CN106366074A - Two-photon fluorescent probe capable of recognizing mitochondrion pH and application - Google Patents

Two-photon fluorescent probe capable of recognizing mitochondrion pH and application Download PDF

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Publication number
CN106366074A
CN106366074A CN201610801435.5A CN201610801435A CN106366074A CN 106366074 A CN106366074 A CN 106366074A CN 201610801435 A CN201610801435 A CN 201610801435A CN 106366074 A CN106366074 A CN 106366074A
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mitochondrion
probe
fluorescent probe
application
recognizing
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林伟英
刘勇
孟芳芳
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University of Jinan
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University of Jinan
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • 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
    • 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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  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The invention discloses a two-photon fluorescent probe capable of recognizing the mitochondrion pH, and further discloses application of the fluorescent probe in mitochondrion pH detection. Experiments prove that the two-photon fluorescent probe detects the pH on the basis of two channels and can be well positioned in a mitochondrion. The fluorescent probe has the outstanding advantage in detection on the pH level of the mitochondrion in a living body, can well penetrate through the tissue and has the potential application value in the field of fluorescent biomarkers.

Description

A kind of two-photon fluorescence probe of identification mitochondrion ph and application
Technical field
The present invention relates to a kind of novel two-photon fluorescent probe of identification mitochondrion ph and its application, more particularly, to a kind of special One identifies mitochondrial phenanthrenequione class compound fluorescent probe and its application；Belong to organic molecule fluorescent probe field.
Background technology
As one of important organelle in human body, mitochondrion is because containing ribosome and itself carries hereditary material and has Particularity, it is the main place that aerobic respiration produces energy, is described as " cellular energy factory ".Human body interior lines under normal circumstances Plastochondria also participates in the processes such as cell differentiation, cellular informatics transmission and apoptosis, and has regulating cell growth and cell The ability in cycle.
A series of forms occurring during apoptosis and Biochemical changes, are to be activated led to cell by apoptotic signal system The change regulation and control of a series of activity of interior enzyme and the expression of apoptosis-associated genes.Research shows, intracellular ph, mitochondrial membrane electricity The changes of biophysicss character such as position (δ ψ m) have close relationship with the regulation and control of antiapoptotic signals system.Mitochondrion is withering Be increasingly subject in research field of dying pay attention to, and in mitochondrion ph because maintain and regulate and control mitochondrial function importance also become research One of focus.
Human body mitochondrion occurs extremely to lead to mitochondrial disease, and mitochondrial disease is a big class inherited metabolic disease, line Plastochondria disease specifically includes that maternal inheritance leigh syndrome, mitochondrial myopathy, multisystem disease, cardiomyopathy etc..Mitochondriopathy Mode of inheritance more complicated, often mainly caused by karyogene and mitochondrial gene the reason lead to disease, and its clinical table Existing complexity, the diagnosis of the definite cause of disease is very difficult, tests and analyzes often through macromole the enzyme activity and combines hereditism's gene The two methodses of analysis assign a cause for an illness.So, the detection of its abnormal level should be caused with the great attention of people.Based on this, open By detecting the change of ph in mitochondrion, the fluorescent probe sending out new, judges that the exception of mitochondrial level becomes urgently to be resolved hurrily Technical problem.
Content of the invention
For the deficiencies in the prior art, the invention provides a kind of luxuriant and rich with fragrance quinoness two-photon of identification mitochondrion ph is glimmering Light probe and its application.
Of the present invention new be suitable to identify mitochondrion ph two-photon fluorescence probe pid it is characterised in that: its chemistry Structural formula such as formula is (i) shown:
(i)
The preparation method of above-claimed cpd is: by 2,3,3- trimethyl -3h- indole 1 and iodoethane under the conditions of acetonitrile solvent It is heated to reflux generating compound 2, then compound 2 and terephthalaldehyde reacts and obtain product 3, product 3 and phenanthrenequione exist Final product pid is reacted to obtain, Fig. 1-2 is shown in by its nmr collection of illustrative plates under the conditions of glacial acetic acid.
Above-claimed cpd to prepare reaction equation as follows:
The application of the above-mentioned fluorescent probe ph change in detection mitochondrion being suitable to single-minded identification mitochondrion ph.
In above-mentioned application: described fluorescent probe is to realize identifying mitochondrion ph in dual pathways mode.
Above-mentioned fluorescent probe energy high selectivity identifies mitochondrion ph, and this probe is under mildly acidic conditions (ph scope is in 4-6) When being excited with 405nm wavelength, in 425-475nm, thus sending blue light, and (ph scope exists its launch wavelength under weak basic condition When 7-8) equally being excited with 405nm wavelength its launch wavelength change red shift to 500-550nm green light.By in difference Ph in send different light to realize with dual pathways form detecting different ph.In cell, mitochondrion is weakly alkaline environment, By acting on green light with alkalescence ph in mitochondrion, realize this probe and identify can mitochondrion to the response of ph and then more indirectly Effect.
Experiment confirms: when detecting that environment is aqueous phase, by the solvation effect of mitochondrion ph fluorescent probe of the present invention Should show, at the fluorescence intensity of water middle probe relatively weak (Fig. 3), its fluorescence quantum yield is 0.02(referring to table 1: compound Photophysical property), this enables probe to be preferably used for the detection of human body environment, greatly reduces the harm to human body for the probe Property, also eliminate the interference of itself simultaneously.In ph titration experiments (Fig. 4), probe occurs bimodal to the response of ph.With molten The increase of liquid ph, certain regularity in probe.In ph scope 4-6, the fluorescence peak at its 546nm is more than at 456nm Peak value, solution shows lighting of 546nm, solution blue light-emitting then.When ph scope 7-8, the peak value at 456nm is higher than 546nm Intensity, and send green glow, the light sending out different in different ph by probe, and well ph can be responded and carry out the dual pathways Detection.In addition cell experiment (Fig. 5) confirms, is excited with 405 wavelength, absorbing wavelength controls in 425-475nm, 1, passage Blue light, when absorbing wavelength controls in 500-550nm, passage 2 green light is it can be verified that this probe is in cell and testing in vitro Equally, enable Dual channel detection.By being fitted with commercial mitochondrial probe mtr, this probe can significantly be positioned line In plastochondria, it is achieved thereby that Intramitochondrial Dual channel detection ph.This probe completes imaging in tissue using the dual pathways simultaneously, Its depth is up to 120 μm (Fig. 6) it is seen that this probe can deep and thorough be organized, and realizes depth detection.
Based on above-mentioned experimental result, may certify that the luxuriant and rich with fragrance quinoness fluorescence of identification mitochondrion ph of the present invention is visited Pin is the fluorescent probe molecule that the new high selectivity of a class identifies mitochondrion ph, and this probe synthesis path simplicity is it is easy to apply. By external titration ph, cell imaging experiment may determine that this probe is capable of the effect of its Dual channel detection mitochondrion ph, And there is certain penetration capacity in the tissue.
The phenanthrenequione class compound fluorescent probe of the identification mitochondrion ph that the present invention provides has its significant advantage, and institute of the present invention State ph selectivity in aqueous phase for the phenanthrenequione Benzazole compounds and synthesizing mean also has novelty and simplicity.Based on the present invention The phenanthrenequione class compound fluorescent probe of the identification mitochondrion ph providing enables Dual channel detection, and realizes organizing interior ph to become Picture, studies for biology and has established theoretical basiss as application, indicate that it has potential using value in fluorescent biolabels field.
Brief description
Fig. 1: pid1h nmr (400 mhz, meod)
Fig. 2: pid13c nmr(100 mhz, dmso-d6)
The solvation effect of Fig. 3: probe.Excitation wavelength 405nm.
Fig. 4: ph titration experiments；Wherein excitation wavelength is 405nm；The concentration of probe mother solution: 10-3M, dilution 5ml is surveyed Examination.
The cell biological imaging applications of Fig. 5: probe.
The imaging of tissue application of Fig. 6: probe.
Specific embodiment
Embodiment 1
The synthesis of compound 2:
It is dissolved in 20ml acetonitrile, 2,3, the 3- trimethyl -3h- indole 1 of 1.0 g (6.25 mmol) by iodoethane 5.3ml (31.25 mmol) drops in mixed liquor, is heated to reflux 15h, and reaction system is changed into bronzing from faint yellow.After reaction terminates It is cooled to room temperature, solvent is evaporated, gained solid washed with ether is filtrated to get red solid.Yield: 86%.
The synthesis of compound 3:
By 0.30g (2mmol) terephthalaldehyde, 0.75 g compound 2 is dissolved in 20ml dehydrated alcohol.Heat under nitrogen protection Overnight, reaction system is changed into red from yellow for backflow.Reaction terminate after, ea extract, with pe:ea(10:1) leacheate cross post purification Obtain red solid.Yield: 80%.
The synthesis of compound pid:
By 0.21g (1mmol) phenanthrenequione, 0.47g(1.1 mmol) compound 3,1.54g ammonium acetate and 10ml glacial acetic acid are blended in In the round-bottomed flask of 50ml, it is heated to reflux 8h under nitrogen protection, reaction is cooled to room temperature after terminating, liquid is poured into 100ml Frozen water in, precipitation is filtered, three drying of washing, ethyl alcohol recrystallization obtains red solid.Yield: 70%.Wherein, Fig. 1: pid1h nmr (400 mhz, meod) ；Fig. 2: pid13c nmr(100 mhz, dmso-d6).
1h nmr (400 mhz, meod): δ 8.85 (s, 1h), 8.72 (s, 1h), 8.59 (s, 1h), 8.55 (s, 1h), 8.51-8.48 (d, j = 12.0 hz, 2h), 8.34–8.32 (d, j = 8.0 hz, 2h), 7.93 – 7.90 (m, 1h), 7.86 – 7.79 (m, 2h), 7.78 – 7.67 (m, 1h), 4.84– 4.78 (q, j = 8.0 hz, 2h), 1.93 (s, 6h), 1.68 – 1.65 (t, j = 7.5 hz, 3h). 13c nmr (100 mhz, dmso-d6): 181.73, 153.37, 148.32, 144.58, 140.90, 138.08, 135.29, 134.65, 131.90, 130.04, 129.66, 128.60, 128.27, 127.74, 127.25, 126.86, 126.41, 126.07, 124.70, 124.35, 123.66, 122.84, 122.69, 122.47, 115.74, 113.17, 63.44, 52.86, 42.88, 30.24, 29.09, 23.46, 23.08, 14.43, 11.49. hrms (m/z): [m-i+h]+calcd for c35h30n3+: 493.2500; found, 493.2124.
Embodiment 2
The solvation effect of compound pid
Prepare the 10 of 1 part of 8 ml in advance-3The n of m probe, n- dimethyl formamide solution, then takes 3 μ l to add seven respectively In identical 5ml volumetric flask, it is diluted to 5ml with dmf, acetonitrile, pbs, methanol, ethanol, chloroform, water respectively, then carries out fluorescence Detection, result is shown in Fig. 3；
Embodiment 3
Quantum yield in each solvent for the probe is calculated according to embodiment 2 data.
Above-mentioned computing formula is as follows:
Wherein,WithThe single photon fluorescence quantum yield of representative sample and reference respectively,WithGeneration respectively Table sample productWithThe absorption values of selected reference,WithRespectively representative sample and reference molecules Fluorescence intensity, ∫ fs and ∫ fr represent the single photon fluorescence integral area of probe molecule and reference respectively.Preferably reference is usually (see Table 1) unrelated with excitation wavelength.
Table 1: the photophysical property of compound pid
Embodiment 4
The titration of ph
Prepare the buffer solution and 10 of 45 ml difference ph-3The probe mother solution of m concentration is as standby.Take 3 μ l probe mother solutions with not It is diluted to 5ml with ph and carry out fluoroscopic examination (λex=405 nm), calculate fluorescence intensity in each system, set up fluorescence intensity and ph Standard curve, standard curve (see Fig. 4).
Embodiment 5
Bio-imaging: living cells Coloration experiment
Probe is made into 1 mm dmf mother solution, is diluted with 1ml culture medium during dyeing.
The cell inoculated is incubated 30 min in probe molecule solutions 37 c of set amount concentration, is washed 3-5 time with pbs, The cell of adherent growth is placed on microscope slide；Then carry out fluorescence imaging with single two-photon fluorescence microscope.Excitation wavelength is 405nm, in 425-475nm, green channel absorbs in 500-550nm the absorbing wavelength of blue channel.
Commercial mitochondrial probe (mtr) carries out common location experiment, and from image under fluorescence microscope, this test proves Probe can be positioned mitochondrion (see figure 5) well.
Mouse tissue is tested
Mouse liver is cut into slices, is washed 3-5 time with pbs, be placed in 1ml culture dish.
30 min are redyed in the section setting in the probe molecule solutions of quantitative concentrations, then uses single two-photon fluorescence Microscope carries out fluorescence imaging, observes the depth (see figure 6) that probe reaches tissue.

Claims (3)

1. a kind of two-photon fluorescence probe of identification mitochondrion ph it is characterised in that, its chemical structural formula such as formula is (i) shown:
().
2. two-photon fluorescence probe according to claim 1 is it is characterised in that be prepared from by following preparation method: will 2,3,3- trimethyl -3h- indole 1 are heated to reflux under the conditions of acetonitrile solvent with iodoethane, then occur with terephthalaldehyde Reaction, reacts to obtain final product under the conditions of glacial acetic acid with phenanthrenequione.
3. the two-photon fluorescence probe identifying mitochondrion ph described in a kind of claim 1 is in abnormal the answering of detection mitochondrial function With.
CN201610801435.5A 2016-09-05 2016-09-05 Two-photon fluorescent probe capable of recognizing mitochondrion pH and application Pending CN106366074A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106928189A (en) * 2017-03-03 2017-07-07 济南大学 A kind of mitochondrial fluorescence probe of identification with larger Stokes displacements
CN108610411A (en) * 2018-04-28 2018-10-02 武汉大学 A kind of tumor-targeting near infrared fluorescent probe and preparation method thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AROCKIAM JESIN BENETO, ET AL: "A tunable ratiometric pH sensor based on phenanthro[9,10-d]imidazole covalently linked with vinylpyridine", 《RSC ADV.》 *
FEI LIU, ET AL: "Ratiometric Detection of Viscosity Using a Two-Photon Fluorescent Sensor", 《CHEM. EUR. J.》 *
TINGTING LI, ET AL: "A colorimetric and fluorescent probe for fluoride anions based on a phenanthroimidazole-cyanine platform", 《ANAL. METHODS》 *
YI ZHANG, ET AL: "Colorimetric and near infrared fluorescent detection of cyanide by a new phenanthroimidazole-indolium conjugated probe", 《RSC ADV.》 *
李杨洁等: "线粒体荧光探针研究新进展", 《有机化学》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106928189A (en) * 2017-03-03 2017-07-07 济南大学 A kind of mitochondrial fluorescence probe of identification with larger Stokes displacements
CN106928189B (en) * 2017-03-03 2019-04-09 济南大学 A kind of fluorescence probe of the identification mitochondria with larger Stokes displacement
CN108610411A (en) * 2018-04-28 2018-10-02 武汉大学 A kind of tumor-targeting near infrared fluorescent probe and preparation method thereof

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Application publication date: 20170201