CN108727223A - One kind can two-photon fluorescence detection nitroreductase NTR probes and preparation method thereof - Google Patents
One kind can two-photon fluorescence detection nitroreductase NTR probes and preparation method thereof Download PDFInfo
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- CN108727223A CN108727223A CN201810817809.1A CN201810817809A CN108727223A CN 108727223 A CN108727223 A CN 108727223A CN 201810817809 A CN201810817809 A CN 201810817809A CN 108727223 A CN108727223 A CN 108727223A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C255/00—Carboxylic acid nitriles
- C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
- C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
- C07C255/42—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms
- C07C255/43—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms the carbon skeleton being further substituted by singly-bound oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C213/02—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton by reactions involving the formation of amino groups from compounds containing hydroxy groups or etherified or esterified hydroxy groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C221/00—Preparation of compounds containing amino groups and doubly-bound oxygen atoms bound to the same carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C253/00—Preparation of carboxylic acid nitriles
- C07C253/30—Preparation of carboxylic acid nitriles by reactions not involving the formation of cyano groups
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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
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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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"
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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
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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Abstract
The present invention relates to one kind can two-photon fluorescence detect nitroreductase (Nitroreductase, NTR) probe preparation, such as scheme (I), belong to organic fluorescence probe field.One kind can two-photon fluorescence detection nitroreductase (NTR) probe structural formula it is as shown.Fluorescence probe of the present invention can accurately detect intracellular nitroreductase (NTR) content and avoid the interference of other intracellular reducing agents.In addition, the probe has the characteristics that preferable chemical stability, bio-compatibility and selectivity.Laser confocal imaging experiment shows that the probe has preferable cell permeability, it has no toxic side effect to cell and organism, the detection of cellular level nitroreductase (NTR) content and indicator cells hypoxemia situation may be implemented, can further apply the research of pre-cancer detection.
Description
Technical field
The present invention relates to one kind can two-photon fluorescence detect nitroreductase NTR probes and preparation method thereof, belong to organic
Fluorescence probe field.
Background technology
Hypoxemia marker one of of the nitroreductase (NTR) as tumour cell is a kind of dependent on flavin mononucleotide
Or the cytoplasm enzyme of flavin adenine dinucleotide (FAD), it is widely present in bacterium.Flavin mononucleotide list is generally comprised in structure
Member or flavin adenine dinucleotide (FAD) unit, it can utilize fast cry of certain animals dinucleotides (NADPH) niacinamide of niacinamide two cores of fast cry of certain animals
Nucleotide (NADPH) is used as coenzyme, realizes the reductive metabolism to a variety of nitro compounds, generates azanol or amino.Tumour is thin
Born of the same parents' anoxic usually may also lead to the increase of intracellular nitroreductase.Therefore, in the presence of electron donor such as NADH/NADPH, nitro
Aromatic nitro compound can be effectively reduced to corresponding amino-compound by reductase.This reduction act can be used to design
Fluorescence probe containing nitro is detected the anoxic conditions of solid tumor cell, directly indicates the presence of tumour cell, has ten
Divide important meaning.
Hypoxemia is one of extremely important feature of tumour cell, and nitroreductase is used as currently as tumour cell
One of mostly important hypoxemia marker, promote it always as the most important thing in scientific research.In the low of tumour cell
In the research of oxygen marker, mostly with fluorescence probe master, an ideal fluorescence probe must have preferable chemical stability, life
The features such as object compatibility and selectivity.So far it has been reported that having come out the hypoxemia marker fluorescence probe of many tumour cells, such as
The fluorescence probe of the biological micromolecules such as nitroreductase, diaphorase, quinone reductase, azo reductase is detected, but these are glimmering
Light probe all have the shortcomings that one it is larger be exactly that probe is water-soluble universal poor, and Stokes shift is smaller, is being imaged
Exciting light can cause different degrees of interference in journey, and the tissue penetration of most of probe is not good enough, and research is caused to tie
Fruit convincingness is insufficient.In order to solve the problems, such as that this exists for a long time, it is badly in need of that research and development are a kind of to have preferable water-soluble, larger this
The fluorescence probe of the two-photon nitroreductase of lentor displacement.
Invention content
More than solving the problems, such as, the present invention provides one kind can two-photon fluorescence detection nitroreductase NTR probes tool
There is the fluorescence probe and preparation method thereof of preferable water-soluble, larger Stokes shift two-photon nitroreductase.
The technical solution adopted by the present invention be one kind can two-photon fluorescence detection nitroreductase NTR probes, feature exists
In the fluorescence probe is the probe XD03 rolled into a ball based on naphthols fluorogen two-photon fluorescence, the structural formula such as (I) of fluorescence probe
It is shown：
Another technical solution that the present invention uses is：The preparation method of the fluorescence probe XD03 includes the following steps：
Two-photon naphthol derivative XD02, malononitrile are added sequentially in ethanol solution, nitrogen protection, are reacted at 0 DEG C
Under the conditions of stir 3-5h, after rotary evaporation removes organic solvent, dissolved with dichloromethane, deionized water be used in combination to be extracted twice, salt
Water extracts, and collects organic phase drying, and rotary evaporation removes solvent, purifies to obtain intermediate product XD03 by plastic column chromatography, above-mentioned
Two-photon naphthol derivative XD02, malononitrile molar ratio be 1：1.0-6.0 reaction equation is as follows:
Preferably, the molar ratio of the XD02 and malononitrile is 1:1；The preparation method of the fluorescence probe XD03
In, dry is to be dried with anhydrous sodium sulfate；In the preparation method of the fluorescence probe XD03, the XD02 per mmol is added
Ethyl alcohol 20-23mL；In reaction process, the equivalent of malononitrile cannot be too many, otherwise will produce by-product, reduces yield, secondly anti-
It should have to keep nitrogen protection and in low temperature environment environment, otherwise can cause to generate other impurity.
Preferably, it is described can two-photon fluorescence detection nitroreductase probe precursor substance XD02 preparation method such as
Under：
(1)XD00：By 2,7- dihydroxy naphthols, 40% aqueous solution of dimethylamine, sodium pyrosulfite, deionized water sequentially adds
Into Shrek bottle, 140-160 DEG C of reaction 7-9h, after being cooled to room temperature, the pH that reaction system is adjusted using hydrochloric acid is 3-5 left
Then the right side is spin-dried for using DCM extractions, purifies to obtain intermediate product white solid XD00 by plastic column chromatography；Above-mentioned 2,7- bis-
Hydroxyl naphthols, 40% aqueous solution of dimethylamine, sodium pyrosulfite molar ratio be 1:4-6:4-6；2,7 dihydroxy naphthalene per mmol
Deionized water 1-6mL is added in phenol, and reaction equation is as follows：
(2)XD01：N,N-Dimethylformamide is added in Shrek bottle to vacuumize, phosphorus oxychloride is added with syringe
In above-mentioned Shrek bottle, stirring to system is sticky mass under the conditions of being placed in 0 DEG C of cold well.The product X D00 of upper step is dissolved in
In appropriate n,N-Dimethylformamide, it is slowly added in Shrek bottle with disposable syringe, 1-3h is stirred under the conditions of 0 DEG C, it will
It pours into the mixture of ice and water of 10 times of volumes and stirs after reaction system cooling, CH is added3COOK after solution turned yellow until filter, warp
Plastic column chromatography is crossed to purify to obtain intermediate product XD01；Above-mentioned XD00, N,N-dimethylformamide, phosphorus oxychloride molar ratio be
1：1:0.4-1.0, reaction equation are as follows：
(4)XD02:By above-mentioned product X D01, to nitro bromobenzyl, that potassium carbonate is added sequentially to N,N-dimethylformamide is molten
In liquid, nitrogen protection is stirred at room temperature 3-5h, is then extracted with dichloromethane, dry, is spin-dried for, is purified by plastic column chromatography
It obtains intermediate product XD02, above-mentioned XD01, be 1 to the molar ratio of nitro bromobenzyl, potassium carbonate:0.9-1.1：3-5, reaction equation is such as
Under：
Preferably, in a series of preparation method of two-photon naphthols derivative compounds, step (1) the intermediate product XD00
In preparation, the molar ratio of 1,6- dihydroxy naphthols, dimethylamine agueous solution, sodium pyrosulfite is 1:4-6:4-6；2,7- per mmol
Deionized water 1-6mL is added in dihydroxy naphthols；
XD00 in the step (2), N,N-dimethylformamide, phosphorus oxychloride molar ratio be 1：1:0.4-1.0, and want
In advance jelly salt is first made in n,N-Dimethylformamide and phosphorus oxychloride in ice-water bath, XD00 is then dissolved into appropriate N, N- bis-
In methylformamide, it is added to and freezes in salt system in the condition of ice-water bath, nitrogen protection；
XD01 in the step (3), it is 1 to the molar ratio of nitro bromobenzyl, potassium carbonate:0.9-1.1：Nitrogen is wanted in 3-5, reaction
It is carried out under body environmental protection.
Advantageous effect：
The fluorescence probe of the present invention has the characteristics that preferable chemical stability, bio-compatibility and selectivity.Laser is total
Focal imaging experiment shows that the probe has preferable cell permeability, has no toxic side effect to cell and organism and nitro restores
The selectivity of enzyme (NTR).Laser confocal imaging experiment shows that the probe has preferable cell permeability, to cell and biology
Body has no toxic side effect.
The fluorescence probe of the present invention, by hypoxemia culture cancer cell population, makes thin itself generate in it can be applied to cell system
A large amount of nitroreductase, and two-photon cell imaging can be carried out, and avoid intracellular autofluorescence and from extraneous
The application of the interference of other reducing substances.
The accurate fluorescence probe for surveying the nitroreductase that hypoxemia cancer cell itself generates of the present invention can be used for living thin
The accurate detection of born of the same parents' line nitroreductase, living cells mainly is Hepg-2 cell strains.
Description of the drawings
1. Fig. 1-a, 1-b, 1-c are the hydrogen spectrum, carbon spectrum, matter of the presoma XD02 of naphthols two-photon fluorescence group devicative respectively
Compose number data.
2. Fig. 2-a, 2-b, 2-c nitroreductase (Nitroreductase, NTR) probe XD03 nuclear magnetic datas, spectra count
According to.
3. Fig. 3 is nitroreductase (Nitroreductase, NTR) probe XD03 to common 14 kind of 10 equivalent in cell
Jamming performance is tested.
4. Fig. 4 be nitroreductase (Nitroreductase, NTR) probe XD03 molecular simulation in NTR enzymatic activitys
The relative position docking results of cavity.
5. Fig. 5 tests for nitroreductase (Nitroreductase, NTR) probe XD03 reaction time dynamic performances.
6. Fig. 6 tests for nitroreductase (Nitroreductase, NTR) probe XD03 concentration gradients.
7. Fig. 7 is the cytotoxicity XTT results of nitroreductase (Nitroreductase, NTR) probe XD03.
8. Fig. 8-a and 8-b are the double light of product after the XD03 reactions of nitroreductase (Nitroreductase, NTR) probe
Sub- fluorescence spectrum and two photon absorption cross section.
9. Fig. 9 be nitroreductase (Nitroreductase, NTR) probe XD03 different oxygen concentrations (1%, 3%,
5%, 10%, 15%, 20%O2) under the conditions of cell imaging figure.
Specific implementation mode
Embodiment 1
Series is based on the preparation of naphthols fluorogen two-photon nitroreductase (Nitroreductase, NTR) probe XD03
Method is as follows：
1.1XD00：By 2,7- dihydroxy naphthols (0.3g, 18.7mmol), dimethylamine (40% aqueous solution, 1.05mL,
9.35mmol), sodium pyrosulfite (0.711g, 3.74mmol), deionized water 2mL are added sequentially in Shrek bottle, and 150 DEG C anti-
8h is answered, after being cooled to room temperature, the pH that reaction system is adjusted using hydrochloric acid is 4 or so, is then spin-dried for using DCM extractions, by glue
Column chromatography purifies to obtain intermediate product white solid XD00.1H-NMR(300MHz,CD3OD)：δ7.57(m,2H),δ6.97-7.01
(m, 1H), δ 6.93-6.94 (d, J=3Hz, 1H), δ 6.67-6.79 (m, 2H), δ 2.98 (S, 6H), reaction equation is as follows：
1.2XD01：N,N-Dimethylformamide (0.4mL) is added in Shrek bottle to vacuumize, with syringe by trichlorine
Oxygen phosphorus (0.2mL) is added in above-mentioned Shrek bottle, is placed under the conditions of 0 DEG C of cold well and stirs 10min.By the product X D00 dissolvings of upper step
It in the n,N-Dimethylformamide of 0.2mL, is slowly added in Shrek bottle with disposable syringe, 2h is stirred under the conditions of 0 DEG C.
It pours into the mixture of ice and water of 10 times of volumes and stirs after reaction system is cooled down, CH is added3COOK until solution turned yellow after filter,
It purifies to obtain intermediate product XD01 by plastic column chromatography.1H-NMR(500MHz,DMSO-d6)：δ10.18(s,1H),δ9.89(s,
1H), δ 8.45 (s, 1H), δ 7.89 (d, J=5Hz, 1H), δ 7.67 (d, J=5Hz, 1H), δ 7.18 (d, J=10Hz, 1H), δ
6.91 (d, J=10Hz, 1H), δ 3.05 (s, 6H)；13CNMR(125MHz,DMSO-d6),δ190.59,δ158.64,δ135.41,
δ 130.06, δ 122.61, δ 115.65, δ 114.79, δ 106.00, δ 45.84. reaction equations are as follows：
1.3XD02:By XD01 (100mg, 0.47mmol), to nitro bromobenzyl (101.67mg 0.47mmol), potassium carbonate
(194.87mg, 1.47mmol) is added sequentially in n,N-Dimethylformamide solution, and 4h is stirred at room temperature in nitrogen protection,
Then extraction is spin-dried for, and purifies to obtain intermediate product XD02 by plastic column chromatography,1H-NMR(500MHz,CDCl3)：δ10.53(s,
1H), δ 8.64 (d, J=5,1H), 8.28 (d, J=5Hz, 2H), 7.92 (d, J=10Hz, 1H), δ 7.73 (m, 3H), 7.33 (d,
J=10Hz, 1H), 7.19 (dd, J1=10Hz, J2=10Hz, 1H), δ 5.35 (s, 2H), δ 3.20 (s, 6H)13C-NMR
(125MHz,CDCl3),δ192.12,δ159.27,δ159.17,δ148.03,δ147.65,δ144.41,δ135.43,δ
133.77,δ128.03,δ123.85δ117.14,116.83,δ115.78,δ104.51,δ68.62,δ46.39.[M+H]+for
C13H13NO2:215.09；Found, 216.00 (attached drawing 1-a, 1-b, 1-c) reaction equations are as follows：
Two-photon naphthol derivative XD02 (100mg), malononitrile (25mg) are added sequentially to ethyl alcohol (3mL) by 1.4XD03
In solution, nitrogen protection stirs 4h under 0 DEG C of reaction condition, is then spin-dried for, and purifies to obtain yellow solid production by plastic column chromatography
Object XD03.XD03,1H NMR(500MHz,CDCl3)：δ 8.41 (s, 1H), 8.31 (d, J=10Hz, 2H), 7.84 (m, 3H), δ
7.72 (m, 3H), 7.23 (d, J=5Hz, 1H), 7.15 (d, J=10Hz, 2H), δ 5.34 (s, 2H), δ 2.98 (s, 6H)13CNMR
(125MHz,DMSO-d6),δ159.79,δ158.06,δ153.68,δ147.11,δ144.84,δ134.35,δ133.99,δ
128.55,δ122.80,δ116.01,δ115.15,δ113.10,δ110.52,δ103.87,δ78.89,δ68.46,δ56.06,δ
43.48,δ18.59.[M+H]+for C23H18N4O3：398.14；Found, 399.21 (attached drawing 1-a, 1-b, 1-c).Reaction equation is such as
Under：
Embodiment 2
Series is based on the preparation of naphthols fluorogen two-photon nitroreductase (Nitroreductase, NTR) probe XD03
Method is as follows：
1.1XD00：By 2,7- dihydroxy naphthols (3g, 187mmol), dimethylamine (40% aqueous solution, 10.5mL,
93.5mmol), sodium pyrosulfite (7.11g, 37.4mmol), deionized water 20mL are added sequentially in Shrek bottle, and 150 DEG C anti-
8h is answered, after being cooled to room temperature, the pH that reaction system is adjusted using hydrochloric acid is 4 or so, is then spin-dried for using DCM extractions, by glue
Column chromatography purifies to obtain intermediate product white solid XD00.
1.2XD01：N,N-Dimethylformamide (4mL) is added in Shrek bottle to vacuumize, with syringe by trichlorine oxygen
Phosphorus (2mL) is added in above-mentioned Shrek bottle, is placed under the conditions of 0 DEG C of cold well and stirs 10min.The product X D00 of upper step is dissolved in
It in the n,N-Dimethylformamide of 2mL, is slowly added in Shrek bottle with disposable syringe, 2h is stirred under the conditions of 0 DEG C.It will be anti-
It pours into the mixture of ice and water of 10 times of volumes and stirs after answering system to cool down, CH is added3COOK after solution turned yellow until filter, process
Plastic column chromatography purifies to obtain intermediate product XD01.
1.3XD02:By XD01 (1g, 4.7mmol), to nitro bromobenzyl (1.0167mg, 4.7mmol), potassium carbonate
(1.9487mg, 14.7mmol) is added sequentially in n,N-Dimethylformamide solution, and 4h is stirred at room temperature in nitrogen protection,
Then extraction is spin-dried for, and purifies to obtain intermediate product XD02. by plastic column chromatography
1.4XD03 by two-photon naphthol derivative XD02 (1g), that malononitrile (250mg) is added sequentially to ethyl alcohol (30mL) is molten
In liquid, nitrogen protection stirs 4h under 0 DEG C of reaction condition, is then spin-dried for, and purifies to obtain yellow solid product by plastic column chromatography
XD03。
Embodiment 3
Series is based on the preparation of naphthols fluorogen two-photon nitroreductase (Nitroreductase, NTR) probe XD03
Method is as follows：
1.1XD00：By 2,7- dihydroxy naphthols (1.5g, 93.5mmol), dimethylamine (40% aqueous solution, 5.25mL,
46.75mmol), sodium pyrosulfite (3.55g, 18.7mmol), deionized water 10mL are added sequentially in Shrek bottle, 150 DEG C
8h is reacted, after being cooled to room temperature, the pH that reaction system is adjusted using hydrochloric acid is 4 or so, is then spin-dried for, passes through using DCM extractions
Plastic column chromatography purifies to obtain intermediate product white solid XD00.
1.2XD01：N,N-Dimethylformamide (2mL) is added in Shrek bottle to vacuumize, with syringe by trichlorine oxygen
Phosphorus (1mL) is added in above-mentioned Shrek bottle, is placed under the conditions of 0 DEG C of cold well and stirs 10min.The product X D00 of upper step is dissolved in
It in the n,N-Dimethylformamide of 1mL, is slowly added in Shrek bottle with disposable syringe, 2h is stirred under the conditions of 0 DEG C.It will be anti-
It pours into the mixture of ice and water of 10 times of volumes and stirs after answering system to cool down, CH is added3COOK after solution turned yellow until filter, process
Plastic column chromatography purifies to obtain intermediate product XD01.
1.3XD02:By XD01 (0.5g, 2.35mmol), to nitro bromobenzyl (0.55mg, 2.35mmol), potassium carbonate
(0.97mg, 7.35mmol) is added sequentially in n,N-Dimethylformamide solution, nitrogen protection, 4h is stirred at room temperature, so
Extraction is spin-dried for afterwards, purifies to obtain intermediate product XD02. by plastic column chromatography
Two-photon naphthol derivative XD02 (0.5g), malononitrile (250mg) are added sequentially to ethyl alcohol (15mL) by 1.4XD03
In solution, nitrogen protection stirs 4h under 0 DEG C of reaction condition, is then spin-dried for, and purifies to obtain yellow solid production by plastic column chromatography
Object XD03.
Embodiment 4
Series is based on the preparation of naphthols fluorogen two-photon nitroreductase (Nitroreductase, NTR) probe XD03
Method is as follows：
1.1XD00：By 2,7- dihydroxy naphthols (0.15g, 9.35mmol), dimethylamine (40% aqueous solution, 0.525mL,
4.675mmol), sodium pyrosulfite (0.355g, 1.87mmol), deionized water 10mL are added sequentially in Shrek bottle, 150 DEG C
8h is reacted, after being cooled to room temperature, the pH that reaction system is adjusted using hydrochloric acid is 4 or so, is then spin-dried for, passes through using DCM extractions
Plastic column chromatography purifies to obtain intermediate product white solid XD00.
1.2XD01：N,N-Dimethylformamide (0.2mL) is added in Shrek bottle to vacuumize, with syringe by trichlorine
Oxygen phosphorus (0.1mL) is added in above-mentioned Shrek bottle, is placed under the conditions of 0 DEG C of cold well and stirs 10min.By the product X D00 dissolvings of upper step
It in the n,N-Dimethylformamide of 0.1mL, is slowly added in Shrek bottle with disposable syringe, 2h is stirred under the conditions of 0 DEG C.
It pours into the mixture of ice and water of 10 times of volumes and stirs after reaction system is cooled down, CH is added3COOK until solution turned yellow after filter,
It purifies to obtain intermediate product XD01. by plastic column chromatography
1.3XD02:By XD01 (0.05g, 0.235mmol), to nitro bromobenzyl (0.055mg, 0.235mmol), potassium carbonate
(0.097mg, 0.735mmol) is added sequentially in n,N-Dimethylformamide solution, and 4h is stirred at room temperature in nitrogen protection,
Then extraction is spin-dried for, and purifies to obtain intermediate product XD02 by plastic column chromatography.
Two-photon naphthol derivative XD02 (0.05g), malononitrile (25mg) are added sequentially to ethyl alcohol by 1.4XD03
In (1.5mL) solution, nitrogen protection stirs 4h under 0 DEG C of reaction condition, is then spin-dried for, and purifies to obtain Huang by plastic column chromatography
Color solid product XD03.
Experimental result：
1. Fig. 1-a, 1-b, 1-c are the hydrogen spectrum, carbon spectrum, matter of the presoma XD02 of naphthols two-photon fluorescence group devicative respectively
Compose number data.
2. Fig. 2-a, 2-b, 2-c nitroreductase (Nitroreductase, NTR) probe XD03 nuclear magnetic datas, spectra count
According to.
3. Fig. 3 is nitroreductase (Nitroreductase, NTR) probe XD03 to common 14 kind of 10 equivalent in cell
Jamming performance is tested.1.Control(XD03+NADH 500μM),2.NaCl(50mM),3.KCl(50mM),4.MgCl2
(50mM),5.CaCl2(50mM),6.tyrosine(Tyr,1mM),7.Glycine(Gly,1mM),8.Glutamic acid
(Glu,1mM),9.Tryptophan(Trp,1mM),10.Arginine(Arg,1mM),11.Glucose(10mM),
12.Vitamin C(1mM),13.H2O2(1mM) 14.BSA (10mg/mL), 15.Nitroreductase 10. (use 5%DMSO+
PBS (0.1M) solution prepares the probe of 10 μM of XD03 respectively, is separately added into from the various interference being configured according to proportioning
The interfering ion final concentration of the solution of substance, addition is 100 μM, respectively fluorescence emission spectrum (exciting light after test response 2h
For 460nm).Only while when NTR and NADH is added there is larger Fluorescence Increasing from the results of view, other interfering ions are without shadow
It rings, it was demonstrated that the material tests NTR of synthesis has very high specificity.
4. Fig. 4 be nitroreductase (Nitroreductase, NTR) probe XD03 molecular simulation in NTR enzymatic activitys
The relative position docking results of cavity.(NTR crystal knots are obtained from PDB (Protein Data Bank) first.Use Autodock
Vina software analyzing crystal structures and interaction parameter, molecular structure use PyMOL program icons.In molecular simulation process
In, polarized hydrogen atom is considered as the proton donor of interaction, and all hydrones are all excluded potential mutual
Except effect.In simulation process, probe molecule is limited in the active chamber of certain protein.It is carried out when using AutoDock
When parameter setting, two kinds of protein are used as reference standard with docking for common substrate tyrosine (tyrasamine), and all rotatable
Chemical bond be arranged to can active twist.In the analysis process, flavin adenine dinucleotide (FAD) (FAD) is measured using PyMOL
Middle C4A atoms and the distance between the N atoms in protein small molecular.After above-mentioned parameter is provided with, NTR and probe XD03
Parameter be configured and optimized.)
5. Fig. 5 tests for nitroreductase (Nitroreductase, NTR) probe XD03 reaction time dynamic performances
It (prepares the probe of 10 μM of XD03 respectively with 5%DMSO+PBS (0.1M) solution, the coenzyme NAD H (500 μM) configured is added
And NTR (0,2,4,6,8,10 μ g/mL) solution of various concentration carries out time dynamics test immediately, obtains the time to the end
Kinetic results (exciting light 440nm, a length of 580-620nm of received wave) test result is aobvious, when XD03 and substrate-function balance
Between be 80 minutes.
6. Fig. 6 tests for nitroreductase (Nitroreductase, NTR) probe XD03 concentration gradients.(use 5%DMSO+
PBS (0.1M) solution prepares the probe of 10 μM of XD03 respectively, be added the coenzyme NAD H (500 μM) configured and NTR (0,2,
4,6,8,10 μ g/mL) solution, reaction 2h obtains the result (exciting light 440nm) of final concentration gradient under the conditions of 37 DEG C.Knot
Fruit is shown in NTR concentration and reaches 10 μ g/mL, and XD03, which reacts, reaches balance.Prove that the enzyme equilibrium that 10 μ g/mL are 10 μM of XD03 is dense
Degree.
7. Fig. 7 is the cytotoxicity XTT results of nitroreductase (Nitroreductase, NTR) probe XD03.(probe
The XTT analytic approach of the toxicity standard of XD03 is analyzed, and by 0.25% trypsin digestion of HepG2 cells, then uses cell
Culture medium is made into cell suspending liquid and is inoculated in 96 orifice plates, is cultivated 24 hours under conditions of 37 DEG C, 5% carbon dioxide, will
Various concentration (0 μM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM) probe be added in orifice plate 24 hours of culture, cultivated with XTT
Four hours, the absorbance value in each hole is tested with microplate reader, while control wells are set).As a result testimonial material XD03 is in cell
The survival rate that interior concentration reaches cell after 50 μM remains to reach 80%, it is seen that the toxicity very little of probe
8. Fig. 8-a and 8-b are the double light of product after the XD03 reactions of nitroreductase (Nitroreductase, NTR) probe
Sub- fluorescence spectrum and two photon absorption cross section.(2.64 μ g/mL of concentration and probe concentration, two-photon excitation emission wavelength are 760nm).As a result
It proves that probe XD03 reaction products have very high two-photon performance, and a length of 760nm of maximum absorption wave, is provided for two photon imaging
Optimum excitation wave elongate member.
9. Fig. 9 be nitroreductase (Nitroreductase, NTR) probe XD03 different oxygen concentrations (1%, 3%,
5%, 10%, 15%, 20%O2) under the conditions of cell imaging figure.It is separately added into XD03 into the culture dish containing Hepg-2 cells
DMSO solution after mixing with cell culture fluid make a concentration of 10 μM of the XD03 in culture solution, then in different oxygen
It is kept in 37 DEG C of environment in the anoxic culture dish of Gas content, cultivates 4h, the buffer solution of pH=7.35 is then used to rinse 3
It is secondary, which is imaged under Laser Scanning Confocal Microscope.(one-photon excitation light is 405nm, and two-photon excitation emission wavelength is
760nm).As a result it being shown under normal oxygen condition, intracellular NTR contents are relatively low, probe XD03 unstressed configurations in cell, and in oxygen
When content is 1%, intracellular NTR contents are higher, and probe XD03 shows stronger fluorescence in cell, it was demonstrated that probe XD03
It is capable of hypoxemia (1%) gas situation of effective indicator cells, two-photon cell imaging result proves that probe XD03 can be in live body
Two photon imaging is realized in cell well.
Claims (5)
1. one kind can two-photon fluorescence detect nitroreductase NTR probes, which is characterized in that the fluorescence probe be based on naphthalene
The structural formula such as (I) of the probe XD03 of phenol fluorogen two-photon fluorescence group, fluorescence probe are shown：
2. it is a kind of as described in claim 1 can two-photon fluorescence detection nitroreductase NTR probes preparation method, feature
It is：The preparation method of the fluorescence probe XD03 includes the following steps：
Two-photon naphthol derivative XD02, malononitrile are added sequentially in ethanol solution, nitrogen protection, in 0 DEG C of reaction condition
Lower stirring 3-5h is dissolved with dichloromethane after rotary evaporation removes organic solvent, deionized water is used in combination to be extracted twice, brine extraction
It takes, collects organic phase drying, rotary evaporation removes solvent, purifies to obtain intermediate product XD03 by plastic column chromatography, above-mentioned is double
Photon naphthol derivative XD02, malononitrile molar ratio be 1：1.0-6.0 reaction equation is as follows:
3. it is according to claim 2 can two-photon fluorescence detection nitroreductase NTR probes preparation method, feature exists
In：The molar ratio of the XD02 and malononitrile is 1:1；In the preparation method of the fluorescence probe XD03, dry is with nothing
Aqueous sodium persulfate is dried；In the preparation method of the fluorescence probe XD03, ethyl alcohol 20-23mL is added in the XD02 per mmol；
In reaction process, the equivalent of malononitrile cannot be too many, otherwise will produce by-product, reduces yield, and secondary response has to keep
Nitrogen protection and in low temperature environment environment, otherwise can cause to generate other impurity.
4. it is according to claim 2 can two-photon fluorescence detection nitroreductase NTR probes preparation method, feature exists
In：It is described can two-photon fluorescence detection nitroreductase probe precursor substance XD02 preparation method it is as follows：
(1)XD00：By 2,7- dihydroxy naphthols, 40% aqueous solution of dimethylamine, sodium pyrosulfite, deionized water is added sequentially to history
In the bottle of Lake, 140-160 DEG C of reaction 7-9h, after being cooled to room temperature, the pH that reaction system is adjusted using hydrochloric acid is 3-5 or so, so
It is spin-dried for afterwards using DCM extractions, purifies to obtain intermediate product white solid XD00 by plastic column chromatography；Above-mentioned 2,7 dihydroxy naphthalene
Phenol, 40% aqueous solution of dimethylamine, sodium pyrosulfite molar ratio be 1:4-6:4-6；2,7 dihydroxy naphthalene phenol per mmol is added
Deionized water 1-6mL, reaction equation are as follows：
(2)XD01：N,N-Dimethylformamide is added in Shrek bottle to vacuumize, phosphorus oxychloride is added with syringe above-mentioned
In Shrek bottle, stirring to system is sticky mass under the conditions of being placed in 0 DEG C of cold well.The product X D00 of upper step is dissolved in right amount
It in n,N-Dimethylformamide, is slowly added in Shrek bottle with disposable syringe, 1-3h is stirred under the conditions of 0 DEG C, will reacted
It pours into the mixture of ice and water of 10 times of volumes and stirs after system cooling, CH is added3COOK after solution turned yellow until filter, by glue
Column chromatography purifies to obtain intermediate product XD01；Above-mentioned XD00, N,N-dimethylformamide, phosphorus oxychloride molar ratio be 1：1:
0.4-1.0, reaction equation are as follows：
(3)XD02:By above-mentioned product X D01, nitro bromobenzyl, potassium carbonate are added sequentially in n,N-Dimethylformamide solution,
Nitrogen protection is stirred at room temperature 3-5h, is then extracted with dichloromethane, dry, is spin-dried for, is purified in obtaining by plastic column chromatography
Between product X D02, above-mentioned XD01, be 1 to the molar ratio of nitro bromobenzyl, potassium carbonate:0.9-1.1：3-5, reaction equation are as follows：
5. it is according to claim 4 it is described can two-photon fluorescence detection nitroreductase NTR probes preparation method,
It is characterized in that：
In a series of preparation method of two-photon naphthols derivative compounds, in prepared by step (1) the intermediate product XD00,1,6-
The molar ratio of dihydroxy naphthols, dimethylamine agueous solution, sodium pyrosulfite is 1:4-6:4-6；2,7 dihydroxy naphthalene phenol per mmol
Deionized water 1-6mL is added；
XD00 in the step (2), N,N-dimethylformamide, phosphorus oxychloride molar ratio be 1：1:0.4-1.0, and to shift to an earlier date
Jelly salt is first made in n,N-Dimethylformamide and phosphorus oxychloride in ice-water bath, XD00 is then dissolved into appropriate N, N- dimethyl
In formamide, it is added to and freezes in salt system in the condition of ice-water bath, nitrogen protection；
XD01 in the step (3), it is 1 to the molar ratio of nitro bromobenzyl, potassium carbonate:0.9-1.1：3-5, reaction want nitrogen to protect
It is carried out under retaining ring border.
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