CN106749153A - The specificity fluorescent probe of nitroreductase and its preparation and the application for cancer target fluorescence imaging and monitoring tumor hypoxia degree - Google Patents
The specificity fluorescent probe of nitroreductase and its preparation and the application for cancer target fluorescence imaging and monitoring tumor hypoxia degree Download PDFInfo
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- CN106749153A CN106749153A CN201611177781.7A CN201611177781A CN106749153A CN 106749153 A CN106749153 A CN 106749153A CN 201611177781 A CN201611177781 A CN 201611177781A CN 106749153 A CN106749153 A CN 106749153A
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- 102000004459 Nitroreductase Human genes 0.000 title claims abstract description 92
- 108020001162 nitroreductase Proteins 0.000 title claims abstract description 92
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- 238000000799 fluorescence microscopy Methods 0.000 title claims abstract description 19
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- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 7
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- NLLHXVBITYTYHA-UHFFFAOYSA-N Nitrofor Chemical compound CCN(CC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O NLLHXVBITYTYHA-UHFFFAOYSA-N 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
- A61K49/0043—Fluorescein, used in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides the specificity fluorescent probe of nitroreductase and its preparation and be applied to cancer target fluorescence imaging and monitoring tumor hypoxia degree reagent, be formed by connecting by chemical bond by tumor biomarker recognition group (sensor), cancer target group (target), fluorophor (dye).The reagent is based on the expression high of the Hypoxic and nitroreductase of tumour in hypoxic tumor, can be applied in micro-environmental hypoxia, and the tumour of expression nitroreductase high, combined with fluorescent imager, can be used for cancer target fluorescence imaging, the application such as the monitoring of tumor hypoxia degree and monitoring metastases.The reagent realizes high sensitivity in the application, high specific, for medically study tumour and clinically monitor, treatment metastases provide effective instrument, therefore in cancer target fluorescence imaging, the monitoring of tumor hypoxia degree and monitor metastases etc. and have a good application prospect.
Description
Technical field
The invention belongs to field of biomedicine technology, it is related to class detection reagent and its preparation method and application, specially
Specificity fluorescent probe of nitroreductase and preparation method thereof and for cancer target fluorescence imaging and monitoring tumor hypoxia journey
The application of degree.
Background technology
The growth of cell and propagation depend on sufficient oxygen and energy supply, in oxygen-supplying amount is normally organized, cell
Energy source about 90% depend on mitochondrial aerobic oxidation.However, in tumor tissues, the runaway growth of tumour cell and increasing
Grow the substantial amounts of nutrition of consumption and oxygen, can not in time, effectively set up new vessels net inside it, or new vessels net structure
And dysfunction, there is temporary stopping or " cecum "；And permeability is higher, intravenous extravasation to tissue space causes blood flow to stick
Resistance hysteresis increase.These factors make inside tumor Oligemia, and, far fewer than the demand of oxygen, tumour cell is in anoxic for the supply of oxygen
State.Additionally, the anaemia caused by the own process and antineoplaston of malignant tumour also exacerbates the anoxic of tumour, tumour
Thus internal micro-environmental hypoxia produces.
Nitroreductase family belongs to flavo-enzyme, and virtue can be metabolized in the presence of coenzyme nicotinamide-adenine dinucleotide phosphate
Fragrant race's nitro compound or the heterocyclic compound of nitro substitution.Research show oxygen-starved tissue or cell and tumour in nitro also
The content of protoenzyme is presented rising trend with respect to normal condition.Under anoxic conditions with NADH
NADH can be catalyzed various external source nitro-aromatic compounds and single electron turn occurs as the intracellular nitroreductase of electron donor
Move, generate Free radicals, be then further reduced into azanol or amino.Research finds that nitroreductase is usual
It is present in bacterium, such as Escherichia coli, is there's almost no in the normal cell of people, and largely exists in malignant tumour.Cause
This, nitroreductase can be as the biomarker of tumour.
Small-molecule fluorescent probe is widely used in chemical analysis, and bioanalysis and biodynamic process are real-time, non-
Invasive is monitored.Therefore, small-molecule fluorescent probe is widely used in drug screening and the assessment of therapeutic effect, realizes tumour and controls
The early monitoring of therapeutic effect.
RGD (Arg-Gly-Asp, arginine-glycine-aspartic acid) amino acid sequence is various biological cell epimatrixs
It is also major site that fibrocyte sticks with basis common in plasma protein structure.The genome of people can produce 24
Individual different known integrin, about 1/3 integrin recognizes RGD sequence to varying degrees.In tumor neogenetic blood vessels
The high-caliber β 1 of 3/ α of α v β 5 of chrotoplast Membrane surface expression and the integrins of 5 β of α 1, therefore the albumen containing RGD sequence, as integration
Plain part, being capable of targeting arrival tumor by local neovascular endothelium.Exogenous rgd peptide and the like can with contain in vivo
The material competition binding of RGD sequence, so as to block courier's transmission of vascular endothelial propagation, terminates cell propagation, makes blood vessel
Can not grow, cause tumor tissues oxygen system to interrupt, final cell atrophy, apoptosis.Research finds that RGD ring type polypeptides compare wire
Polypeptide has receptor binding specificities higher.For these reasons, a large amount of linear or ring type polypeptides containing RGD sequence are by as α v
The antagonists of β 3 synthesize, and part has been successfully made fluorescence probe.
The content of the invention
Regarding to the issue above, it is an object of the invention to be prepared for the specificity fluorescent probe and system of a class nitroreductase
Preparation Method and application.The probe bodies and the product after being reduced by nitroreductase, fluorescence intensity have obvious difference, and produce
Thing fluorescence can detect by fluorescence detector, can be to the distribution of nitroreductase in various living things systems and anti-using the probe reaction
Efficiency is answered to be evaluated.
Second purpose of the present invention provides the reagent of a kind of cancer target fluorescence imaging and monitoring metastases.
The specificity fluorescent probe that the 3rd purpose of the present invention provides the nitroreductase is being prepared for cancer target
Application on the reagent of fluorescence imaging and monitoring metastases.
The 4th purpose of the present invention provides a kind of reagent for monitoring tumor hypoxia degree.
The specificity fluorescent probe that the 5th purpose of the present invention provides the nitroreductase is being prepared for monitoring tumour
Application on the reagent of degree of oxygen deficiency.
Technical scheme is as follows：
The specificity fluorescent probe of nitroreductase, the probe is by detection moiety sensor, fluorophor dye, tumor target
It is formed by connecting by chemical bond to group target；Its structure is：Structure I type sensor-dye or structure I I types sensor-
dye-target。
Further, the detection moiety sensor structures are：
The fluorophor dye can for fluoresceins like thing, its structure is：
The structure of the cancer target group target is：
The specificity fluorescent probe of nitroreductase of the present invention, it is preferable that the structure of the structure I type sensor-dye
Shown in following formula FBN-1 or FBN-2：
The structure of the structure I I types sensor-dye-target is shown in following formula FBN-3 or FBN-4：
The specificity fluorescent probe of nitroreductase of the present invention,, without fluorescence, the nitro in probe is by nitro for itself
Reductase specific reduction, the fluorescence-causing substance after reduction has fluorescence quantum yield very high, can be realized using fluorescence detector
Quick, the Sensitive Detection of Substrate fluorescence change.
The specificity fluorescent probe of nitroreductase of the present invention, as the specific substrate of nitroreductase, passes through
The production rate of substrate elimination factor or its product in the quantitative determination unit interval come nitro in quantitative determining different living things systems also
The activity of protoenzyme.
The present invention also provides the preparation method of the specificity fluorescent probe of the nitroreductase, and the method is：A) pass through
Fluorescein (compound (1)) hydroxy alkylated is prepared into the special of nitroreductase with to nitrobenzyl bromine in the basic conditions
Property fluorescence probe FBN-1；B) after obtaining hydroxyl by reducing the ketone carbonyl of FBN-1, then it is alkylated with to nitrobenzyl bromine
To the specificity fluorescent probe FBN-2 of nitroreductase.C) FBN-3 and FBN-4 are respectively by FBN-1, the contracting of FBN-2 and RGD cyclic peptide
Conjunction is formed.
The present invention also provides the reagent of a kind of cancer target fluorescence imaging and monitoring metastases, and the reagent contains above-mentioned
The specificity fluorescent probe of nitroreductase.
The specificity fluorescent probe that the present invention also provides the nitroreductase is being prepared for cancer target fluorescence imaging
With the application on the reagent of monitoring metastases.
The specificity fluorescent probe that the present invention also provides the nitroreductase is being prepared for cancer target fluorescence imaging
With monitoring metastases reagent on application, can be used as the reagent of surgical navigational.
The present invention also provides a kind of reagent for monitoring tumor hypoxia degree, and the reagent contains the spy of above-mentioned nitroreductase
Anisotropic fluorescent probe.
The specificity fluorescent probe that the present invention also provides the nitroreductase is being prepared for monitoring tumor hypoxia degree
Reagent on application.
Detailed description of the invention
The probe is formed by connecting by detection group and fluorophor by chemical bond, is summarized as structure I type, and it is represented by
sensor-dye。
The specificity fluorescent probe instantiation of nitroreductase is：
Structure I type：dye-sensor
I types probe, in itself due to the strong sucting electronic effect of nitro, causes d- as the specific substrate of nitroreductase
PET effects and quenching molecules fluorescence, after it is reduced by nitroreductase, discharge compound (1), and fluorescence intensity increases greatly
By force.The biological sample such as enzyme or cell preparation solution and cell are determined by the growing amount of reduzate in the quantitative determination unit interval
The activity of middle nitroreductase.
Recognition mechanism is as follows：
1) in the conventional buffer solution such as PBS or Tris-HCl, reaction temperature is between 20 DEG C to 45 DEG C, preferably 37 DEG C for most
The excellent reaction time；Between 5.0~8.5, preferably pH 7.4 is peak optimization reaction pH value to incubation system pH；Reaction time be 5~
30 minutes, preferred reaction time was 20min.
2) by the activity that multiple evaluates nitroreductase that increases of analytical unit time fluorescence intensity, can be examined using fluorescence
Survey the rapid sensitive detection that device realizes product and substrate；Fluoroscopic examination condition is：Excitation wavelength 490nm, enters in 510~550nm
The detection of row fluorescence emission spectrum.
The fluorescence probe of the nitroreductase in the present invention has the characteristics that：
Only there is reduction reaction, other common organisms with nitroreductase in organism in the Specific probe
Be matrix and impurity for example：Inorganic salts, carbohydrate, amino acid, vitamin, biological sulfydryl species, oxide species do not produce interference.Should
The relative fluorescence quantum efficiency of probe reachesFluorescence quantum yield is higher, and excitation wavelength is 490nm, the wavelength
More typically, can detect that determination limit is up to 0.65ng/mL with fluorescent spectrometry.The probe is relatively low to the toxicity of organism, can be used for
The detection of nitroreductase content is produced in animal organism and in bacteria growth process.
A further object of the present invention, there is provided the reagent and method of a kind of surgical navigational.The reagent can be connected by I types reagent
Connect cancer target group (target) to be prepared from, can in real time target fluorescence imaging tumour cell or tissue, the reagent can be summarized
It is structure I I types, it is represented by sensor-dye-target.
The reagent instantiation of targeting fluorescence imaging tumour cell or tissue is in real time：
Structure I I types：target-dye-sensor
The preparation method of above-mentioned fluorescence probe is as follows：
The preparation method of FBN-1：
The preparation method of FBN-2：
The preparation method of FBN-3：
The preparation method of FBN-4：
Research finds that anoxic is the feature microenvironment of tumour, nitroreductase expression high in hypoxic tumor, without hair
Existing its is present in normal cell and tissue.In organism, II type reagents are both the specific substrate of nitroreductase, again may be used
Gather tumor tissues with selectively targeted.So during II type reagents can be applicable to surgical tumor inadvertent closure, tumour side
Boundary differentiates that the tracking of tumors remaining tissue identification and metastases lymph node is that the fluorescence navigation system of cancer surgery is carried
A kind of new fluorescence navigation reagent is supplied.After the reagent injector enters the live body with tumor disease, body surgical field of view is miscellaneous specific
Under wavelength light-wave irradiation, tumour cell or tissue can send the fluorescence of certain wavelength, instruct doctor to carry out under imaging system outer
Section performs the operation, and effectively improves surgical result, reduces the tumor patient death rate.
Reagent of the present invention is as follows as the technical scheme that surgical operation fluorescence navigation reagent is implemented in tumor-bearing mice：
1) II types reagent dissolves in PBS solution or normal saline solution containing DMSO 0%-5%, is configured to working solution, tail
In intravenous injection or local injection to Mice Body；
2) II types reagent is through blood circulation, can specific accumulation be targeted to tumor locus；
3) II types reagent is weaker in nonneoplastic tissue region unstressed configuration signal or fluorescence signal intensity, at tumor tissues position
Fluorescence signal intensity is very strong.
4) living body fluorescent imaging technique is utilized, according to fluorescence signal, accurately image knub position is led for surgical operation is provided
Boat.
Another object of the present invention provides a kind of reagent for targetting monitoring tumour cell or histanoxia degree.Utilize
Structure I I types probe of the invention detects that the nitroreductase content in tumor hypoxia region can be in evaluation by Imaging-PAM
With the anoxic level in research tumor hypoxia region.
Anoxic is the feature microenvironment of tumour, and research finds that the content of nitroreductase and the degree of oxygen deficiency of tumour are in positive
Pass relation, so II type reagents can be used to monitor the degree of oxygen deficiency of tumour.II types reagent as nitroreductase specific bottom
Thing, in organism, tumor tissues is targeted to by targetting group, in the different tumour of degree of oxygen deficiency, nitroreductase
Concentration is also different, and the also commercial weight of fluorescence probe is also different.By the fluorescence intensity of reduzate in quantitative determination same time come
The degree of oxygen deficiency of indirect determination tumour cell and tissue.Respectively with human lung carcinoma cell (A549), Proliferation of Human Ovarian Cell (SKOV-3)
With human liver cancer cell (HepG-2) as tumor models, live body hepatic carcinoma bearing mouse model is used as living body biological model
Probe into the ability that II types reagent detects cancer cell degree of oxygen deficiency.
Specific assay method in tumour cell is：
1) inoculating cell in four copolymerization Jiao's culture dishes, overnight, makes its adherent；
2) II types reagent pre-incubation cell 30min；
3) four ware cells are put into the different environment of degree of oxygen deficiency respectively and cultivate 8h；
4) fluorescence intensity of the ware cell of fluorescence microscope four is strong and weak.
Specific assay method in live body tumor-bearing mice is：
1) II types reagent dissolves in PBS solution or normal saline solution containing DMSO 0%-5%, is configured to working solution, tail
In intravenous injection into mice body；
2) II types reagent is through blood circulation, can specific accumulation be targeted to tumor locus；
3) because tumor hypoxia degree is different, the fluorescence intensity of tumor locus is also different, is examined using living body fluorescent imaging technique
Survey the fluorescence intensity of knub position；
4) positive correlation of the strong and weak and tumor hypoxia degree according to fluorescence intensity, the fluorescence intensity of different tumours it is strong
The size of weak its degree of oxygen deficiency of secondary indication.
Advantageous Effects of the present invention：
The invention provides the specificity fluorescent probe of nitroreductase, the specific probe can be by living things system
Nitroreductase is reduced, therefore can be used to detect the nitroreductase in organism；The probe can be selectively targeted to tumor group
Knit, after other nitroreductase reduction, Fluorescence Increasing, therefore the probe can be used for cancer target fluorescence imaging, and because not
With in degree of oxygen deficiency, the expression quantity of nitroreductase is different, and the probe can be used for detecting tumor hypoxia degree again.Experiment hair
Existing, probe fluorescence intensity in itself is weaker, and after being reduced by nitroreductase, fluorescence is significantly increased；In degree of oxygen deficiency ring higher
In border, when probe is by nitroreductase, the intensification factor of fluorescence intensity is higher
The expression high of Hypoxic and nitroreductase of the present invention based on tumour in hypoxic tumor, can be micro- with anoxic
Applied in environment, and the tumour of expression nitroreductase high, combined with fluorescent imager can be used for cancer target fluorescence imaging, swell
The application such as metastases is monitored and monitored to knurl degree of oxygen deficiency.
In the present invention, fluorescence signal change after detection cancer target reagent identification tumour is simple to operate, harmless to the body；
The reagent realizes high sensitivity in the application, and high specific is medically research tumour and clinically monitoring, treatment tumour turn
The effective instrument that provides is moved, therefore in cancer target fluorescence imaging, the tool such as the monitoring of tumor hypoxia degree and monitoring metastases
There is good application prospect.
Brief description of the drawings
Fig. 1 is the nuclear-magnetism of probe FBN-11H NMR spectras；
Fig. 2 is the nuclear-magnetism of probe FBN-113C NMR spectras；
Fig. 3 is the high performance liquid chromatography (HPLC) and flying time mass spectrum analysis spectrogram of probe FBN-3；
Fig. 4 is probe FBN-1 (5 μM) abosrption spectrogram front and rear with nitroreductase reaction；
Fig. 5 is probe FBN-1 (5 μM) launching light spectrogram front and rear with nitroreductase reaction；
Fig. 6 is probe FBN-2 (5 μM) abosrption spectrogram front and rear with nitroreductase reaction；
Fig. 7 is probe FBN-2 (5 μM) launching light spectrogram front and rear with nitroreductase reaction；
Fig. 8 is the dynamic experiment of probe FBN-1 and nitroreductase effect.Wherein excitation wavelength is 490nm；Probe
Concentration：5μM.Nitroreductase concentration is respectively 0,0.01,0.02,0.04,0.07,0.1,0.15,0.2,0.25,0.3,
0.35、0.4、0.45、0.5、0.55μg/mL；
Fig. 9 is the dynamic experiment of probe FBN-2 and nitroreductase effect.Wherein excitation wavelength is 490nm；Probe
Concentration：5μM.Nitroreductase concentration is respectively 0,0.01,0.03,0.06,0.10,0.15,0.2,0.25,0.3,0.4,0.5 μ
g/mL；
Figure 10 is the fluorescence emission spectrum that probe FBN-1 reacts with various interference species and nitroreductase；
Figure 11 be probe FBN-3 20%, with human lung carcinoma cell under 15%, 8%, 0.1% Oxygen Condition, HOC is thin
Born of the same parents and human liver cancer cell are incubated the fluorescence co-focusing figure of 8 hours altogether；
Figure 12 be probe FBN-3 in liver cancer bearing mouse model, to the Time Dependent fluorescence of diameter 6mm and 14mm tumour
Imaging.
Specific embodiment
For a better understanding of the present invention, the content that the present invention is furture elucidated with reference to the accompanying drawings and examples, but this
The content of invention is not limited solely to the following examples.
Agents useful for same of the present invention：
Instrument of the present invention：
| Instrument | Producer |
| High performance liquid chromatograph | GE,AKTA |
| XRF | Varian Cary Eclipse |
| Matrix-assisted laser desorption ionization instrument | AB SCIE,4800Plus |
| Living imaging instrument | PerkinElmer |
Embodiment 1.
The synthesis technique of FBN-1 fluorescence probes is：
Weigh 0.40mmol compounds (1), 0.60mmol compounds (2), 1.20mmol, K2CO3In 50mL there-necked flasks,
12mL DMF dissolvings are added, Ar is protected, 40 DEG C are stirred 5 hours.Vacuum distillation, removes solvent, and crude product is purified by silicagel column,
Obtain product FBN-1. yields 81.8%.1H NMR(400MHz,CDCl3) δ 8.29 (d, J=8.7Hz, 2H), 8.03 (s, 1H),
8.00 (d, J=7.9Hz, 1H), 7.64 (d, J=8.7Hz, 2H), 7.25 (d, J=7.9Hz, 1H), 7.03 (d, J=2.4Hz,
1H), 6.94 (d, J=8.9Hz, 1H), 6.88 (d, J=9.7Hz, 1H), 6.84 (dd, J=8.9,2.4Hz, 1H), 6.56 (dd,
J=9.7,1.9Hz, 1H), 6.45 (d, J=1.8Hz, 1H), 5.30 (s, 2H), 2.13 (s, 3H), 1.65 (s, 9H)；13C NMR
(101MHz,CDCl3)δ185.76,165.13,162.63,158.59,154.41,147.95,147.89,142.77,
136.58,136.54,133.30,131.53,130.56,130.18,129.30,129.22,127.76,127.22,124.05,
118.50,114.48,113.83,106.13,101.61,81.71,69.29,28.20,19.61；FT-IR:ν[cm-1]3425,
3045,2978,2922,1712,1643,1600,1513,1442,1347,1300,1260,1205,1165,1100,1030,
905,850,764,725,600,495；
Embodiment 2.
The synthesis technique of FBN-3 fluorescence probes is：
Weigh 0.20mmol RGD cyclic peptide, 0.20mmol FBN-1,0.6mmol DIEA, 0.6mmol EDCHCl in
In 50mL there-necked flasks, 25mL DMF dissolvings, Ar protections is added to be stirred at room temperature 10 hours.Vacuum distillation, removes solvent, crude product
96% trifluoroacetic acid is added, after being stirred at room temperature 3 hours, vacuum distillation removes solvent, obtains yellow, viscous crude product, efficient liquid
Phase chromatographic purification, is purified by silicagel column, obtains product FBN-1. yields 50.1%.HPLC purity 99%, MALDI-TOF
(m/z):Theoretical value 1082.4134, experiment value [M+H]+1083.55。
Embodiment 3.
Probe FBN-1 changes with the front and rear absorption spectrum of nitroreductase reaction with fluorescence emission spectrum
1.9mg reagent FBN-1 are weighed, the 2mM dimethyl sulfoxide (DMSO) mother liquors of 2ml are made into.The above-mentioned mother liquor of 2.5 μ l is added drop-wise to
It is a certain amount of containing 500 μM of 10mM PBSs of NADH (NADH) in, be subsequently adding 60 μ
The nitroreductase solution of the 100 μ g/mL of L, then with the PBS constant volume of 10mM to 1ml.25min is reacted at 37 DEG C
Afterwards, its absorption spectrum and fluorescence emission spectrum are measured.Excitation wavelength is 490nm when fluorescence emission spectrum is determined；Excite and launch
Slit width be 5nm；Voltage 500V.
Fig. 4 and Fig. 5 results show that the reagent FBN-1 in the present invention has the characteristics that：
1) probe is in the solution faint yellow and unstressed configuration, and absorption spectrum has two absorptions at 456nm and 479nm
Band, fluorescence spectrum has weaker fluorescence at 515nm.
2) with the addition of nitroreductase, original intensity of absorption bands is died down in absorption spectrum, and one is generated at 493nm
New absorption band；The fluorescence spectrum of the probe produces green fluorescence at 515nm, and fluorescence intensity enhances about 11 times.
Embodiment 4.
Probe FBN-2 changes with the front and rear absorption spectrum of nitroreductase reaction with fluorescence emission spectrum
2.8mg reagent FBN-1 are weighed, the 2mM dimethyl sulfoxide (DMSO) mother liquors of 2ml are made into.The above-mentioned mother liquor of 2.5 μ l is added drop-wise to
It is a certain amount of containing 500 μM of 10mM PBSs of NADH (NADH) in, be subsequently adding 60 μ
The nitroreductase solution of the 100 μ g/mL of L, then with the PBS constant volume of 10mM to 1ml.25min is reacted at 37 DEG C
Afterwards, its absorption spectrum and fluorescence emission spectrum are measured.Excitation wavelength is 490nm when fluorescence emission spectrum is determined；Excite and launch
Slit width be 5nm；Voltage 600V.
Fig. 6 and Fig. 7 results show that the reagent FBN-1 in the present invention has the characteristics that：
1) probe is in the solution faint yellow and unstressed configuration, and absorption spectrum has two absorptions at 460nm and 485nm
Band, fluorescence spectrum is almost without Fluorescence Fluorescence.
2) with the addition of nitroreductase, original intensity of absorption bands is died down in absorption spectrum, and one is generated at 497nm
New absorption band；The fluorescence spectrum of the probe produces green fluorescence at 515nm, and fluorescence intensity enhances about 255 times.
Embodiment 5.
The fluorescent spectroscopic properties that FBN-1 reacts with various concentrations nitroreductase
By the above-mentioned mother liquor of 2.5 μ L be added drop-wise to it is a certain amount of containing 500 μM of 10mM PBSs of NADH in,
It is subsequently adding the nitroreductase solution of various concentrations, then with the PBS constant volume of 10mM to 1ml.Reacted at 37 DEG C
After 20min, its absorption spectrum and fluorescence emission spectrum are measured.Excitation wavelength is 490nm when fluorescence emission spectrum is determined；Excite and
The slit width of transmitting is 5nm；Voltage 500V.
Fig. 8 results show that the reagent FBN-1 in the present invention has the characteristics that：
1) probe is in the solution faint yellow and unstressed configuration, but with the addition of nitroreductase, the probe is in 515nm
Place produces green fluorescence；
2) at 515nm, green fluorescence intensity increases with the increase of nitroreductase concentration；
3) when using 5 μM of FBN-1, Fluorescence Increasing is linear in the range of 0-100ng/mL with the concentration of nitroreductase
Relation.
Embodiment 6.
The fluorescent spectroscopic properties that FBN-2 reacts with various concentrations nitroreductase
By the above-mentioned mother liquor of 2.5 μ L be added drop-wise to it is a certain amount of containing 500 μM of 10mM PBSs of NADH in,
It is subsequently adding the nitroreductase solution of various concentrations, then with the PBS constant volume of 10mM to 1ml.Reacted at 37 DEG C
After 20min, its absorption spectrum and fluorescence emission spectrum are measured.Excitation wavelength is 490nm when fluorescence emission spectrum is determined；Excite and
The slit width of transmitting is 5nm；Voltage 600V.
Fig. 9 results show that the reagent FBN-1 in the present invention has the characteristics that：
1) probe is in the solution faint yellow and unstressed configuration, but with the addition of nitroreductase, the probe is in 515nm
Place produces green fluorescence；
2) at 515nm, green fluorescence intensity increases with the increase of nitroreductase concentration；
3) when using 5 μM of FBN-1, Fluorescence Increasing is linear in the range of 0-150ng/mL with the concentration of nitroreductase
Relation.
Embodiment 7.
Reagent FBN-1 and other species response situations (selection Journal of Sex Research) in organism
Various materials are separately added into 5 μM of probe FBN-1 solution：Calcium chloride (2.5mM), sodium chloride (2.5mM), chlorine
Change potassium (50mM) glucose (10mM), vitamin C (1mM), vitamin B6 (1mM), human serum albumins (100 μM), hydrogen peroxide
(10 μM), hydroxyl radical free radical (10 μM), glutamic acid (1mM), arginine (1mM), tyrosine (1mM), glutamic acid (1mM), silk ammonia
Sour (1mM), cysteine (1mM), dithiothreitol (DTT) (1mM), glutathione reductase (5mM), bovine serum albumin (100mg/
ML, potassium superoxide (1mM), hydrogen peroxide (1mM) sodium hypochlorite (1mM) and nitroreductase (0.6 μ g/mL).Reacted at 37 DEG C
After 20min, its fluorescence emission spectrum is measured.It is 490nm that fluorescence emission spectrum determines excitation wavelength；The slit for exciting and launching is wide
It is 5nm to spend；Voltage is 500V.
Figure 10 results show that only nitroreductase can cause probe FBN-1 to produce obvious optical signal to respond, it was demonstrated that should
Reagent has the selectivity of height to nitroreductase, and the measure of nitroreductase is not disturbed in the presence of other species.
Embodiment 8.
Detection of the FBN-3 reagents to hypoxic tumor cell degree
It is 3 × 10 by density5The cancer cell inoculation of individual/mL is covered with four 35mm culture dishes of cover glass to sterilizing,
In CO2(temperature is 37 DEG C, 5%CO to incubator2) in overnight incubation, after after cell attachment, 10 μM of FBN-3 preincubate 30min, so
Four ware cells are respectively in four kinds of conditions (37 DEG C, 5%CO afterwards2, 20%O2；37 DEG C, 5%CO2, 15%O2；) middle culture；37 DEG C,
5%CO2, 8%O2；) middle culture；37 DEG C, 5%CO2, 0.1%O2；) in culture) in culture 8 hours, discard cell culture fluid,
After PBS washed cell 5 times, it is imaged under confocal microscope, compares the fluorescence intensity of four ware cells, Tu11Jie
Fruit shows in 37 DEG C, 5%CO2, 0.1%O2Under the conditions of cultivate cell fluorescence intensity it is most strong.
Embodiment 9
It is scarce with tissue come indirect determination tumour cell by the fluorescence intensity of reduzate in quantitative determination same time
Oxygen degree.Respectively with human lung carcinoma cell (A549), Proliferation of Human Ovarian Cell (SKOV-3) and human liver cancer cell (HepG-2) are used as swollen
Oncocyte model, live body hepatic carcinoma bearing mouse model probes into II types reagent detection cancer cell anoxic as living body biological model
The ability of degree.
Specific assay method in tumour cell is：
1) inoculating cell in four copolymerization Jiao's culture dishes, overnight, makes its adherent；
2) II types reagent pre-incubation cell 30min；
3) four ware cells are put into the different environment of degree of oxygen deficiency respectively and cultivate 8h；
4) fluorescence intensity of the ware cell of fluorescence microscope four is strong and weak.
Specific assay method in live body tumor-bearing mice is：
1) II types reagent dissolves in PBS solution or normal saline solution containing DMSO 0%-5%, is configured to working solution, tail
In intravenous injection into mice body；
2) II types reagent is through blood circulation, can specific accumulation be targeted to tumor locus；
3) because tumor hypoxia degree is different, the fluorescence intensity of tumor locus is also different, is examined using living body fluorescent imaging technique
Survey the fluorescence intensity of knub position；
4) positive correlation of the strong and weak and tumor hypoxia degree according to fluorescence intensity, the fluorescence intensity of different tumours it is strong
The size of weak its degree of oxygen deficiency of secondary indication.
Embodiment 10
Targeted imaging of the FBN-3 reagents in liver cancer bearing mouse model to tumour and its to different size tumor hypoxia journey
The detection of degree
Human lung cancer mouse model is initially set up, mass propgation human lung cancer HepG-2 cells collect the cell of exponential phase,
Removal nutrient solution, is washed twice with PBS, and cell density is adjusted into 1 × 107/ ml, 0.2ml is injected in the right side oxter of nude mice
About 2 × 106Cell, then proceedes to raise, daily the growing state of observation nude mice, and has seen whether that solid tumor is generated.Confirm
After mouse-borne tumor, after diameter of tumor respectively reaches 6mm and 14mm, tail vein injection FBN-3 probes.Respectively after probe is injected
1min, 5min, 10min, 15min, 20min, 25min carry out fluorescence imaging to mouse, and mouse tumour fluorescence intensity is compared in observation,
Result is shown in Figure 12.Gradually strengthen as data can be seen that tumor tissues fluorescence intensity from fluorescence, the tumour of diameter 14mm is finally glimmering
Fluorescence intensity of the luminous intensity more than diameter 6mm tumours.Result illustrates that probe FBN-3 can be in living body biological aspect as operation
Navigation reagent, and the degree of oxygen deficiency of tumor tissues can be characterized.
Reagent of the present invention is as follows as the technical scheme that surgical operation fluorescence navigation reagent is implemented in tumor-bearing mice：
1) II types reagent dissolves in PBS solution or normal saline solution containing DMSO 0%-5%, is configured to working solution, tail
In intravenous injection or local injection to Mice Body；
2) II types reagent is through blood circulation, can specific accumulation be targeted to tumor locus；
3) II types reagent is weaker in nonneoplastic tissue region unstressed configuration signal or fluorescence signal intensity, at tumor tissues position
Fluorescence signal intensity is very strong.
4) living body fluorescent imaging technique is utilized, according to fluorescence signal, accurately image knub position is led for surgical operation is provided
Boat.
Claims (10)
1. the specificity fluorescent probe of nitroreductase, it is characterised in that the probe is by detection moiety sensor, fluorophor
Dye, cancer target group target are formed by connecting by chemical bond；Its structure is：Structure I type sensor-dye or structure I I types
sensor-dye-target。
2. the specificity fluorescent probe of nitroreductase according to claim 1, it is characterised in that
The detection moiety sensor structures are：
The fluorophor dye can for fluoresceins like thing, its structure is：
The structure of the cancer target group target is：
3. the specificity fluorescent probe of nitroreductase according to claim 1, it is characterised in that the structure I type
The structure of sensor-dye is shown in following formula FBN-1 or FBN-2：
The structure of the structure I I types sensor-dye-target is shown in following formula FBN-3 or FBN-4：
4. according to claim any one of 1-3 nitroreductase specificity fluorescent probe, it is characterised in that the probe
Without fluorescence, by nitroreductase specific reduction, the fluorescence-causing substance after reduction has very high glimmering the nitro in probe for itself
Quantum yield, can realize quick, Sensitive Detection that Substrate fluorescence changes using fluorescence detector.
5. according to claim any one of 1-3 nitroreductase specificity fluorescent probe, it is characterised in that the probe
As the specific substrate of nitroreductase, by substrate elimination factor or the production rate of its product in the quantitative determination unit interval
To quantitative determine the activity of nitroreductase in different living things systems.
6. according to claim any one of 1-3 the specificity fluorescent probe of nitroreductase preparation method, its feature exists
In：FBN-1 is by the basic conditions being prepared fluorescein hydroxy alkylated with to nitrobenzyl bromine；FBN-2 by FBN-1 also
After original, then it is alkylated with to nitrobenzyl bromine；FBN-3 and FBN-4 are respectively by FBN-1, the condensation of FBN-2 and RGD cyclic peptide
Into.
7. a kind of cancer target fluorescence imaging and monitoring metastases reagent, it is characterised in that the reagent containing has the right will
Seek the specificity fluorescent probe of nitroreductase described in 1.
8. the specificity fluorescent probe of nitroreductase described in claim 1 is being prepared for cancer target fluorescence imaging and monitoring
Application on the reagent of metastases, can be used as the reagent of surgical navigational.
9. a kind of reagent for monitoring tumor hypoxia degree, it is characterised in that the reagent contains described in claim 1 nitro reduction
The specificity fluorescent probe of enzyme.
10. the specificity fluorescent probe of nitroreductase described in claim 1 is preparing the examination for monitoring tumor hypoxia degree
Application in agent.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107266407A (en) * | 2017-06-08 | 2017-10-20 | 浙江工业大学 | A kind of nitroreductase that responds kills photaesthesia targeting anti-tumor prodrug of tumour cell and preparation method and application |
| CN107954994A (en) * | 2017-12-14 | 2018-04-24 | 大连理工大学 | Long-life phosphors element derivative with the targeting of weary oxygen, its synthesis and biologic applications |
| CN109942608A (en) * | 2019-02-19 | 2019-06-28 | 中国药科大学 | A kind of more boron phenylalanine class compounds and its preparation method and application containing nitroimidazole |
| CN110845556A (en) * | 2019-11-27 | 2020-02-28 | 济南大学 | Target tumor β -galactosidase near-infrared fluorescent probe and preparation method thereof |
| WO2021120653A1 (en) * | 2019-12-16 | 2021-06-24 | 大连理工大学 | Cofactor-substrate probe platform for fast quantitative detection of tumor hypoxia related enzyme |
| CN113563279A (en) * | 2021-08-25 | 2021-10-29 | 安徽大学 | Two-photon fluorescent probe for detecting nitroreductase and preparation method and application thereof |
| NL2032012B1 (en) * | 2022-05-28 | 2023-02-24 | Shandong First Medical Univ & Shandong Academy Of Medical Sciences | Rhodamine - based near - infrared fluorescent probe as well as preparation method and application thereof |
| CN116925025A (en) * | 2023-08-09 | 2023-10-24 | 陕西理工大学 | Near infrared fluorescent probe for rapidly detecting nitroreductase and preparation and application thereof |
| CN116925025B (en) * | 2023-08-09 | 2024-05-14 | 陕西理工大学 | Near infrared fluorescent probe for rapidly detecting nitroreductase and preparation and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006019105A1 (en) * | 2004-08-17 | 2006-02-23 | Daiichi Pure Chemicals Co., Ltd. | Fluorescent labeling agents |
| WO2006100417A1 (en) * | 2005-03-22 | 2006-09-28 | Ge Healthcare Uk Limited | Enzyme detection method and reagent |
| US20150191768A1 (en) * | 2010-11-16 | 2015-07-09 | Enzo Life Sciences, Inc. C/O Enzo Biochem, Inc. | Self-immolative probes for enzyme activity detection |
| CN104861039A (en) * | 2015-05-14 | 2015-08-26 | 山东大学 | Phthalocyanine compound, preparation method and application as single/two-photon fluorescent probe in cancer targeting and mitochondria labeling |
| CN105732564A (en) * | 2016-01-26 | 2016-07-06 | 济南大学 | Two-photon fluorescence probe and application thereof in detecting anoxic-zone nitroreductase |
-
2016
- 2016-12-19 CN CN201611177781.7A patent/CN106749153B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006019105A1 (en) * | 2004-08-17 | 2006-02-23 | Daiichi Pure Chemicals Co., Ltd. | Fluorescent labeling agents |
| WO2006100417A1 (en) * | 2005-03-22 | 2006-09-28 | Ge Healthcare Uk Limited | Enzyme detection method and reagent |
| US20150191768A1 (en) * | 2010-11-16 | 2015-07-09 | Enzo Life Sciences, Inc. C/O Enzo Biochem, Inc. | Self-immolative probes for enzyme activity detection |
| CN104861039A (en) * | 2015-05-14 | 2015-08-26 | 山东大学 | Phthalocyanine compound, preparation method and application as single/two-photon fluorescent probe in cancer targeting and mitochondria labeling |
| CN105732564A (en) * | 2016-01-26 | 2016-07-06 | 济南大学 | Two-photon fluorescence probe and application thereof in detecting anoxic-zone nitroreductase |
Non-Patent Citations (2)
| Title |
|---|
| ZHAO LI等: "in vivo imaging and detection of nitroreductase in zebrafish by a new near-infrared fluorescence off-on probe", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 万琼琼等: "硝基还原酶荧光探针的研究进展", 《分析科学学报》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107266407A (en) * | 2017-06-08 | 2017-10-20 | 浙江工业大学 | A kind of nitroreductase that responds kills photaesthesia targeting anti-tumor prodrug of tumour cell and preparation method and application |
| CN107266407B (en) * | 2017-06-08 | 2020-08-21 | 浙江工业大学 | Photosensitive targeted anti-tumor prodrug capable of killing tumor cells in response to nitroreductase and preparation method and application thereof |
| CN107954994A (en) * | 2017-12-14 | 2018-04-24 | 大连理工大学 | Long-life phosphors element derivative with the targeting of weary oxygen, its synthesis and biologic applications |
| CN109942608A (en) * | 2019-02-19 | 2019-06-28 | 中国药科大学 | A kind of more boron phenylalanine class compounds and its preparation method and application containing nitroimidazole |
| CN110845556A (en) * | 2019-11-27 | 2020-02-28 | 济南大学 | Target tumor β -galactosidase near-infrared fluorescent probe and preparation method thereof |
| WO2021120653A1 (en) * | 2019-12-16 | 2021-06-24 | 大连理工大学 | Cofactor-substrate probe platform for fast quantitative detection of tumor hypoxia related enzyme |
| CN113563279A (en) * | 2021-08-25 | 2021-10-29 | 安徽大学 | Two-photon fluorescent probe for detecting nitroreductase and preparation method and application thereof |
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| CN116925025A (en) * | 2023-08-09 | 2023-10-24 | 陕西理工大学 | Near infrared fluorescent probe for rapidly detecting nitroreductase and preparation and application thereof |
| CN116925025B (en) * | 2023-08-09 | 2024-05-14 | 陕西理工大学 | Near infrared fluorescent probe for rapidly detecting nitroreductase and preparation and application thereof |
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