CN106496102A - Mitochondrial two-photon fluorescence viscosity probe and preparation method thereof - Google Patents
Mitochondrial two-photon fluorescence viscosity probe and preparation method thereof Download PDFInfo
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- 239000000523 sample Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 230000002438 mitochondrial effect Effects 0.000 title abstract description 9
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 13
- 230000015572 biosynthetic process Effects 0.000 claims description 11
- 238000003786 synthesis reaction Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000013078 crystal Substances 0.000 claims description 7
- -1 dimethyl hydroxyethyl amine Chemical class 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 229960004756 ethanol Drugs 0.000 claims description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 5
- UOQXIWFBQSVDPP-UHFFFAOYSA-N 4-fluorobenzaldehyde Chemical compound FC1=CC=C(C=O)C=C1 UOQXIWFBQSVDPP-UHFFFAOYSA-N 0.000 claims description 4
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 208000035126 Facies Diseases 0.000 claims description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000010410 layer Substances 0.000 claims description 3
- XKBGEWXEAPTVCK-UHFFFAOYSA-M methyltrioctylammonium chloride Chemical compound [Cl-].CCCCCCCC[N+](C)(CCCCCCCC)CCCCCCCC XKBGEWXEAPTVCK-UHFFFAOYSA-M 0.000 claims description 3
- 239000012044 organic layer Substances 0.000 claims description 3
- 150000003053 piperidines Chemical class 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 238000001953 recrystallisation Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 238000010792 warming Methods 0.000 claims description 3
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims 2
- BSKHPKMHTQYZBB-UHFFFAOYSA-N 2-methylpyridine Chemical compound CC1=CC=CC=N1 BSKHPKMHTQYZBB-UHFFFAOYSA-N 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 229910000027 potassium carbonate Inorganic materials 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 16
- 238000003384 imaging method Methods 0.000 abstract description 11
- 238000011161 development Methods 0.000 abstract description 6
- 230000008859 change Effects 0.000 abstract description 4
- 238000012800 visualization Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000000975 dye Substances 0.000 description 6
- GRQHQQCBHTVBPI-UHFFFAOYSA-N [N+](=O)(O)[O-].C(=C)C1=NC=CC=C1 Chemical class [N+](=O)(O)[O-].C(=C)C1=NC=CC=C1 GRQHQQCBHTVBPI-UHFFFAOYSA-N 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- OKJPEAGHQZHRQV-UHFFFAOYSA-N Triiodomethane Natural products IC(I)I OKJPEAGHQZHRQV-UHFFFAOYSA-N 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
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- 239000001569 carbon dioxide Substances 0.000 description 2
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- 238000004624 confocal microscopy Methods 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
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- 239000011358 absorbing material Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000000680 avirulence Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000013332 literature search Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 230000008965 mitochondrial swelling Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- CAIZIGKCPQJNCW-UHFFFAOYSA-N nitric acid;pyridine Chemical compound O[N+]([O-])=O.C1=CC=NC=C1 CAIZIGKCPQJNCW-UHFFFAOYSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- MYFATKRONKHHQL-UHFFFAOYSA-N rhodamine 123 Chemical compound [Cl-].COC(=O)C1=CC=CC=C1C1=C2C=CC(=[NH2+])C=C2OC2=CC(N)=CC=C21 MYFATKRONKHHQL-UHFFFAOYSA-N 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
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- 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
- C07D213/36—Radicals substituted by singly-bound nitrogen atoms
- C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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Abstract
The invention discloses a mitochondrial two-photon fluorescence viscosity probe and a preparation method thereof, wherein the structural formula of the mitochondrial two-photon fluorescence viscosity probe is as follows:
Description
Technical field
The present invention relates to a kind of mitochondrion two-photon fluorescence viscosity probe and preparation method thereof, be a kind of have avirulence,
Identification single-minded to cell mitochondrial and the good two-photon absorbing material vinylpyridine nitric acid salt derivative of light stability.
Background technology
Mitochondrion (Mitochondria) is the key cells device of cell survival, produces the energy of the somagenic need more than 90%
Amount is grown with sustaining life and supporting, in many important physiological process, such as apoptosis, differentiation, signal transduction and activity
Indispensable effect is played in the generation of oxygen (ROS).In the different phase of cell division and metabolism and in cell development
In atomization, mitochondrial size, quantity and form can all occur different changes.Work in recent years it has been shown that
Mitochondrial form and quantity, such as neurodegenerative disease parkinson (Parkinson ' s related to numerous disease
Disease), A Zihaimoshi diseases (Alzheimer ' s disease), atherosclerosiss, heart disease, apoplexy and cancer, its
In be mainly shown as that the viscosity that shown by mitochondrial swelling increases.Therefore, by imaging in fluorescent probe observation living cells
Mitochondrial metamorphosis, understand effect of the mitochondrion in bio-metabolic process, have in terms of disease early diagnosis and therapy
There is directive significance.
However, the organic molecule mitochondrial probe that has commercially produced at present, is by fluorescence and phosphorescence skill mostly
Art carries out imaging analysis, such as Rhodamine123, Mito to mitochondrion quantity and formGreenFM and MitoRed FM, they are broadly divided into the mitochondrion of the fluorescent probe and dependent/non-dependent that depend on mitochondrial membrane potential
Probe, although these dyestuffs can carry out living cells imaging, but their cytotoxicity is big, photo-labile, poorly water-soluble,
Stokes displacements are less, it is impossible to the mitochondrion viscosity increase produced because of disease is identified, so as to limit making for they
With.Therefore, develop the high mitochondrion fluorescent probe of a kind of nontoxic, good water solubility, light stability have important scientific meaning and
Obvious application prospect.
Applicant has carried out following literature search to the theme of the application：
1st, scholar.glgoo.org nets retrieval result：(2016/9/29)
2nd, middle National IP Network's retrieval result：
Retrieval mode one：
Piece name-mitochondrion two-photon fluorescence viscosity probe：Without pertinent literature.
A kind of piece name-mitochondrion two-photon fluorescence viscosity probe vinylpyridine nitric acid salt derivative and its preparation side
Method：Without pertinent literature.
Retrieval mode two：
In full-mitochondrion two-photon fluorescence viscosity probe：Without pertinent literature.
A kind of in full-mitochondrion two-photon fluorescence viscosity probe vinylpyridine nitric acid salt derivative and its preparation side
Method：Without pertinent literature.
Content of the invention
The present invention is intended to provide a kind of mitochondrion two-photon fluorescence viscosity probe and preparation method thereof, is with iodomethane, first
Yl pyridines, dimethyl hydroxyethyl amine, 4-Fluorobenzaldehyde are raw material, have simply efficiently synthesized organic pyridine nitric acid of D- π-A configurations
Salt.Target molecule of the present invention has good cellular affinity, can carry out double light of long-time stable to living cells mitochondria
Son imaging, and mitochondrion viscosity B coefficent can be recognized.Effective visualization tool is provided for tracking change of the mitochondrion in cell,
There are good development and application values.
Mitochondrion two-photon fluorescence viscosity probe of the present invention is vinylpyridine nitric acid salt derivative, and its structural formula is：
The preparation method of mitochondrion two-photon fluorescence viscosity probe of the present invention, comprises the steps：
1st, the synthesis of intermediate 1
Iodomethane (14.40g, 100mmol) is added in 50mL round-bottomed flasks, is slowly dropped under stirring and is dissolved in 4mL ethanol
4-methyl pyridine (6.20g, 65mmol), be heated to back flow reaction 20min, separate out a large amount of white crystals, filter, washing with alcohol
And after drying, obtain intermediate 1,14.60g, yield 96%.
2nd, the synthesis of intermediate 2
DMSO (200mL), dimethyl hydroxyethyl amine (165g, 2200mmol), Carbon Dioxide is added in 500mL round-bottomed flasks
Potassium (166g, 1200mmol) and 3 drop Aliquat-336, are warming up to 95 DEG C of reaction 10min, add 4-Fluorobenzaldehyde
(124g, 1000mmol), continues reaction three days at 95 DEG C；Reaction is cooled to room temperature after terminating, and reactant liquor is poured in frozen water, uses
Salt acid for adjusting pH value collects water layer to 2, after being stirred vigorously, with NaOH solution regulation pH value to 11, collected organic layer, repeatable operation
3 times, extracted with DCM and collect organic faciess, add anhydrous magnesium sulfate to dry, intermediate 2 is obtained after removing solvent, be faint yellow solid,
142g, yield 79%.
3rd, the synthesis of target product B1
Intermediate 2 (1.80g, 10mmol), intermediate 1 (2.40g, 20mmol), 30mL is added in 100mL round-bottomed flasks
Dehydrated alcohol and 3 drop piperidines, stirring reaction 24h at 60 DEG C obtain dark red solution；To in the dark red solution, Deca contains
AgNO3The 30mL ethanol solutions of (1.70g, 10mmol), back flow reaction 2h, reaction are cooled down after terminating, and are filtered, washing with alcohol
And dry, then DCM recrystallization are used, there are red crystals to separate out, obtain target product 2.00g, yield 60%.
4th, the bio-imaging research of target molecule B1 of the present invention
In living cells co-focusing imaging, the target molecule B1 and human hepatocarcinoma cells HepG2 of low concentration (1-10 μm of ol)
After the co-cultivation of 10 minutes, PBS buffer solution is cleaned 2-3 time, then the Mitotracker Far Red cultures 10 with 500nM
Minute.By two-photon laser confocal microscopy, target molecule and mitochondrion commercial dyes Mitotracker are found
Far Red are overlapped well, and there is the effect for responding that mitochondrion viscosity (viscosity) changes.
Compared with the prior art, beneficial effects of the present invention are embodied in：
1st, the vinylpyridine nitrate B1 of present invention synthesis is that a kind of two-photon absorption with biological developing function has
Machine small molecule material, is capable of identify that the change of active somatic cell Mitochondria viscosity, can be used as mitochondrion two-photon fluorescence viscosity
Probe；
2nd, compared with document report or commercial dye, the target product B1 of the present invention has two-photon absorption feature, to thin
The features such as born of the same parents' not damaged, good water solubility, high light stability, and there is obvious using value；
3rd, target product B1 of the invention single-minded can develop to mitochondrion, and its extremely low cytotoxicity and
High light stability is applicable to long-time and follows the trail of form and viscosity B coefficent of the mitochondrion in living cells.
4th, target product B1 of the invention is the new mitochondrial dye of a class.
5th, the raw material of target product B1 of the invention be easy to get, synthetic route brief, synthesis condition is gentle.
Description of the drawings
Fig. 1 is the crystal structure figure of target product B1 of the present invention, illustrates that B1 is the novel substance for having clear and definite structure.
Fig. 2 is the water solublity test of target product B1 of the present invention, illustrates that B1 has excellent water solublity.
Fig. 3 is real-time monitoring results of the target product B1 of the present invention to the cell proliferation activity influence of HepG-2 and 3T3, table
Bright B1 to two kinds of cytotoxics, with good biocompatibility.
Fig. 4 is that the single, double photon of target product B1 of the present invention develops and 3D images, (a) single photon (b) two-photon (c)
Overlay chart (d) three-dimensional imaging, shows that B1 can enter intracellular and with the development of single, double photon function.
Fig. 5 is the development figure of target product B1 of the present invention and commercial dyes Mitotracker Far Red common locations,
Pearson ' s Rr=0.8921 illustrate that B1 has overlapping for height with commercial dyes MTFR.
Fig. 6 is development figure and intracellular Fluorescence imaging region of the target product B1 of the present invention in HepG-2 cell Mitochondrias
Microviscosity Gradient distribution image, show that B1 has response mitochondrion viscosity (viscosity) effect that changes.
Fig. 7 is microviscosity Gradient distribution images of the target product B1 of the present invention to the imaging region of variety classes cell, says
Bright B1 can be with a wide range of applications as mitochondrion two-photon fluorescence viscosity probe.
Fig. 8 is the development detected to cell in 0-72h by target product B1 of the present invention, illustrates that the chemical combination object light is steady
Qualitative height, can be used for long-time and the mitochondrion of living cells is tracked.
Specific embodiment
The preparation of the present embodiment Mitochondria two-photon fluorescence viscosity probe and biological developing method are as follows：
1st, the synthesis of intermediate 1
Iodomethane (14.40g, 100mmol) is added in 50mL round-bottomed flasks, is slowly dropped under stirring and is dissolved in 4mL ethanol
4-methyl pyridine (6.20g, 65mmol), be heated to 78 DEG C of back flow reaction 20min, separate out a large amount of white crystals, filter, ethanol
Intermediate 1,14.60g, yield 96% is obtained after washing and drying.
2nd, the synthesis of intermediate 2
DMSO (200mL), dimethyl hydroxyethyl amine (165g, 2200mmol), Carbon Dioxide is added in 500mL round-bottomed flasks
Potassium (166g, 1200mmol) and 3 drop Aliquat-336, are warming up to 95 DEG C of reaction 10min, add 4-Fluorobenzaldehyde
(124g, 1000mmol), continues reaction three days at 95 DEG C；Reaction is cooled to room temperature after terminating, and reactant liquor is poured in frozen water, uses
Salt acid for adjusting pH value collects water layer to 2, after being stirred vigorously, with NaOH solution regulation pH value to 11, collected organic layer, repeatable operation
3 times, extracted with DCM and collect organic faciess, add anhydrous magnesium sulfate to dry, intermediate 2 is obtained after removing solvent, be faint yellow solid,
142g, yield 79%.
3rd, the synthesis of target product
Intermediate 2 (1.80g, 10mmol), intermediate 1 (2.40g, 20mmol), 30mL is added in 100mL round-bottomed flasks
Dehydrated alcohol and 3 drop piperidines, are heated to 60 DEG C of stirring reactions 24h, obtain dark red solution；To in the dark red solution, Deca contains
There is AgNO3The 30mL ethanol solutions of (1.70g, 10mmol), back flow reaction 2h, reaction are cooled down after terminating, and are filtered, and ethanol is washed
Wash and dry, then use DCM recrystallization, there are red crystals to separate out, obtain target product 2.00g, yield 60%.
1H NMR(400MHz,d6- DMSO) δ=8.67 (d, 2H), 8.02 (d, 2H), 7.89 (d, 1H), 7.56 (d, 2H),
7.14(d,1H),6.79(d,2H),4.76(s,1H),4.17(s,3H),3.53(d,4H),3.03(d,3H).
13C NMR(101MHz,d6- DMSO) δ=153.34,151.06,144.25,141.38,130.20,122.04,
116.77,111.67,58.14,53.83,46.25,38.70.
Anal.Calcd.for C17H21N3O4:C,61.62；H,6.39；N, 12.68%.Found:C,56.08；H,
7.052；N, 11.64%.IR (KBr, cm-1):3390(m),2925(w),1640(m),1584(s),1518(s),1377(vs),
1176(s),1040(w),818(w),535(m).
M.p.=120 DEG C of .ESI:m/z,cal:269.36,found:269.42[M+]
4th, the bio-imaging research of target molecule B1 of the present invention
In living cells co-focusing imaging, the target molecule B1 and human hepatocarcinoma cells HepG2 of low concentration (1-10 μm of ol)
After the co-cultivation of 10 minutes, PBS buffer solution is cleaned 2-3 time, then the Mitotracker Far Red cultures 10 with 500nM
Minute.By two-photon laser confocal microscopy, target molecule and mitochondrion commercial dyes Mitotracker are found
Far Red are overlapped well, and there is the effect for responding that mitochondrion viscosity (viscosity) changes.
Claims (2)
1. a kind of mitochondrion two-photon fluorescence viscosity probe, it is characterised in that its structural formula is：
2. the preparation method of the mitochondrion two-photon fluorescence viscosity probe described in a kind of claim 1, it is characterised in that include as
Lower step：
(1) synthesis of intermediate 1
Iodomethane 100mmol is added in 50mL round-bottomed flasks, and the 65mmol for being dissolved in 4mL ethanol is slowly added dropwise under stirring to methyl
Pyridine, is heated to back flow reaction 20min, separates out a large amount of white crystals, filters, and obtains intermediate 1 after washing with alcohol drying；
(2) synthesis of intermediate 2
To in 500mL round-bottomed flasks add DMSO 200mL, dimethyl hydroxyethyl amine 2200mmol, Anhydrous potassium carbonate 1200mmol with
And 3 drop Aliquat-336, be warming up to 95 DEG C reaction 10min, add 4-Fluorobenzaldehyde 1000mmol, at 95 DEG C continue reaction
Three days；Reaction is cooled to room temperature after terminating, and reactant liquor is poured in frozen water, with salt acid for adjusting pH value to 2, collects water after stirring
Layer, adjusts pH value to 11 with NaOH solution, and collected organic layer collects organic faciess with DCM extractions, adds anhydrous magnesium sulfate to dry,
Intermediate 2 is obtained after removing solvent；
(3) synthesis of target product B1
2 10mmol of intermediate, 1 20mmol, 30mL dehydrated alcohol of intermediate and 3 drop piperidines are added in 100mL round-bottomed flasks,
Stirring reaction 24h at 60 DEG C, obtains dark red solution；To in the dark red solution, Deca contains 10mmol AgNO330mL without
Hydrous ethanol solution, back flow reaction 2h, reaction are cooled down after terminating, and are filtered, and washing with alcohol is simultaneously dried, then use DCM recrystallization, has redness
Crystal is separated out, and obtains target product.
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