CN110156839A - A kind of Mitochondrially targeted hypochlorite two-photon fluorescence probe and its preparation method and application - Google Patents
A kind of Mitochondrially targeted hypochlorite two-photon fluorescence probe and its preparation method and application Download PDFInfo
- Publication number
- CN110156839A CN110156839A CN201910436820.8A CN201910436820A CN110156839A CN 110156839 A CN110156839 A CN 110156839A CN 201910436820 A CN201910436820 A CN 201910436820A CN 110156839 A CN110156839 A CN 110156839A
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- Prior art keywords
- hypochlorite
- compound
- crude product
- added
- water
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- 239000000523 sample Substances 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 66
- 150000001875 compounds Chemical class 0.000 claims description 62
- 239000000243 solution Substances 0.000 claims description 35
- 238000006243 chemical reaction Methods 0.000 claims description 34
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- 239000012043 crude product Substances 0.000 claims description 29
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 20
- 239000000741 silica gel Substances 0.000 claims description 20
- 229910002027 silica gel Inorganic materials 0.000 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 16
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
- C07F9/65615—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing a spiro condensed ring system of the formula where at least one of the atoms X or Y is a hetero atom, e.g. S
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- 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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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Optics & Photonics (AREA)
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Abstract
The invention discloses a kind of Mitochondrially targeted hypochlorite two-photon fluorescence probes and its preparation method and application, belong to technical field of analytical chemistry.Technical solution of the present invention main points are as follows: a kind of Mitochondrially targeted hypochlorite two-photon fluorescence probe, structural formula are as follows:The application of hypochlorite the invention further particularly discloses the preparation method of the Mitochondrially targeted hypochlorite two-photon fluorescence probe and its in selective enumeration method water environment, biological cell system, mouse tissue.Double fluorescence probes of the invention have many advantages, such as that the remote red sector of two-photon excitation is got well and responded rapid to the transmitting of near infrared region, high sensitivity, selectivity.
Description
Technical field
The invention belongs to technical field of analytical chemistry, and in particular to a kind of Mitochondrially targeted hypochlorite two-photon fluorescence
Probe and its preparation method and application.
Background technique
Hypochlorous acid (HClO) is a kind of active oxygen (ROS), it is related with a variety of physiology and pathologic process, as pathogen is anti-
It answers, anti-inflammatory is adjusted, aging.Hypochlorous acid is usually by the peroxide of the chloride ion of myeloperoxidase (MPO) catalysis in cell
Change what reaction generated.Due to hypochlorous pKaIt is 7.59, can be partially dissociated into hypochlorite (ClO under physiological ph conditions-)。
Maintain the HClO/ClO of proper level-Function normal for cell is of great significance, and intracellular excessive HClO/ClO-
Then it is considered related to a variety of diseases, such as arthritis, atherosclerosis, nerve retrograde affection, cancer.Mitochondria is cell
A kind of middle organelle for being capable of providing energy, plays an important role in various important life process.As cell oxygen
Main consumer, mitochondria are the main sources of cellular reactive oxygen species (ROS), and it is dead that the accumulation of reactive oxygen species can lead to cell
It dies and various diseases.As one of mitochondria active oxygen, HClO/ClO-It can induce mitochondrial permeability, inducing cell withers
It dies.Therefore, exploitation is able to detect HClO/ClO in mitochondria-Effective ways be particularly important.
Fluorescent molecular probe has high sensitivity, high-spatial and temporal resolution, not damaged sample preparation and original position real-time, quickly
The advantages that detection, it has also become be the effective tool that related substances detect in life entity, and further applied in organelle and divided
The detection for analysing object, achieves satisfactory result.In recent years, researcher reports a large amount of HClO/ClO-Fluorescence is visited
Needle, some of fluorescence probes are successfully applied to HClO/ClO in mitochondria-Real-time detection and imaging.However, these
Fluorescence probe is designed based on single photon fluorescence imaging technique mostly.Since their excitation wavelength is shorter, in vivo
Practical application performance is restricted, and will lead to background fluorescence serious interference, fluorogen light big to the phototoxicity of biological sample floats
The problems such as Bai Yanchong.And two-photon fluorescence imaging technology obtains the excitation state of fluorogen using two lower photons of energy,
Thus it is shown that the advantage bigger than single photon fluorescence imaging technique, for example lesser background fluorescence interference, to biological sample compared with
Low photo-toxicity, better three dimension location ability, deeper penetration depth, lower self-absorption.It in recent years, is understanding
HClO/ClO in mitochondria-Biological action, scientific research personnel has developed several for HClO/ClO in mitochondria-It is double
Photon fluorescence probe.However, these two-photon fluorescence probes can only solve the problems, such as that the short excitation wavelength of single photon probe (uses
The two-photon excitation of near infrared region), they still have the defect that launch wavelength is located at short wavelength region (< 550nm), this is still
The problems such as so will lead to background fluorescence interference and lower tissue penetration depths.The short wavelength of these two-photon fluorescence probes emits,
So that their advantages in bio-imaging are weakened, because some biomolecule (such as NADH, riboflavin and folic acid) also can
It is excited by two-photon laser, generates the background fluorescence interference of blue green regions.In addition, penetrate can quilt for the bob of above-mentioned two-photon probe
Such as hemoglobin (Hb) and oxyhemoglobin (HbO2) etc. biomolecule absorb, it reduce deep tissue imaging in fluorescence
The collection efficiency of signal.Therefore, there is the remote red two-photon probe near infrared region emission characteristics to be of great significance for development,
Transmitting light can less be absorbed by biomolecule, and the deep enough deep tissues of energy, to further decrease background fluorescence
Interference improves collecting efficiency.However, having no that the Mitochondrially targeted hypochlorite with far infrared near infrared emission is double at present
The report of photon fluorescence probe.
Summary of the invention
The present invention is directed to current hypochlorite two-photon fluorescence probe problem encountered and status, provides a kind of line grain
The hypochlorite two-photon fluorescence probe of body targeting, the two-photon fluorescence probe is using triphenylphosphine cation as mitochondria target
To seeking group, for ClO in cell mitochondrial-Imaging, which has the remote red transmitting near infrared region, sensitive
It spends high, selective good and responds the features such as rapid.
The present invention also provides the preparation method of above-mentioned Mitochondrially targeted hypochlorite two-photon fluorescence probe and its
The application of hypochlorite in selective enumeration method water environment, biological cell system or mouse tissue.
The present invention adopts the following technical scheme that solve above-mentioned technical problem, a kind of double light of Mitochondrially targeted hypochlorite
Sub- fluorescence probe, it is characterised in that the structural formula of the two-photon fluorescence probe is as follows:
The preparation method of Mitochondrially targeted hypochlorite two-photon fluorescence probe of the present invention, it is characterised in that tool
Body step are as follows:
Step S1: by 325mg 6- amino -1,2,3,4- tetrahydro -1- naphthalenone, 430mg iodomethane, 830mg potassium carbonate with
5mL DMF is added into dry 250mL round-bottomed flask, is stirred to react overnight, then reaction solution is cooled to room temperature, adds in 45 DEG C
Enter 50mL water, solution 100mL ethyl acetate is extracted, organic phase is collected, continues after organic phase is washed with water with saturation
The washing of NaCl aqueous solution obtains crude product with solvent is spin-dried for after anhydrous sodium sulfate drying, crude product is obtained with silica gel column purification
1 160mg of compound as white solid, yield 42%, eluant, eluent volume proportion used are petrol ether/ethyl acetate=3:1,
Synthetic route is as follows:
Step S2: under conditions of ice-water bath, by 189mg compound 1,313mg 4- lignocaine ketone acid and the dense sulphur of 15mL
Acid is added into 50mL round-bottomed flask, and reaction is transferred in oil bath pan after 1 hour is heated to 100 DEG C of reactions 4 hours, by reaction solution
It is cooled to room temperature, pours into mixture of ice and water, 2mL perchloric acid is added, the reaction solution that precipitating is precipitated is filtered and is rinsed with cold water
Filter cake dissolves filter cake with methylene chloride, is washed with water after washing and continues to be washed with saturation NaCl aqueous solution, anhydrous sodium sulfate is dry
Decompression is spin-dried for solvent and obtains crude product afterwards, and crude product is obtained 2 490mg of compound, yield 86%, institute with silica gel column purification
It is methylene chloride/methanol=15:1 with eluant, eluent volume proportion, synthetic route is as follows:
Step S3: 339mg compound 2,312mg PyBOP and 20mL methylene chloride are added in 100mL round-bottomed flask, so
The hydrazine hydrate that 1mL mass concentration is 85% is added in round-bottomed flask afterwards, reaction solution reacts to 3 hours at room temperature, then
Continue to be washed with saturation NaCl aqueous solution after reaction solution is washed with water, vacuum distillation removes dichloromethane after anhydrous sodium sulfate is dry
Alkane mutually obtains crude product, and crude product purified by silica gel column purification is obtained 3 246mg of compound, yield 85%, eluant, eluent body used
Product proportion dichloromethane/ethyl acetate=10:1, synthetic route are as follows:
Step S4: under conditions of ice-water bath, by 200mg compound 3,30mL methylene chloride and 45 μ L triethylamines be added to
In the round-bottomed flask of 100mL, then to 25 μ L bromoacetyl chlorides are added dropwise in round-bottomed flask, reaction solution is reacted at room temperature 1 hour,
Reaction solution is evaporated under reduced pressure removing methylene chloride again and obtains the crude product of compound 4, by crude product purified by silica gel column column chromatographic purifying
4 136.8mg of compound is obtained, yield 68.4%, synthetic route is as follows:
Step S5: 140mg compound 4,280mg triphenylphosphine and 20mL acetonitrile are poured into the round-bottomed flask of 50mL, in
90 DEG C back flow reaction 24 hours, by reaction solution it is cooling after solvent be removed under reduced pressure obtain crude product, by crude product purified by silica gel column column layer
Analysis purifying obtains target product two-photon fluorescence probe Mito-TP-ClO 90.1mg, and yield 40.7%, synthetic route is such as
Under:
Mitochondrially targeted hypochlorite two-photon fluorescence probe of the present invention is in selective enumeration method water environment, biology
The application of hypochlorite in cell system or mouse tissue, wherein detection include aqueous solution in fluorescence detection, cell imaging detection,
Imaging of tissue detection.
Compared with the prior art, the invention has the following beneficial effects: the synthesis of (1) two-photon fluorescence probe compares
It is easier to, and last handling process is relatively easy；(2) two-photon fluorescence probe realizes the highly selective Gao Ling of hypochlorite ion
Sensitivity quickly detects, and has the ability for resisting other molecule interference in life entity；(3) two-photon fluorescence probe has close excellent
Mitochondrially targeted ability, the remote red transmitting near infrared region of two-photon excitation can be applied to hypochlorite in tissue
Image checking.The two-photon fluorescence probe is by reducing autofluorescence background interference in life entity, reducing the light to biological sample
Damage improves the features such as tissue penetration depths, to obtain more accurate and stable optical signalling and imaging effect.Therefore, originally
Two-photon fluorescence probe in invention has broad application prospects in hypochlorite detection field, to hypochlorite in organism
The research of physiology and the mechanism of action of pathologic process is of great significance.
Detailed description of the invention
Fig. 1 is fluorescent probe compounds Mito-TP-ClO made from embodiment 1 after various concentration hypochlorite is added
Ultraviolet-visible absorption spectroscopy figure；
Fig. 2 is fluorescent probe compounds Mito-TP-ClO made from embodiment 1 after various concentration hypochlorite is added
Fluorescence spectra；
It is glimmering at 650nm that Fig. 3, which is fluorescent probe compounds Mito-TP-ClO made from embodiment 1 in launch wavelength,
The graph of relation that luminous intensity changes with (0-70 μM) of hypochlorite concentration, illustration are fluorescent probe compounds Mito-TP-ClO
It is the graph of relation that the fluorescence intensity at 650nm changes with (0-10 μM) of hypochlorite concentration in launch wavelength；
Fig. 4 is selective column of the fluorescent probe compounds Mito-TP-ClO made from embodiment 1 to different ions and molecule
Shape figure, wherein 1, PBS；2,K+；3,Ca2+；4,Mg2+；5,Zn2+；6,Fe3+；7,Cu2+；8,CH3COO-；9,NO3 -；10,Cl-；11,
F-；12,I-；13,Cys；14,GSH；15,Glucose；16,ATP；17,ADP；18,t-BuOO.；19,NO2 -；20,H2O2；21,
ONOO-；22,O2 .-；23,.OH；24,1O2；25,NO；26,t-BuOOH；27,NaClO；
Fig. 5 be fluorescent probe compounds Mito-TP-ClO made from embodiment 1 under condition of different pH to hypochlorite
Response condition；
Fig. 6 is the Two-photon excitation spectra figure of fluorescent probe compounds Mito-TP-ClO made from embodiment 1；
Fig. 7 is that the lysosome of fluorescent probe compounds Mito-TP-ClO made from embodiment 1 and mitochondria common location are real
It tests；
Fig. 8 is endogenous hypochlorite in the detection of fluorescent probe compounds Mito-TP-ClO made from embodiment 1 HeLa
Fluorescence imaging figure；
Fig. 9 and Figure 10 is that fluorescent probe compounds Mito-TP-ClO made from embodiment 1 is detected in mouse liver slice
The Z axis depth scan fluorescence imaging figure of hypochlorite.
Specific embodiment
Above content of the invention is described in further details by the following examples, but this should not be interpreted as to this
The range for inventing above-mentioned theme is only limitted to embodiment below, and all technologies realized based on above content of the present invention belong to this hair
Bright range.
Embodiment 1
The synthesis of fluorescent probe compounds Mito-TP-ClO
(1) synthesis of compound 1
325mg 6- amino -1,2,3,4- tetrahydro -1- naphthalenone, 430mg iodomethane, 830mg potassium carbonate and 5mL DMF are added
Enter into dry 250mL round-bottomed flask, be stirred to react under the conditions of 45 DEG C overnight, reaction solution is cooled to room temperature, is added
Solution 100mL ethyl acetate is extracted, collects organic phase by 50mL water, after organic phase is washed with water (50mL × 2), is continued
(50mL × 1), anhydrous Na are washed with saturation NaCl aqueous solution2SO4Dry, Rotary Evaporators are evaporated off solvent and obtain crude product, will slightly produce
Product silica gel column purification, silica gel particle size are 200-300 mesh, and eluant, eluent volume proportion is petrol ether/ethyl acetate=3:1,
Obtain compound as white solid 1,160mg, yield 42%.Its synthetic route is as follows:
(2) synthesis of compound 2
Under conditions of ice-water bath, 189mg compound 1,313mg 4- lignocaine ketone acid and the 15mL concentrated sulfuric acid are added
Into 50mL round-bottomed flask, reaction is transferred in oil bath pan after 1 hour is heated to 100 DEG C of reactions 4 hours, and reaction solution is cooled to
Room temperature is poured into mixture of ice and water, and the perchloric acid of 2mL is added, and the reaction solution that precipitating is precipitated is filtered and rinses filter cake with cold water,
Filter cake is dissolved with methylene chloride, (50mL × 2) continue to wash (50mL × 1), nothing with saturation NaCl aqueous solution after being washed with water
Water Na2SO4Dry, decompression obtains crude product after being spin-dried for solvent, and by crude product silica gel column purification, silica gel particle size is 200-
300 mesh, eluant, eluent volume proportion are methylene chloride/methanol=15:1, obtain compound 2,490mg, yield 86%.It is synthesized
Route is as follows:
(3) synthesis of compound 3
339mg compound 2,312mg PyBOP and 20mL methylene chloride are added in the round-bottomed flask of 100mL, then
The hydrazine hydrate that 1mL mass concentration is 85% is added in round-bottomed flask, reaction solution is reacted at room temperature 3 hours, then will
After reaction solution is washed with water (50mL × 2), continue to wash (50mL × 1), anhydrous Na with saturation NaCl aqueous solution2SO4It is dry, subtract
Pressure is distilled off methylene chloride and mutually obtains crude product.By crude product purified by silica gel column purification, silica gel particle size is 200-300 mesh,
Eluant, eluent volume proportion dichloromethane/ethyl acetate=10:1, obtains compound 3,246mg, yield 85%.Its synthetic route
It is as follows:
(4) synthesis of fluorescent probe compounds 4
Under conditions of ice-water bath, 200mg compound 3,30mL methylene chloride and 45 μ L triethylamines are added to 100mL's
In round-bottomed flask, then to continuing that 25 μ L bromoacetyl chlorides are slowly added dropwise in round-bottomed flask, it is small that reaction solution is reacted to 1 at room temperature
When, reaction solution is evaporated under reduced pressure removing methylene chloride and obtains the crude product of compound 4, by crude product silica gel column purification, silica gel
Granular size is 200-300 mesh, and eluant, eluent volume proportion is petrol ether/ethyl acetate=1:1, and purifying obtains compound
4136.8mg, yield 68.4%, synthetic route is as follows:
(5) synthesis of fluorescence probe Mito-TP-ClO
140mg compound 4,280mg triphenylphosphine and 20mL acetonitrile are poured into the round-bottomed flask of 50mL, returned in 90 DEG C
Stream reaction obtains crude product for 24 hours, by solvent is removed under reduced pressure after reaction solution cooling, by crude product silica gel column purification, silica gel particle
Size is 200-300 mesh, and eluant, eluent volume proportion is methylene chloride/methanol=10:1, and it is green to obtain target fluorescent probe compound
Color solid Mito-TP-ClO, 90.1mg, yield 49.2%.Its synthetic route is as follows:
Embodiment 2
The ultraviolet-visible absorption spectroscopy figure that fluorescent probe compounds Mito-TP-ClO and various concentration hypochlorite act on
Measurement
After Fig. 2 is fluorescent probe compounds Mito-TP-ClO made from embodiment 1 and the hypochlorite effect of various concentration
Ultraviolet-visible absorption spectroscopy figure, the additional amount of hypochlorite is 0-70 μM.From in figure 2 as can be seen that 605nm can from
To an absorption peak, with the increase of hypochlorite concentration, the absorbance at 605nm is gradually increased.Ultraviolet-visible absorption spectroscopy
The instrument of measurement is TU-1900 type ultraviolet-uisible spectrophotometer (Beijing Purkinje General Instrument
Co.,Ltd.)。
Embodiment 3
The measurement of fluorescent probe compounds Mito-TP-ClO and the fluorescence spectra of various concentration hypochlorite effect
Fluorescent probe compounds Mito-TP-ClO prepared by Example 1 is dissolved in system in N,N-dimethylformamide (DMF)
It at 50 μM of stock solution, is added in the centrifuge tube of 5mL from 0.8mL is taken out in stock solution, time of different equivalents (0-7) is added
Chlorate anions standard solution, with the solution of PBS buffer solution (10mM, pH=7.4) be diluted to 4mL (DMF/PBS volume ratio be 1:
4), using 605nm as exciting light, slit width is set as 2.5nm/5nm, measures its fluorescence spectrum.Fluorescence spectrum as shown in Figure 1,
As hypochlorite is added, the fluorescence at 650nm is gradually increased, the relationship of fluorescence intensity and hypochlorite concentration such as Fig. 3
It is shown, 6.0 × 10-8-1.0×10-5In a linear relationship in the range of mol/L, fluoremetry instrument used is Perkin
Elmer LS55 sepectrophotofluorometer.
Embodiment 4
Selectivity of the fluorescent probe compounds Mito-TP-ClO to different molecular or ion
It takes out 0.8mL from the fluorescence probe stock solution in embodiment 3 to be added in the centrifuge tube of 5mL, addition needs checking
The standard solution for examining competition substance, is diluted to 4mL (DMF/PBS volume ratio with the solution of PBS buffer solution (10mM, pH=7.4)
For 1:4), 26 kinds of competition substances have been investigated altogether, configure 27 samples altogether, the last one is sodium hypochlorite.It is excitation with 605nm
Light, slit width are set as 2.5nm/5nm, measure its fluorescence spectrum, as a result as shown in Figure 4.It is by Fig. 4 it can be found that intracellular
Metal ion, anion, neutral molecule, active nitrogen, other active oxygens are to fluorescent probe compounds Mito-TP-ClO in 650nm
The fluorescence at place has little effect, and the addition of liquor natrii hypochloritis keeps fluorescence of the probe Mito-TP-ClO at 650nm significant
Enhancing.
Embodiment 5
Influence of the solution ph to the fluorescence response of fluorescent probe compounds Mito-TP-ClO measurement hypochlorite
The present embodiment has investigated pH value to blank fluorescence probe compound Mito-TP-ClO and Mito-TP-ClO+ respectively
The influence of fluorescence intensity in the case of two kinds of hypochlorite (20 μM), as a result such as Fig. 5.Within the scope of pH=5-9, pH is glimmering to blank
The fluorescence intensity of light probe compound Mito-TP-ClO is substantially without influence.And in the presence of sodium hypochlorite (20 μM), when
When pH=9, the fluorescence intensity of detection architecture is maximum, shows that fluorescence probe is best to the response of hypochlorite at this time.Mitochondria pH
About position 7.8 is within the scope of 7-9 in pH as can be known from Fig. 5, fluorescent probe compounds Mito-TP-ClO to hypochlorite all
It shows preferable response, shows that fluorescent probe compounds Mito-TP-ClO can satisfy what hypochlorite in mitochondria detected
Demand.
Embodiment 6
The Two-photon excitation spectra of fluorescent probe compounds Mito-TP-ClO and hypochlorite reaction product compound 2
Measurement
Using two-photon activity absorption cross-section (Φ δ) of the femtosecond fluorescence spectrometry compound 2 in the section 760-860nm,
And draw the Two-photon excitation spectra of its compound 2.It is 5.0 × 10 that compound 3, which is configured to concentration,-6The 25mM PBS of M is buffered
Liquid (pH=7.4, containing the reference material in 1wt%DMF), measured using rhodamine B as two photon absorption cross section.Two-photon absorption
Section is using formula δ=δ r (SsΦrφrcr)/(SrΦsφscs) calculate, wherein subscript s and r representative sample and reference point
Son.The intensity of two-photon fluorescence excitation is expressed as S.Φ is fluorescence quantum yield, and φ is total phosphor collection effect of experimental provision
Rate.Molecules in solution number density is expressed as c, and δ r is the known two photon absorption cross section of reference material.
Embodiment 7
The mitochondria common location of fluorescent probe compounds Mito-TP-ClO is tested
In HeLa cell, by fluorescent probe compounds Mito-TP-ClO and business lysosome dyestuff LysoTracker
Green DND-26 carries out common location experiment (as shown in Figure 7).Specific steps are as follows: by fluorescent probe compounds Mito-
The DMSO solution and commercialization lysosome dyestuff of TP-ClO is added in the culture solution for giving birth to HeLa cell, the two final concentration difference
For 10 μM and 500nM.After cultivating 30 minutes in carbon dioxide incubator, a certain amount of liquor natrii hypochloritis is added into system,
Make 20 μM of system sodium hypochlorite concentration, waiting is imaged with Laser Scanning Confocal Microscope after twenty minutes, while with 488nm and
635nm excitation, in green channel it can be observed that there is green fluorescence (505-545nm) sending, this is LysoTracker Green
The light of DND-26 transmitting, and it has been observed that red fluorescence (650-720nm) is issued in red channel, this is fluorescence probe
The feux rouges issued after object Mito-TP-ClO and hypochlorite response is closed, the two is handled with software it can be concluded that two kinds of dyes
The common location coefficient of material is 0.35, while the significantly different step of variation of the fluorescence intensity distribution of the target red line in two channels.With
Afterwards, in another ware in HeLa cell, by fluorescent probe compounds Mito-TP-ClO and commercialization mitochondrial dye
MitoTracker Green FM carries out common location experiment (as shown in Figure 7).Specific steps are as follows: by fluorescence probe chemical combination
The DMSO solution and commercialization lysosome dyestuff of object Mito-TP-ClO is added in the culture solution for giving birth to HeLa cell, and the two is whole
Concentration is respectively 10 μM and 500nM.After cultivating 30 minutes in carbon dioxide incubator, a certain amount of hypochlorous acid is added into system
Sodium solution, makes 20 μM of system sodium hypochlorite concentration, and waiting is imaged with Laser Scanning Confocal Microscope after twenty minutes, is used simultaneously
488nm and 635nm is excited, in green channel it can be observed that having green fluorescence (505-545nm) sending, this is
The light of MitoTracker Green FM transmitting, and it has been observed that red fluorescence (650-720nm) is issued in red channel,
This is the feux rouges issued after fluorescent probe compounds Mito-TP-ClO and hypochlorite response, and the two is handled with software
It can be concluded that the common location coefficient of two kinds of dyestuffs is 0.94, while the variation of the fluorescence intensity distribution of the target red line in two channels
Tend to be synchronous.The above result shows that Mito-TP-ClO has excellent Mitochondrially targeted ability.
Embodiment 8
Fluorescence imaging of the fluorescent probe compounds Mito-TP-ClO to endogenous cellular hypochlorite
In HeLa cell, investigate fluorescent probe compounds Mito-TP-ClO to the fluorescence of endogenic hypochlorite at
As situation (Fig. 8).Specific steps are as follows: four ware cells are prepared, the DMSO of fluorescent probe compounds Mito-TP-ClO is molten
Liquid is added in cell ware, and final concentration of 10 μM of probe.After cultivating 30min in carbon dioxide incubator, four ware cells are carried out
Different subsequent operations.No. 1 ware directly carries out two-photon fluorescence imaging, as shown, without obvious fluorescence.No. 2 wares continue with LPS (1
μ g/mL) with after PMA (1 μ g/mL) culture 1 hour, it is then imaged, as shown, No. 2 wares show that apparent fluorescence increases
By force.In order to prove the enhancing of cell fluorescence caused by the reacting of hypochlorite and probe, our further progresss pair
Than test.By No. 3 wares and No. 4 wares respectively with para aminobenzoyl hydrazine ABH (500 μM) and N-acetyl-L-cysteine NAC
After (3mM) is cultivated 1 hour, it is imaged.As shown, two wares all show faint fluorescence, hence it is evident that it is lower than No. 2 wares, this
It is to be able to suppress the generation of hypochlorite because ABH is a kind of inhibitor of myeloperoxidase (MPO).And NAC can be with secondary chlorine
Acid reaction, the hypochlorous acid that a large amount of consumption generate.The above result shows that in fluorescent probe compounds Mito-TP-ClO is successfully realized
The fluorescence imaging of source property hypochlorite detects.
Embodiment 9
Fluorescence imaging of the fluorescent probe compounds Mito-TP-ClO in mouse liver histotomy
Two pieces of nude mice liver frozen tissue sections are prepared, No. 1 and No. 2 slices are named as.Will wherein No. 1 slice only with
Mito-TP-ClO (20 μM) cultivates 1h together, as control sample.No. 2 slices are trained with Mito-TP-ClO (20 μM) together first
1h is supported, is then washed 3 times with DPB, continues to cultivate 1h together with NaClO (300 μM), as target sample.Then 820nm's
Two pieces of slices are imaged under two-photon excitation, collect the transmitting in the section 605-680nm.Imaging results such as Fig. 9 and Figure 10
It is shown.It can be seen that probe Mito-TP-ClO can carry out fluorescence imaging, imaging depth to the hypochlorite in tissue
Up to 230 μm.
Embodiment above describes basic principles and main features of the invention and advantage, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, under the range for not departing from the principle of the invention, various changes and improvements may be made to the invention, these changes and improvements are each fallen within
In the scope of protection of the invention.
Claims (3)
1. a kind of Mitochondrially targeted hypochlorite two-photon fluorescence probe, it is characterised in that the structure of the two-photon fluorescence probe
Formula is as follows:
2. a kind of preparation method of Mitochondrially targeted hypochlorite two-photon fluorescence probe described in claim 1, feature
It is specific steps are as follows:
Step S1: by 325mg6- amino -1,2,3,4- tetrahydro -1- naphthalenone, 430mg iodomethane, 830mg potassium carbonate and 5mL DMF
It is added into dry 250mL round-bottomed flask, is stirred to react in 45 DEG C overnight, then reaction solution is cooled to room temperature, 50mL is added
Solution 100mL ethyl acetate is extracted, collects organic phase by water, is continued after organic phase is washed with water water-soluble with saturation NaCl
Liquid washing obtains crude product with solvent is spin-dried for after anhydrous sodium sulfate drying, crude product is obtained white solid with silica gel column purification
1 160mg of compound, yield 42%, eluant, eluent volume proportion used are petrol ether/ethyl acetate=3:1, synthetic route
It is as follows:
Step S2: under conditions of ice-water bath, 189mg compound 1,313mg 4- lignocaine ketone acid and the 15mL concentrated sulfuric acid are added
Enter into 50mL round-bottomed flask, reaction is transferred in oil bath pan after 1 hour is heated to 100 DEG C of reactions 4 hours, and reaction solution is cooling
It to room temperature, pours into mixture of ice and water, 2mL perchloric acid is added, the reaction solution that precipitating is precipitated is filtered and rinse filter cake with cold water,
Filter cake is dissolved with methylene chloride, is washed with water after washing and continues to be washed with saturation NaCl aqueous solution, subtract after anhydrous sodium sulfate is dry
Pressure is spin-dried for solvent and obtains crude product, crude product is obtained 2 490mg of compound with silica gel column purification, yield 86% is used to wash
De- agent volume proportion is methylene chloride/methanol=15:1, and synthetic route is as follows:
Step S3: 339mg compound 2,312mg PyBOP and 20mL methylene chloride are added in 100mL round-bottomed flask, then will
The hydrazine hydrate that 1mL mass concentration is 85% is added in round-bottomed flask, reaction solution is reacted at room temperature 3 hours, then will be anti-
Continue to be washed with saturation NaCl aqueous solution after answering liquid to be washed with water, vacuum distillation removes methylene chloride phase after anhydrous sodium sulfate is dry
Crude product is obtained, crude product purified by silica gel column purification is obtained into 3 246mg of compound, yield 85%, eluant, eluent volume used is matched
Than dichloromethane/ethyl acetate=10:1, synthetic route is as follows:
Step S4: under conditions of ice-water bath, by 200mg compound 3,30mL methylene chloride and 45 μ L triethylamines be added to
In the round-bottomed flask of 100mL, then to 25 μ L bromoacetyl chlorides are added dropwise in round-bottomed flask, reaction solution is reacted at room temperature 1 hour,
Reaction solution is evaporated under reduced pressure removing methylene chloride again and obtains the crude product of compound 4, by crude product purified by silica gel column column chromatographic purifying
4 136.8mg of compound is obtained, yield 68.4%, synthetic route is as follows:
Step S5: 140mg compound 4,280mg triphenylphosphine and 20mL acetonitrile are poured into the round-bottomed flask of 50mL, in 90 DEG C
Back flow reaction 24 hours, crude product is obtained by solvent is removed under reduced pressure after reaction solution cooling, crude product purified by silica gel column column is chromatographed pure
Change obtains target product two-photon fluorescence probe Mito-TP-ClO 90.1mg, and yield 40.7%, synthetic route is as follows:
3. Mitochondrially targeted hypochlorite two-photon fluorescence probe described in claim 1 is in selective enumeration method water environment, life
The application of hypochlorite in object cell system or mouse tissue, wherein detection includes fluorescence detection in aqueous solution, cell imaging inspection
It surveys, imaging of tissue detection.
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| CN111849460A (en) * | 2020-06-19 | 2020-10-30 | 嘉兴学院 | Near-infrared ratiometric fluorescent probe for detecting biological mercaptan based on fluorescence resonance energy transfer and preparation and application thereof |
| CN112442056A (en) * | 2020-11-03 | 2021-03-05 | 上海应用技术大学 | Fluorescent probe for simultaneously detecting hypochlorous acid and peroxynitrite anions, and synthetic method and application thereof |
| CN112779001A (en) * | 2019-11-02 | 2021-05-11 | 复旦大学 | Preparation and application of near-infrared viscosity fluorescent probe |
| CN112920081A (en) * | 2021-02-06 | 2021-06-08 | 许昌学院 | Fluorescent probe for selectively identifying hypochlorous acid and preparation method and application thereof |
| CN113603701A (en) * | 2021-08-11 | 2021-11-05 | 中国矿业大学 | Colorimetric/fluorescent probe for detecting hypochlorite ions and preparation method and application thereof |
| CN114195774A (en) * | 2021-11-04 | 2022-03-18 | 徐州医科大学 | Photosensitizer with hypochlorous acid activated fluorescence and mitochondrion targeting functions and preparation method and application thereof |
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| CN112779001A (en) * | 2019-11-02 | 2021-05-11 | 复旦大学 | Preparation and application of near-infrared viscosity fluorescent probe |
| CN112779001B (en) * | 2019-11-02 | 2022-08-26 | 复旦大学 | Preparation and application of near-infrared viscosity fluorescent probe |
| CN111849460A (en) * | 2020-06-19 | 2020-10-30 | 嘉兴学院 | Near-infrared ratiometric fluorescent probe for detecting biological mercaptan based on fluorescence resonance energy transfer and preparation and application thereof |
| CN111849460B (en) * | 2020-06-19 | 2022-04-22 | 嘉兴学院 | Near-infrared ratiometric fluorescent probe for detecting biological mercaptan based on fluorescence resonance energy transfer and preparation and application thereof |
| CN112442056A (en) * | 2020-11-03 | 2021-03-05 | 上海应用技术大学 | Fluorescent probe for simultaneously detecting hypochlorous acid and peroxynitrite anions, and synthetic method and application thereof |
| CN112442056B (en) * | 2020-11-03 | 2022-08-23 | 上海应用技术大学 | Fluorescent probe for simultaneously detecting hypochlorous acid and peroxynitrite anions, and synthetic method and application thereof |
| CN112920081A (en) * | 2021-02-06 | 2021-06-08 | 许昌学院 | Fluorescent probe for selectively identifying hypochlorous acid and preparation method and application thereof |
| CN113603701A (en) * | 2021-08-11 | 2021-11-05 | 中国矿业大学 | Colorimetric/fluorescent probe for detecting hypochlorite ions and preparation method and application thereof |
| CN114195774A (en) * | 2021-11-04 | 2022-03-18 | 徐州医科大学 | Photosensitizer with hypochlorous acid activated fluorescence and mitochondrion targeting functions and preparation method and application thereof |
| CN114195774B (en) * | 2021-11-04 | 2023-09-26 | 徐州医科大学 | Photosensitizer with hypochlorous acid activated fluorescence and mitochondrial targeting functions and preparation method and application thereof |
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