as UV-light or visible light undergoing photo-oxidative dehydrocyclization.
- A method of preparation of the photoactivatable probes of any of claims 1-4, wherein the photoactivatable probes are constructed based on RIR, TICT and Photo-oxidative Dehydrocyclization.
- Photoactivated product of the photoactivatable probes of any of claims 1-4, wherein the photoactivated product is a photocyclized product.
- The photoactivated product of claim 7, wherein as compared to the structure of the photoactivatable probe as defined above, at least one single bond is newly produced between pyridinium ring and phenyl ring of the photocyclized product under photoactivation.
- Use of these photoactivatable probes in spatiotemporal selective cells (e.g. HeLa cells), organelles and macromolecules imaging and super-resolved fluorescence imaging.
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CN108982460A (en) * | 2018-08-22 | 2018-12-11 | 深圳大学 | A kind of super-resolution imaging method, device and terminal device |
WO2019161626A1 (en) * | 2018-02-21 | 2019-08-29 | The Hong Kong University Of Science And Technology | Corannulene-incorporated aie nanodots with highly suppressed nonradiative decay for boosted cancer phototheranostics in vivo |
CN112047977A (en) * | 2020-08-20 | 2020-12-08 | 宁波大学 | Mitochondrial targeting fluorescent probe and synthetic method and application thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102858911A (en) * | 2010-03-01 | 2013-01-02 | 香港科技大学 | Light emitting tetraphenylene derivatives, its method for preparation and light emitting device using the same derivatives |
CN103842472A (en) * | 2011-09-01 | 2014-06-04 | 香港科技大学 | Biocompatible nanoparticles with aggregation induced emission characteristics as fluorescent bioprobes and methods of using the same for in vitro and in vivo imaging |
US20140212359A1 (en) * | 2013-01-29 | 2014-07-31 | The Hong Kong University Of Science And Technology | Photostable aie luminogens for specific mitochondrial imaging and its method of manufacturing thereof |
-
2015
- 2015-12-29 WO PCT/CN2015/099420 patent/WO2016070854A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102858911A (en) * | 2010-03-01 | 2013-01-02 | 香港科技大学 | Light emitting tetraphenylene derivatives, its method for preparation and light emitting device using the same derivatives |
CN103842472A (en) * | 2011-09-01 | 2014-06-04 | 香港科技大学 | Biocompatible nanoparticles with aggregation induced emission characteristics as fluorescent bioprobes and methods of using the same for in vitro and in vivo imaging |
US20140212359A1 (en) * | 2013-01-29 | 2014-07-31 | The Hong Kong University Of Science And Technology | Photostable aie luminogens for specific mitochondrial imaging and its method of manufacturing thereof |
Non-Patent Citations (3)
Title |
---|
GU XINGGUI ET AL.: "Mitochondrion-Specific Live- Cell Bioprobe Operated in a Fluorescence Turn-On Manner and a Well-Designed Photoactivatable Mechanism", ADV. MATER., vol. 27, 7 October 2015 (2015-10-07), pages 7093 - 7100, XP055278311 * |
PRADEEP P. KAPADIA ET AL.: "Tetrapyridyl tetraphenylethylenes: supramolecular building blocks with aggregation-induced emission properties", TETRAHEDRON LETTERS, vol. 52, no. 19, 12 March 2011 (2011-03-12), pages 2519 - 2522, XP028188669 * |
SIEGFRIED HUÈNIG ET AL.: "Violene/Cyanine Hybrids as Electrochromic Systems. Part 3:1 Heterocyclic Onium End Groups", TETRAHEDRON, vol. 56, no. 25, 16 June 2000 (2000-06-16), pages 4203 - 4211, XP004201672 * |
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WO2018210334A1 (en) * | 2017-05-19 | 2018-11-22 | The Hong Kong University Of Science And Technology | Aiegens for cancer cells and gram-positive bacteria discrimination and killing |
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CN107383094A (en) * | 2017-09-04 | 2017-11-24 | 中山大学 | A kind of novel chiral gathering induced luminescence material and its preparation method and application |
WO2019161626A1 (en) * | 2018-02-21 | 2019-08-29 | The Hong Kong University Of Science And Technology | Corannulene-incorporated aie nanodots with highly suppressed nonradiative decay for boosted cancer phototheranostics in vivo |
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