CN106117291B - A kind of fluorescence nucleosides and its preparation method and application for cell imaging - Google Patents
A kind of fluorescence nucleosides and its preparation method and application for cell imaging Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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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
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1055—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with other heteroatoms
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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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Abstract
The fluorescence nucleosides and its preparation method and application that the invention discloses a kind of for cell imaging.Such nucleosides fluorescent dye has lower background fluorescence in the few cell compartment of rna content and tissue, there is stronger fluorescence signal in the high cell compartment of rna content and tissue, and when the viscosity of cell changes, fluorescence intensity and service life also change therewith.Since granulophilocyte only contains RNA, so such fluorescent molecule can filter out granulophilocyte from haemocyte, and multi-modal imaging is carried out to it.This kind of compound has certain solubility in water, while having good water oil amphipathic, can pass through cell membrane；And also there is biggish effective two photon absorption cross section.In addition, this kind of compound of the invention also has lower bio-toxicity, photobleaching and high photostability, and the spectral region of itself of its spectral region and biological sample has sufficiently large difference.
Description
Technical field
The present invention relates to the synthetic methods of a kind of fluorescence nucleosides, and in particular to a kind of fluorescence nucleosides for cell imaging and
Preparation method and application.
Background technique
In recent years, due to various reasons, the disease incidence of cardiovascular and cerebrovascular disease is higher and higher, and gradually rejuvenation.Heart and brain
Vascular diseases have the features such as " disease incidence is high, disability rate is high, the death rate is high, high recurrence rate, and complication is more ".Currently, the whole world
The number for dying of cardiovascular and cerebrovascular disease every year is up to 15,000,000 people, and it is the first to occupy the various causes of the death.Blood viscosity increases, and causes human body newly old
Accretion rate slows down, and velocity of blood flow can slow down, and causes cardiac and cerebral blood supply insufficiency and then causes the hearts such as coronary heart disease, hypertension, cerebral thrombosis
Cranial vascular disease.So establish it is a kind of simplicity, efficiently, efficiently, sensitively blood viscosity detection technique be prevention and inspection
One important work of thought-read cranial vascular disease.Viscosity test method for the sticky fluid of the large volume of macroscopic view is to utilize
Viscosmeter.But for microenvironment, (viscosity in especially intracellular or biological tissue, is that can not achieve it using viscosity apparatus
The measurement of viscosity.At present, it has been reported that viscosity rotor mainly have: it is fluorescence intensity viscosity rotor, scale fluorescence viscosity rotor, glimmering
Light service life viscosity rotor, Fluorescence Ratio and service life double mode viscosity rotor.However, these rotors all cannot granulophilocyte very
Viscosity is imaged and detected in red blood cell.
With the development of two-photon technology, two-photon laser confocal microscopic image in the research of life science at
For most important imaging technique.Compared with traditional single-photon laser confocal microscopic image, Laser scanning confocal microscopy tool
Have a clear superiority, including near-infrared excitation, dark-field imaging, avoid fluorescent bleach and it is photic poison, targeting excitation, high lateral resolution
With longitudinal resolution, reduce biological tissue's absorptivity and reduce tissue autofluorescence interference etc. (Helmchen F, Svoboda
K,Denk W et al.Nature 1999,2,989-996.Zhang,H.；Fan,J.L.；Wang,J.Y.；Zhang,S.Z.；
Dou,B.R.；Peng,X.J.J.Am.Chem.Soc.2013,135,11663-11669.Zhang,H.；Fan,J.L.；Wang,
K.；Li,J.；Wang,C.X.；Nie,Y.M.；Jiang,T.；Mu,H.Y.；Peng,X.J.；Jiang,
K.Anal.Chem.2014,86,9131-9138), therefore two-photon micro-imaging technique makes bio-imaging research enter one
Brand-new stage.And also to the fluorescence nucleosides of the specificity multi-modal imaging of the detection of granulophilocyte imaging and its viscosity variation
Not yet someone reports, the good screening granulophilocyte of exploitation specificity and the fluorescence nucleosides for detecting its viscosity change are one and start
The work of property.
Summary of the invention
In order to solve the deficiencies in the prior art, the present invention provides a kind of for the fluorescence nucleosides of cell imaging and its preparation
Methods and applications.
The technical scheme is that a kind of fluorescence nucleosides for cell imaging, the fluorescence nucleosides is tied with general formula I
Structure:
Wherein:
B is selected from dA, dG, dC, dT or dU, wherein R7It is independently selected from alkyl chain ,-H ,-TBDMS ,-Boc ,-Ac or-DMTr；
R1Selected from-Ph ,-Ph (CH3)n、-Ph(CH2CH3)n、-Ph((OCH2CH2)nOCH3)m、-Ph((OCH2CH2CH2)nOCH3)m,-Ph((OCH2CH2CH2)nOCH3)2Or-PhN ((OCH2CH2CH2)nOCH3)2, wherein n is selected from the integer of 1-8；M selection
The integer of 1-3；
R2、R3、R4、R5It is each independently selected from-CH3Or-(CH2)nCH3, wherein n is selected from the integer of 1-8；
R6Independently selected from-N (CH3)2Or-N (CH2)nCH3, wherein n is selected from the integer of 1-8；
Wherein n is selected from the integer of 1-3；
L is selected from alkyl chain ,-Ph- ,-CH2(CH=CH)n-(C≡C)m-CH2-、-(CH2)nCH=CH (CH2)nOr-
(CH2)nC≡C(CH2)n, wherein n, m are independently selected from the integer of 0-4.
Include: as a further improvement of the present invention
The fluorescence nucleosides is selected from following compounds:
Another object of the present invention is to provide a kind of methods for preparing above-mentioned fluorescence nucleosides, comprising the following steps:
) compound a reacts with trimethyl silicane ethyl-acetylene according to molar ratio 1:1-1:6, prepare compound b:
Reaction temperature is 0-50 DEG C, and Pd, Cu and amine are catalyzed simultaneously, and the reaction time is 6-24 hours, and reaction dissolvent is selected from two
At least one of chloromethanes, tetrahydrofuran, ether or dioxanes；
2) compound b is reacted with tetrabutyl ammonium fluoride according to molar ratio 1:0.8-1:4, prepare compound c:
Reaction temperature is 0-50 DEG C, reaction time 10min-5h, and reaction dissolvent is selected from methylene chloride, tetrahydrofuran, second
At least one of ether or dioxanes；
3) by step 1) or 2) in the compound b or c that are prepared, it is anti-according to molar ratio 1:1-3 with compound dA respectively
It answers, prepare compound dABp-1-dABp-4:
Reaction temperature is 0-50 DEG C, and Pd, Cu and amine are catalyzed simultaneously, reaction time 8-24h, and reaction dissolvent is selected from dichloromethane
At least one of alkane, tetrahydrofuran, ether or dioxanes.
Application of the above-mentioned fluorescence nucleosides in bio-imaging.
Application of the fluorescence nucleosides in bio-imaging, biological sample be include that haemocyte or granulophilocyte exist
Interior living cells.
The present invention improves existing viscosity fluorescence nucleosides in the deficiency of application, designs and synthesizes out two-photon excitation, is applicable in
In granulophilocyte (in haemocyte) of effective, the single-minded identification containing RNA and detection cell viscosity and it is imaged
Fluorescence nucleosides.Such nucleosides fluorescent dye has lower background fluorescence in the few cell compartment of rna content and tissue,
There is stronger fluorescence signal in the high cell compartment of rna content and tissue, and be that fluorescence is strong when the viscosity of cell changes
Degree and service life also change therewith.
Since granulophilocyte only contains RNA, so such fluorescent molecule can filter out granulophilocyte from haemocyte,
And multi-modal imaging is carried out to it.This kind of compound has certain solubility in water, while having good water oil amphipathic,
Cell membrane can be passed through；And also there is biggish effective two photon absorption cross section.In addition, this kind of compound of the invention also has
There are lower bio-toxicity, photobleaching and high photostability, and the spectrum model of itself of its spectral region and biological sample
It is with sufficiently large difference.
Fluorescence nucleosides of the present invention has following advantages: the compound introduces specific recognition and positioning base
Group, improves the specificity to cell and tissue identification, specificity；The compound has good two-photon performance, application
With low biological photobleaching, light injury and bio-toxicity when biological sample imaging；The fluorescence of the compound moieties
Launch wavelength is longer, can be used for living cells imaging；The compound can be used as ratio and service life fluorescence nucleosides net is knitted it is red thin
Born of the same parents are imaged, and exclude interference of the environmental factor to fluorescence intensity；The compound side effect is small, and raw material is easy to get, and structure is simple,
Easily preparation, easy industrialization.
Detailed description of the invention
Fig. 1 is that embodiment 2 characterizes the response of fluorescence nucleoside compound dABp-1 of the invention to viscosity.Measuring method are as follows:
Compound dABp-1 is added separately in the mixed solution of the different proportion of water and glycerol, measures fluorescence emission spectrum.
Fig. 2 is that embodiment 3 characterizes the fluorescence lifetime of fluorescence nucleoside compound dABp-1 of the invention under different viscositys
Variation.Measuring method are as follows: compound dABp-1 is added separately in the mixed solution of the different proportion of water and glycerol, is measured glimmering
The light service life.
Fig. 3 is that embodiment 4 characterizes the fluorescence nucleoside compound dABp-1 of the invention different imaging moulds in granulophilocyte
Imaging picture under formula.Measuring method are as follows: the granulophilocyte for being added to the DMSO solution for the dABp-1 that 10 μ L concentration are 1mM
In, at 37 DEG C, 5%CO2It is lower to be incubated for 50 minutes, a certain region is chosen, is observed with oil mirror (60 ×), in triplicate.Picture is collected
Wavelength 490-520nm, 550-580nm.
Fig. 4 is the water-soluble that embodiment 6 characterizes fluorescence nucleoside compound dABp-2 of the invention.Test method: with not
With the aqueous solution of the dABp-2 of concentration, its fluorescent emission intensity under absorption maximum is tested.
Fig. 5 is that embodiment 7 characterizes the response of fluorescence nucleoside compound dABp-2 of the invention to cell viscosity.Test side
Method: by 2 cell of HepG that is added to of DMSO solution for the dABp-1 that 10 μ L concentration are 1mM, at 37 DEG C, 5%CO2Lower incubation
30 minutes, a certain region is chosen, respectively at 25 DEG C and 37 DEG C, with oil mirror (60 ×) observation, is taken pictures, in triplicate.Picture is received
Collect wavelength 490-520nm, 550-580nm.
Fig. 6 is the parameter in bio kinetic model that embodiment 8 characterizes fluorescence nucleoside compound dABp-3 of the invention.Test method:
The DMSO solution of the dABp-3 of various concentration gradient is added separately to A549 cell, BEL-7402 cell, HepG2 cell, RH-
In 373 cell of 35 cells, BRL3A cell and NIH, at 37 DEG C, 5%CO2It is lower to be incubated for 24 hours.Three are washed with certain volume PBS
Time, MTT is then added, is tested with microplate reader.
Fig. 7 is the measurement knot for the two photon absorption cross section that embodiment 9 characterizes fluorescence nucleoside compound dABp-3 of the invention
Fruit.Measure solvent are as follows: dimethyl sulfoxide.Measuring method are as follows: use femtosecond two-photoninduced fluorescence method, utilize fluorescein
NaOH solution (pH=11) is used as reference, A used1Solution concentration is all 1 × 10-4M, laser pulse width 70fs, repetition rate
80MHz, the average output power 1.5W (780nm) of laser, wavelengthtunable 700~980nm of range, in an experiment femtosecond laser
Wavelength is adjusted to required test wavelength.
Fig. 8 a is the solvation effect that embodiment 10 characterizes fluorescence nucleoside compound dABp-3 of the invention.Test method:
DABp-3 is added separately to n-hexane, methylene chloride, acetone, DMF, DMSO, ethyl alcohol, in Tris-HCl, measures UV absorption
Spectrum.
Fig. 8 b is the solvation effect that embodiment 10 characterizes fluorescence nucleoside compound dABp-3 of the invention.Test method:
DABp-3 is added separately to n-hexane, methylene chloride, acetone, DMF, DMSO, ethyl alcohol, in Tris-HCl, measures fluorescent emission
Spectrum.
Fig. 9 is that embodiment 11 characterizes the launch wavelength extraction of fluorescence nucleoside compound dABp-3 of the invention in living cells
Picture.By in 2 cell of HepG that is added to of DMSO solution for the dABp-3 that 10 μ L concentration are 1mM, at 37 DEG C, 5%CO2Under incubate
It educates 30 minutes, chooses a certain region, do wavelength surface sweeping, step-length: 2nm, bandwidth: 2nm；Extract wave-length coverage 490-610nm.
Figure 10 is the photostability that embodiment 13 characterizes fluorescence nucleoside compound dABp-4 of the invention.Test method: it uses
500W iodine-tungsten lamp irradiating sample is surveyed once every half an hour, surveys 7 hours.
Figure 11 is the change that embodiment 14 characterizes fluorescence nucleoside compound dABp-4 emission spectrum at different pH of the invention
Change.Test method: adjusting pH under conditions of compound dABp-4 concentration is certain, tests compound of the pH range in 4-10
The variation of the fluorescence emission spectrum of dABp-4.
Figure 12 is that embodiment 15 characterizes the lifetime change of fluorescence nucleoside compound dABp-4 of the invention under different viscositys.
Measuring method are as follows: compound dABp-4 is added separately in the mixed solution of the different proportion of water and glycerol, measures the fluorescence longevity
Life.
Specific embodiment
It elaborates below with reference to embodiment to the present invention.
Unless otherwise stated, term used herein has following meanings.
Term " alkyl " used herein includes straight chained alkyl and branched alkyl.For example, " C1-6Alkyl " includes C1-4Alkane
Base, C1-3Alkyl, methyl, ethyl, n-propyl, isopropyl and tert-butyl.Similar rule is also applied for used in this specification
Other groups.
In general formula compound of the invention, the R that is addressed1Selected from-Ph ,-Ph (CH3)n、-Ph(CH2CH3)n、-Ph
((OCH2CH2)nOCH3)m、-Ph((OCH2CH2CH2)nOCH3)m、-Ph((OCH2CH2CH2)nOCH3)2Or-PhN
((OCH2CH2CH2)nOCH3)2, wherein n is selected from the integer of 1-8, and m is selected from the integer of 1-3；R in preferred embodiment1Selected from-Ph or-Ph
(OCH2CH2)nOCH3, integer of the n selected from 1-8；More preferable scheme R1Selected from-Ph or-Ph (OCH2CH2)nOCH3, n is selected from 1-6's
Integer；Most preferably scheme R1Selected from-Ph or-Ph (OCH2CH2)4OCH3, integer of the n selected from 2-5；
The R2、R3、R4、R5It is each independently selected from-CH3、-(CH2)nCH3, integer of the n selected from 1-8；In preferred embodiment
R2、R3、R4、R5It is each independently selected from-CH3、-(CH2)nCH3, integer of the n selected from 1-6；More preferable scheme R2、R3、R4、R5Respectively
Independently selected from-CH3、-(CH2)nCH3, integer of the n selected from 1-4；Most preferably scheme R2、R3、R4、R5Be each independently selected from-
CH3、-(CH2)nCH3, integer of the n selected from 1-2；
The R addressed6It is selected from
-N(CH3)2、-N(CH2)nCH3、Wherein n is selected from the whole of 1-3
Number；R in preferred embodiment6Selected from-N (CH3)2、N is selected from the integer of 1-3；More preferable scheme R6Selected from-N
(CH3)2、N is selected from the integer of 1-3；Most preferably scheme R6It is selected fromN is selected from 1-3's
Integer；
The L addressed is selected from alkyl chain ,-Ph- ,-CH2(CH=CH)n-(C≡C)m-CH2-、-(CH2)nCH=CH
(CH2)n-、-(CH2)nC≡C(CH2)n, wherein n, m are independently selected from the integer of 0-4；L is selected from-Ph- ,-CH in preferred embodiment2-
(CH=CH)n-(C≡C)m-CH2-、-(CH2)nC≡C(CH2)n, n, m are independently selected from the integer of 0-4；More preferable scheme L is selected from-
Ph-、-(CH2)nC≡C(CH2)n, integer of the n selected from 0-4；Most preferably scheme L is selected from-Ph- ,-(CH2)nC≡C(CH2)n, n choosing
From the integer of 0-2；
The B addressed is selected from dA, dG, dC, dT and dU, wherein R7It is independently selected from alkyl chain ,-H ,-TBDMS ,-Boc ,-Ac
With-DMTr；B is selected from dA, dG, dC, dT and dU in preferred embodiment, wherein R7Be independently selected from-H ,-TBDMS ,-Boc ,-Ac and-
DMTr；B is selected from dA, dG, dC and dT in more preferable scheme, wherein R7It is independently selected from-H ,-TBDMS ,-Boc and-Ac；It is most preferably square
B is selected from dA, dG, dC and dT in case, wherein R7It is independently selected from-H ,-TBDMS and-Ac.
In addition, including the following steps: the present invention provides above-mentioned fluorescence nucleosides synthetic method
) compound a reacts with trimethyl silicane ethyl-acetylene according to molar ratio 1:1-1:6, prepare compound b:
Reaction temperature is 0-50 DEG C, and Pd, Cu and amine make catalyst, and the reaction time is 6-24 hours, and reaction dissolvent is selected from two
Or mixtures thereof chloromethanes, tetrahydrofuran, ether, dioxanes；
Preferred embodiment: reaction temperature is 0-30 DEG C, PdCl2(PPh3)2, CuI and amine make catalyst, reaction time
It is 8-18 hours, reaction dissolvent is selected from or mixtures thereof tetrahydrofuran, ether, mole of compound a and trimethyl silicane ethyl-acetylene
Compare 1:1-1:4；
Further preferred embodiment: reaction temperature is 10-30 DEG C, PdCl2(PPh3)2, CuI and NEt3Make catalyst,
Reaction time is 8-16 hours, reaction dissolvent tetrahydrofuran, the molar ratio 1:1-1:3 of compound a and trimethyl silicane ethyl-acetylene；
The most preferred embodiment: reaction temperature is 20-30 DEG C, PdCl2(PPh3)2, CuI and NEt3Make catalyst, reacts
Time is 8-16 hours, reaction dissolvent tetrahydrofuran, the molar ratio 1:1-1:2 of compound a and trimethyl silicane ethyl-acetylene；
2) compound b is reacted with tetrabutyl ammonium fluoride according to molar ratio 1:0.8-1:4, prepare compound c:
Reaction temperature is 0-50 DEG C, reaction time 10min-5h, and reaction dissolvent is selected from methylene chloride, tetrahydrofuran, second
Or mixtures thereof ether, dioxanes；
Preferred embodiment: reaction temperature is 0-40 DEG C, reaction time 10min-3h, and reaction dissolvent is selected from dichloromethane
Alkane, tetrahydrofuran, or mixtures thereof, the molar ratio 1:0.8-1:3 of compound b and tetrabutyl ammonium fluoride；
Further preferred embodiment: reaction temperature is 10-40 DEG C, reaction time 10min-2h, reaction dissolvent four
The molar ratio 1:0.8-1:2 of hydrogen furans, compound b and tetrabutyl ammonium fluoride；
The most preferred embodiment: reaction temperature is 20-40 DEG C, reaction time 20min-1.5h, reaction dissolvent tetrahydro
The molar ratio 1:0.8-1:1.8. of furans, compound b and tetrabutyl ammonium fluoride
3) by step 1) or 2) in the compound b or c that are prepared, react, make according to molar ratio 1:1-1:3 with dA respectively
Standby compound dABp-1-dABp-4:
Reaction temperature is 0-50 DEG C, and Pd, Cu and amine are catalyzed simultaneously, and dA, Pd and Cu, N is first added in when reaction2Protection is lower to be added
Amine and another raw material, reaction time 8-24h, reaction dissolvent be selected from methylene chloride, tetrahydrofuran, ether, dioxanes or its
Mixture；
Preferred embodiment: reaction temperature is 10-50 DEG C, PdCl2(PPh3)2, CuI and amine make catalyst, when reaction, is first
DA, Pd and Cu, N is added2The lower addition amine of protection and another raw material, reaction time 10-24h, reaction dissolvent are selected from tetrahydro furan
It mutters, or mixtures thereof ether, the molar ratio 1:1-1:3 of compound b or c and compound dA；
Further preferred embodiment: reaction temperature is 20-50 DEG C, PdCl2(PPh3)2、CuI、NEt3Or TBAF is urged
DA, Pd and Cu, N is first added in agent, when reaction2Protection is lower to be added TBAF and another raw material, reaction time 10-18h, reaction
Solvent is tetrahydrofuran, the molar ratio 1:1-1:2 of compound b or c and compound dA；
The most preferred embodiment: reaction temperature is 20-40 DEG C, PdCl2(PPh3)2, CuI and TBAF make catalyst, react
When dA, PdCl is first added2(PPh3)2And CuI, in N2Protection is lower to be added TBAF and other raw material b or c, reaction time 8-
20h, reaction dissolvent are methylene chloride, the molar ratio 1:1.2-1:2 of compound b or c and compound dA.
It is above-mentioned to the synthesis of the invention in the ucleosides fluorescence nucleosides that two pyrroles of fluorine boron is parent, base is recognition group
In the description of method, each substituent group (R1、R2、R3、R4、R5、R6And R7) definition and preferably, in the present invention to chemical combination
Definition in the description of object and preferably identical.
To the present invention using the ucleosides fluorescence nucleoside compound of above method synthesis, composed using nuclear magnetic resonance spectroscopy, carbon
Or mass spectrum confirms its structure.
Fluorescence nucleosides of the present invention has following advantages:
The compound introduces specific recognition and seeking group, improve the specificity to cell and tissue identification,
Specificity；
The compound has good two-photon performance, floats when applied to biological sample imaging with low bio-light
White, light injury and bio-toxicity；
The fluorescence emission wavelengths of the compound moieties are longer, can be used for living cells imaging；
The compound can be used as ratio and granulophilocyte is imaged in service life fluorescence nucleosides, exclude environmental factor
Interference to fluorescence intensity；
The compound side effect is small, and raw material is easy to get, and structure is simple, easily prepares, easy industrialization；
In consideration of it, ucleosides fluorescence nucleoside compound of the present invention can be used for viscosity detection and imaging in living cells,
In addition, ucleosides fluorescence nucleosides of the present invention can also screen granulophilocyte from haemocyte and it is imaged.This hair
It include ucleosides fluorescence nucleoside compound provided by the present invention in the bright composition.
The present invention also provides use the ucleosides fluorescence nucleoside compound of aforementioned present invention to can be used for viscosity in living cells to examine
The method for surveying and being imaged and screening granulophilocyte from haemocyte and it is imaged, this method include making the compound and life
The step of object sample contacts.Term " contact " used herein may include contacting in solution or solid phase.
Following non-limiting embodiments can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.
Embodiment 1
The synthesis of rotor compound dABp-1
(1) synthesis of intermediate 1 and 3
The synthesis of intermediate 1 is referring to document V á zquez-Romero, A.；Kielland,N.；Arévalo,M.J.；
Preciado,S.；Mellanby,R.J.；Feng,Y.；Lavilla,R.；Vendrell,M.J.Am.Chem.Soc.2013,
135,16018-16021 (synthesis of intermediate 1)；Moreau,C.；Wagner,G.K.；Weber,K.；Guse,A.H.；
Potter, B.V.L.J.Med.Chem.2006,49,5162-5176 (synthesis of intermediate 3).
(2) synthesis of intermediate 2
The TBAF of 0.8mmol is added in the THF solution of intermediate 1 of 0.4mmol, is stirred at room temperature 30 minutes,
Stop reaction, decompression steams solvent, adds suitable methylene chloride dissolved solid, and suitable water is added to extract 3 times, merges organic
Phase, is evaporated under reduced pressure away solvent, and column separation obtains red solid, yield 77%.1H NMR(400MHz,CDCl3):δ
(ppm) 8.02 (s, 1H), 7.98 (s, 1H), 7.64-7.50 (m, 5H), 7.02 (d, J=4.3Hz, 1H), 6.99 (s, 1H),
6.60 (d, J=4.2Hz, 1H), 3.09 (s, 1H)；13C NMR(100MHz,CDCl3):δ(ppm)147.8,146.3,145.3,
133.3,133.1,132.7,131.2,130.5,128.6,119.7,79.8,76.4.
(3) synthesis of rotor compound dABp-1
By the intermediate 2 of 0.1mmol, the PdCl of 0.005mmol2(PPh3)2Schlenk is added to the CuI of 0.01mmol
In reaction tube, 2mL THF and 1mL NEt are then added under nitrogen protection3；Finally the intermediate 3 of 0.11mmol is separately taken to dissolve
Into 2mL THF, the THF dissolved with intermediate 3 is added in reaction tube with syringe.It stirs 19 hours, stops at room temperature
Reaction.Reaction solution is crossed diatomite, removes catalyst, decompression steams solvent.Column chromatography for separation, obtains red solid, and yield is
21%.1H NMR(400MHz,CDCl3):δ(ppm)8.28(s,1H),8.09(s,2H),7.68-7.52(m,5H),7.09(d,J
=6.8Hz, 2H), 6.66 (d, J=4.3Hz, 1H), 6.51 (t, J=6.9Hz, 1H), 5.83 (s, 2H), 4.80 (dt, J=
5.8Hz, 3.1Hz, 1H), 4.01-3.90 (m, 2H), 3.70 (dd, J=10.1Hz, 4.7Hz, 1H), 3.54-3.45 (m, 1H),
2.22 (m, 1H), 0.89 (s, 9H), 0.85 (s, 9H), 0.11 (d, J=3.1Hz, 6H), 0.01 (s, 3H), -0.01 (s, 3H)；13C NMR(100MHz,CDCl3):δ(ppm)155.0,153.2,149.5,147.6,135.4,133.2,131.4,130.5,
128.8,87.9,85.4,72.8,63.1,37.1,25.9,25.8,18.4,18.0,-4.9,-4.7,-5.4,-5.4；HRMS:
m/z calcd for C39H51BF2N7O3Si2[M+H]770.3655,found:770.3644.
Embodiment 2
Response of the fluorescence nucleoside compound dABp-1 to viscosity
Take concentration be 30 μ L 1mM dABp-1 DMSO solution, be separately added into 10mL to different proportion water and glycerol group
At mixed solution in, ultrasound 20 minutes then stands 24 hours at 0 DEG C, with its fluorescent emission of fluorescent spectrophotometer assay
Spectrum.Test result shows that, with the increase of the viscosity of solution, the enhancing of dABp-1 fluorescence intensity is detailed in Fig. 1.
Embodiment 3
The variation of fluorescence lifetime of the fluorescence nucleoside compound dABp-1 under different viscositys
Take concentration be 30 μ L 1mM dABp-1 DMSO solution, be separately added into 10mL to different proportion water and glycerol group
At mixed solution in, ultrasound 20 minutes then stands 24 hours at 0 DEG C, with transient state/steady state time resolved fluorometric luminosity measurement
Determine fluorescence lifetime.Test result is shown, with the increase of the viscosity of solution, dABp-1 fluorescence lifetime increases, and is detailed in figure Fig. 2.
Embodiment 4
Fluorescence nucleoside compound dABp-1 in the normal and granulophilocyte of thrombus model under difference imaging patterns at
Picture
In the granulophilocyte for the normal and thrombus model that the DMSO solution that 10 μ L concentration are 1mM is added to, 37
DEG C, 5%CO2Lower cell incubation 50 minutes by addition rotor dABp-1.Then, PBS concussion rinsing 5min × 3, add life
Salt water is managed, is imaged under laser confocal microscope.Representative area is chosen, is observed with oil mirror (100 ×), in triplicate.Picture
Collect wavelength 490-520nm, 550-580nm.Test result shows that dABp-1 can be imaged in granulophilocyte, and when net is knitted
When the viscosity variation of red blood cell, the ratio of fluorescence intensity also changes.It is detailed in Fig. 3.
Embodiment 5
The synthetic method of fluorescence nucleoside compound dABp-2
The synthesis of intermediate 4 is referring to document V á zquez-Romero, A.；Kielland,N.；Arévalo,M.J.；
Preciado,S.；Mellanby,R.J.；Feng,Y.；Lavilla,R.；Vendrell,M.J.Am.Chem.Soc.2013,
135,16018-16021。
(2) synthesis of compound dABp-2: synthetic method is similar with the synthesis of compound dABp-1, only intermediate 2
Change intermediate 4 into.Obtained yield is 17%.1H NMR(400MHz,CDCl3):δ(ppm)8.30(s,1H),7.55-7.49
(m, 3H), 7.28 (m, 2H), 6.55-6.50 (m, 1H), 6.09 (s, 1H), 5.72 (s, 2H), 4.74 (d, J=2.5Hz, 1H),
3.97-3.89(m,2H),3.69-3.63(m,2H),2.72(s,3H),2.60(s,3H),2.15(m,1H),1.53(s,3H),
1.42(s,3H),0.88(s,9H),0.85(s,9H),0.09(s,6H),0.01(s,3H),-0.01(s,3H)；13C NMR
(100MHz,CDCl3):δ(ppm)155.0,153.1,149.6,135.9,133.4,131.4,130.5,128.5,120.4,
87.9,85.6,72.9,63.1,37.4,25.9,25.8,18.4,18.0,-4.7,-5.4,-5.5；HRMS:m/z calcd
for C41H55BF2N7O3Si2[M+H]798.3968,found:798.3960.
Embodiment 6
The water-soluble of fluorescence nucleoside compound dABp-2
The DMSO solution of dABp-2 that 3 μ L concentration are 1mM is added in 3mL water, with fluorescent spectrophotometer assay its
Next intensity of emission spectra adds the DMSO solution of 3 μ L dABp-2 every time, and is mapped with fluorescence intensity to concentration, work as appearance
Concentration when inflection point is the concentration of ordinary dissolution of dABp-2 in water.Test result show when compound dABp-2 concentration be 5 μM when,
Fluorescence intensity does not shift, i.e., the solubility of compound dABp-2 in water is 5 μM.It is detailed in Fig. 4.
Embodiment 7
Response of the fluorescence nucleoside compound dABp-2 to cell viscosity
In 2 cell of HepG for taking 10 μ L concentration to be added to for the DMSO solution of the dABp-2 of 1mM, it will be added dABp-2's
2 cell of HepG is at 37 DEG C, 5%CO2It is lower to be incubated for 30 minutes, the good region of cellular morphology is chosen, respectively at 25 DEG C and 37 DEG C,
Co-focusing imaging is carried out, picture collects wavelength 490-520nm, 550-580nm.Test result is shown, when the viscosity of cell changes
When, the ratio of dABp-2 fluorescence intensity also changes, and is detailed in Fig. 5.
Embodiment 8
The parameter in bio kinetic model of fluorescence nucleoside compound dABp-3
It is 10 cell density5A549 cell, BEL-7402 cell, HepG2 cell, RH-35 cell, BRL3A cell
It with 373 cell of NIH, is inoculated on 96 orifice plates, adds appropriate culture medium, at 37 DEG C, 5%CO2It is incubated for 24 hours in incubator, point
Not Jia Ru various concentration gradient dABp-3 DMSO solution, at 37 DEG C, 5%CO2Incubator in be incubated for again 24 hours.With
PBS is washed three times, and MTT is then added, is tested with microplate reader, is handled data by the method for the prior art, is obtained the knot of cytotoxicity
Fruit, test result show that compound dABp-3 has lower bio-toxicity, are detailed in Fig. 6.
Embodiment 9
The measurement of two photon absorption cross section under different viscositys of fluorescence nucleoside compound dABp-3
Changed using femtosecond two-photoninduced fluorescence method using the NaOH solution (pH 11) of fluorescein as reference
Close the measurement of the two-photon cross-sections of object dABp-3.DABp-3 is added separately in the water of different proportion and the mixed liquor of glycerol,
Make concentration 10-4M, ultrasound 20 minutes, then 24 hours are stood at 0 DEG C, then carry out the test of two photon absorption cross section.With calculating
Shown in formula 2.1:
C in formula is the concentration of solution, and n is the refractive index of solvent, can table look-up to obtain.F is up-conversion fluorescence intensity, by
Experiment measures.δ is two photon absorption cross section.The physical quantity of reference solution is indicated with subscript r.Two-photon absorption can be obtained to cut
Face amount.The excitaton source of the two-photon fluorescence excitation spectrum used is a stand lock mould femto second titanium sapphire laser, laser pulse width
70fs, repetition rate 80MHz, the average output power 1.5W (780nm) of laser, wavelengthtunable 700~980nm of range,
Femtosecond laser wavelength is adjusted to required test wavelength in experiment.Test result shows, the two-photon cross-sections of dABp-3 increase with viscosity
Increase greatly, is detailed in Fig. 7.
Embodiment 10
The solvation effect of fluorescence nucleoside compound dABp-3
DABp-3 is added separately to n-hexane, methylene chloride, acetone, DMF, DMSO, ethyl alcohol, is surveyed in Tris-HCl respectively
Try its absorption spectrum and emission spectrum.Test result is shown, but when solvent polarity increase, fluorescence intensity weakens, and blue shift occurs,
It is detailed in Fig. 8 a and Fig. 8 b.
Embodiment 11
Launch wavelength of the fluorescence nucleoside compound dABp-3 in living cells is extracted
In 2 cell of HepG for taking 10 μ L concentration to be added to for the DMSO solution of the dABp-3 of 1mM, dABp-3 will be added
2 cell of HepG at 37 DEG C, 5%CO2It is incubated for 30 minutes in incubator, chooses the preferable region of cellular morphology, do wavelength and sweep
Face, step-length: 2nm, bandwidth: 2nm；Extract wave-length coverage 490-610nm.The results show that dABp-3 also has obviously in living cells
The double transmittings in ground, are detailed in Fig. 9.
Embodiment 12
The synthesis of fluorescence nucleoside compound dABp-4
(1) synthesis of intermediate 5: the synthetic method of intermediate 5 is referring to document V á zquez-Romero, A.；Kielland,
N.；Arévalo,M.J.；Preciado,S.；Mellanby,R.J.；Feng,Y.；Lavilla,R.；Vendrell,
M.J.Am.Chem.Soc.2013,135,16018-16021。
(2) synthesis of intermediate 6: the synthetic method of intermediate 6 is similar with the synthesis of compound dABp-1, only centre
Body 1 changes intermediate 5 into, and obtained yield is 29%.
(3) synthesis of compound dABp-4: 0.21mmol intermediate 6 being dissolved in THF, TBAF is added into solution,
The reaction of stopping in 30 minutes is stirred at room temperature, decompression steams solvent, adds suitable methylene chloride dissolved solid, and add suitable water extraction
3 times, merges organic phase, be evaporated under reduced pressure away solvent, column separation obtains red solid, yield 62%.1H NMR
(400MHz,CDCl3): δ (ppm) 8.29 (s, 1H), 7.15 (d, J=8.7Hz, 2H), 7.05 (d, J=8.7Hz, 2H), 6.77
(d, J=11.7Hz, 1H), 6.68 (dd, J=9.8,5.4Hz, 1H), 6.09 (s, 1H), 5.83 (s, 2H), 4.76 (d, J=
4.7Hz, 1H), 4.20 (d, J=5.4Hz, 3H), 4.00-3.89 (m, 3H), 3.76 (dd, J=5.9,3.1Hz, 3H), 3.73-
3.63 (m, 8H), 3.55 (dd, J=5.7,3.5Hz, 2H), 3.37 (s, 3H), 3.06-2.97 (m, 1H), 2.71 (s, 3H),
2.59 (s, 3H), 2.46 (s, 1H), 2.26 (dd, J=13.2,5.5Hz, 1H), 1.56 (s, 3H), 1.47 (s, 3H)；13C NMR
(100MHz,CDCl3):δ(ppm)159.7,156.0,155.3,152.8,148.6,146.5,143.0,142.7,135.2,
133.8,130.2,129.0,126.4,123.1,120.6,115.5,90.8,89.7,87.5,83.7,73.6,71.9,70.9,
70.6,70.5,69.7,67.6,63.5,59.1,40.6,29.7,15.0,13.7,13.5.
Embodiment 13
The photostability of fluorescence nucleoside compound dABp-4
The DMSO solution for taking 9 μ L dABp-4, is added in 3mL water.Stability testing device: 500W iodine-tungsten lamp is assembled,
Glass jar (filter action) apart from 25-30 centimetres of iodine-tungsten lamp of splendid attire saturation sodium nitrite.Sample is put into inner glass jar 10-
15 centimeters, with iodine-tungsten lamp irradiating sample.Its primary transmitting is surveyed every half an hour, surveys 7 hours.Test result shows, dABp-
The 4 molecules influence very little to light to its stability in water, is detailed in Figure 10.
Embodiment 14
The variation of fluorescence nucleoside compound dABp-4 emission spectrum at different pH
Preparing 21mL concentration is 3 × 10-6The compound dABp-4 aqueous solution of M keeps continuously adjusting under conditions of concentration is constant
Its pH value is saved, the variation of the fluorescence emission spectrum of compound dABp-4 of the pH range in 4-10 is tested.Test result shows, pH
Change within the scope of 4-10 and the emissive porwer of dABp-4 is had little effect, is detailed in Figure 11.
Embodiment 15
Lifetime change of the fluorescence nucleoside compound dABp-4 under different viscositys
Take concentration be 30 μ L 1mM dABp-4 DMSO solution, be separately added into 10mL to different proportion water and glycerol group
At mixed solution in, ultrasound 20 minutes then stands 24 hours at 0 DEG C, with transient state/steady state time resolved fluorometric luminosity measurement
Determine fluorescence lifetime.Test result is shown, with the increase of the viscosity of solution, dABp-4 fluorescence lifetime increases, and is detailed in Figure 12.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (4)
1. a kind of fluorescence nucleosides for cell imaging, which is characterized in that its structural formula is as follows:
2. a kind of method for preparing fluorescence nucleosides as described in claim 1, which comprises the following steps:
1) compound a is reacted with trimethyl silicane ethyl-acetylene according to molar ratio 1:1-6, prepare compound b:
Reaction temperature is 0-50 DEG C, and Pd, Cu and amine are catalyzed simultaneously, and the reaction time is 6-24 hours, and reaction dissolvent is selected from dichloromethane
At least one of alkane, tetrahydrofuran, ether or dioxanes；
2) compound b is reacted with tetrabutyl ammonium fluoride according to molar ratio 1:0.8-4, prepare compound c:
Reaction temperature be 0-50 DEG C, reaction time 10min-5h, reaction dissolvent be selected from methylene chloride, tetrahydrofuran, ether or
At least one of dioxanes；
3) by step 1) or 2) in the compound b or c that are prepared, react, make according to molar ratio 1:1-3 with compound dA respectively
Standby compound dABp-1-dABp-4:
The structural formula of the compound dA is as follows:
Reaction temperature be 0-50 DEG C, Pd, Cu and amine are catalyzed simultaneously, reaction time 8-24h, reaction dissolvent selected from methylene chloride,
At least one of tetrahydrofuran, ether or dioxanes.
3. fluorescence nucleosides as described in claim 1 is preparing the application in bio-imaging drug, which is characterized in that the fluorescence
Nucleosides are as follows:
4. fluorescence nucleosides according to claim 3 is preparing the application in bio-imaging drug, which is characterized in that biological sample
Product are the living cells including haemocyte or granulophilocyte.
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| CN105237570A (en) * | 2014-05-28 | 2016-01-13 | 中国科学院化学研究所 | Biological reagent used for DNA fluorescence in-situ hybridization (FISH), and preparation and applications thereof |
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