CN104592984A - Nitroreductase specific fluorescent probe and application thereof - Google Patents
Nitroreductase specific fluorescent probe and application thereof Download PDFInfo
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- CN104592984A CN104592984A CN201410836056.0A CN201410836056A CN104592984A CN 104592984 A CN104592984 A CN 104592984A CN 201410836056 A CN201410836056 A CN 201410836056A CN 104592984 A CN104592984 A CN 104592984A
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Abstract
The invention relates to a nitroreductase specific fluorescent probe and application thereof. The specific probe substrate is 5-nitroacenaphthenequinone and can be used for determining enzyme activity of nitroreductase in a biological system. The process for determining enzyme activity of nitroreductase comprises the following steps: selecting nitro reduction reaction as probe reaction, and determining the activity of nitroreductase in a biological sample, cell, carrier or integral organ by quantitatively detecting the reduction product generation amount in unit time. The probe reaction can also be used for vitro fast screening of the nitroreductase inhibitor and evaluation on inhibiting capacity. The invention provides a 5-nitroacenaphthenequinone nitroreductase fluorescent probe and application thereof. The 5-nitroacenaphthenequinone can be metabolized by the nitroreductase to generate the metabolite with two-photon fluorescence property. The enzymatic reaction has the characteristics of high selectivity and the like, and can easily detect the metabolite and quickly and efficiently evaluate the enzyme activity and inhibiting activity.
Description
Technical field
The invention belongs to medical art, be specifically related to specificity fluorescent probe and the application thereof of nitroreductase.
Background technology
Nitroreduction enzyme family belongs to flavo-enzyme, usually containing vitamin B2 phosphate unit or flavin adenine dinucleotide unit, can metabolism aromatic nitro compound or nitro replace under coenzyme Triphosphopyridine nucleotide, reduced exists heterogeneous ring compound, two classes can be divided into: a class is oxygen non-sensitive type according to and the different nitroreductase of transfer transport mode whether responsive to oxygen, this kind of nitroreductase to be existed at oxygen by bielectron tranfer system or all nitro-compound can be reduced to nitroso-group and azanol intermediate and final aminocompound by the effect of NADPH under anoxia condition, another kind of is oxygen sensitive type, this class nitroreductase only has under anaerobic could nitro compound reducing, this is because the Free radicals obtained by one-electron reduction nitro-compound in the presence of oxygen can directly and oxygen to react generation superoxide radical, regenerate the nitro-compound started most.
Nitroreductase is present in bacterium usually as in intestinal bacteria.Research shows that the relative standard state of content of nitroreductase in oxygen-starved tissue or cell and tumour presents rising trend.Under anoxic conditions can catalysis multiple external source nitro-aromatic compound generation single electron transfer using nicotinamide adenine dinucleotide reduced NADH as the intracellular nitroreductase of electron donor, generate Free radicals, be reduced into azanol or amino further subsequently.Therefore, nitroreductase can as the important indicator of measure of cell or organism anoxic condition.On the other hand, nitroreductase also plays an important role in the activation of medicine and the biological degradation of nitro-aromatic compound, and has good application prospect in biotechnology oncotherapy inhibitor field.Therefore, the nitroreductase measured in living things system is extremely important to understanding its biological function further.
Summary of the invention
The object of the present invention is to provide a kind of specificity fluorescent probe and application thereof of nitroreductase, the fluorescence properties of this substrate prototype and hydrolysate has notable difference, and product more easily detects.Utilize this probe reaction can carry out quantitative evaluation to the distribution of nitroreductase in multiple living things system and function.
The invention provides a kind of specificity fluorescent probe of nitroreductase, the nitro of this substrate can be corresponding aminocompound by nitroreduction enzymes metabolism, the emission spectrum that product display is different from substrate; This substrate is raw material with acenaphthenequinone, and obtained by nitration reaction, its general structure is as shown in (1).
The invention provides the application of the specificity fluorescent probe of nitroreductase, this substrate is as the specific substrate of nitroreductase, there is reduction reaction, measure by the growing amount of reduzate in the detection by quantitative unit time activity that enzyme or cell prepare nitroreductase in the biological samples such as liquid and cell.Concrete measuring method is:
---using 5-nitro acenaphthenequinone as Specific probe in system; Concentration of substrate selects 1/10 ~ 10K
m; Concentration of substrate preferred K during single point assay
m.
---in the conventional damping fluids such as PBS or Tris-HCl, temperature of reaction is between 20 DEG C to 60 DEG C, and preferably 37 DEG C is the peak optimization reaction time; Incubation system pH is between 5.5 ~ 10.5, and preferred pH 7.4 is peak optimization reaction pH value;
---the reaction times is 5 ~ 120 minutes, the termination reaction when guaranteeing that the corresponding hydrolysate of above substrate reaches quantitative limit and substrate conversion efficiency is no more than 20%;
---in the analytical unit time, hydrolysate growing amount is as the evaluation index of Nitroreductase Activity.
The application of nitroreductase specificity fluorescent probe substrate provided by the invention, the production rate of described substrate elimination factor or hydrolysate should between 0.1% ~ 20%.
The application of nitroreductase specificity fluorescent probe substrate provided by the invention, probe substrate and hydrolysate thereof all have fluorescence properties, and the rapid sensitive that fluorimetric detector can be adopted to realize product and substrate detects; Fluoroscopic examination condition is: excitation wavelength 430nm, carries out the detection of fluorescence emission spectrum at 460 ~ 660nm.In addition, this Specific probe and corresponding Nitroreductase Activity testing process can not be subject to the interference of living things system matrix and impurity, can be used for the quantitative assay of Nitroreductase Activity in various living things system.
The reaction of this specific probe can be used for the quantitative assay that Nitroreductase Activity in liquid and various is prepared by restructuring Procaine esterase, people and animal tissues, also can be used for the inhibitor of rapid screening nitroreductase and the quantitative evaluation of rejection ability.
As the fluorescent probe substrate of the nitroreductase of high specific, this compound can be used for detecting the activity of nitroreductase, be especially suitable for the enzyme assay to the nitroreductase recombinase that bacterium, insect cell, mammalian cell and yeast clonal expression system produce, and in the prepared product such as tissue microsomal, S-9 of multiple mammalian tissues organ origin, the activity of nitroreductase is demarcated.
The invention provides the application of a kind of 5-nitro acenaphthenequinone as nitroreductase fluorescent probe substrate, it can generate the meta-bolites with two-photon fluorescence attribute after nitroreduction enzymes metabolism.This enzymatic reaction has that selectivity is high, meta-bolites easily detects, enzymic activity and the inhibit activities evaluation feature such as rapidly and efficiently.
Select the Specific probe of nitroreductase of the present invention to detect nitroreductase external activity and there is following outstanding advantage:
(1) high specific: 5-nitro acenaphthenequinone can be metabolized to a meta-bolites with high specificity by nitroreductase, namely nitroreduction becomes amino product.
(2) cheap and easy to get: 5-nitro acenaphthenequinone and meta-bolites thereof all can obtain through chemosynthesis, and synthesis technique is simple.
(3) highly sensitive: 5-nitro acenaphthenequinone is metabolized to the amino acenaphthenequinone of corresponding 5-by nitroreductase, there is good fluorescence emission spectral property (460 ~ 660nm), this substrate does not have fluorescence, product has good fluorescence properties, differentiation can be carried out preferably detect, carry out quantitative assay by drawing standard curve simultaneously.Product has two-photon fluorescence attribute simultaneously, can reduce biological context fluorescence.
Accompanying drawing explanation
The people of Fig. 1 5-nitro acenaphthenequinone recombinates single enzyme shaker test result;
The fluorogram that Fig. 2 5-nitro acenaphthenequinone increases with nitroreductase protein concentration;
The relation of Fig. 3 5-nitro acenaphthenequinone hydrolysate growing amount and incubation time;
Fig. 4 5-nitro acenaphthenequinone and nitroreduction enzyme kinetics figure;
The general structure of Fig. 5 5-nitro acenaphthenequinone.
Embodiment
The following examples will be further described the present invention, not thereby limiting the invention.
Embodiment 1 nitroreductase selectivity test
(1) 190 μ L nitroreductase metabolic reaction systems are prepared in advance, comprise the Tris-HCl damping fluid (50mM) of pH 7.4, nitroreductase (20 μ g/mL), ammoxidation single enzyme (20 μ g/mL), N-acetyltransferase (20 μ g/mL), sulfonic acid lytic enzyme (20 μ g/mL), Final substrate concentrations is 10 μMs, shakes and incubate 3 minutes in advance under 37 DEG C of conditions;
(2) in reaction system, add the NAD initial action that 10 μ L final concentrations are 5mM (final concentration 250 μMs);
(3) react after 1 hour and carry out fluorescent scanning detection (Ex=430nm), see Fig. 1.
Embodiment 2 nitroreductase concentration curve
(1) 190 μ L nitroreductase metabolic reaction systems are prepared in advance, comprise the Tris-HCl damping fluid (50mM) of pH 7.4, nitroreductase (1-20 μ g/mL), Final substrate concentrations is 10 μMs, shakes and incubate 3 minutes in advance under 37 DEG C of conditions;
(2) in reaction system, add the NAD initial action that 10 μ L final concentrations are 5mM (final concentration 250 μMs);
(3) react after 1 hour and carry out fluorescent scanning detection (Ex=430nm), see Fig. 2.
Embodiment 3 nitroreductase reaction times curve
(1) 190 μ L nitroreductase metabolic reaction systems are prepared in advance, comprise the Tris-HCl damping fluid (50mM) of pH 7.4, nitroreductase (20 μ g/mL), Final substrate concentrations is 10 μMs, shakes and incubate 3 minutes in advance under 37 DEG C of conditions;
(2) in reaction system, add the NAD initial action that 10 μ L final concentrations are 5mM (final concentration 250 μMs);
(3) carry out fluorescent scanning detection (Ex=430nm) every 10 minutes, see Fig. 3.
Embodiment 4 nitroreduction enzyme dynamics
(1) prepare 190 μ L nitroreductase metabolic reaction systems in advance, comprise the Tris-HCl damping fluid (50mM) of pH 7.4, nitroreductase (5 μ g/mL), different concns substrate, shakes and incubates 3 minutes in advance under 37 DEG C of conditions;
(2) in reaction system, add the NAD initial action that 10 μ L final concentrations are 5mM (final concentration 250 μMs);
After (3) 30 minutes, add 100 μ L ice acetonitriles, after concuss, termination reaction;
(4) fluoroscopic examination (Ex=430nm, Em=524nm) is carried out; Matching kinetic curve, calculating the maximum catalytic rate of nitroreductase to this probe compound is 0.47 ± 0.03nmol/min/mg.(see Fig. 4)
The activity of CES2 in embodiment 5 quantitative assay human pulmonary epithelial cells
(1) A549 cell strain is inoculated in 24 orifice plates equably, at aerobic (20%pO
2) cultivate under condition 24 hours adherent.A549 cell is placed in aerobic conditions (20%pO respectively
2) and weary oxygen (5% or l%pO
2) cultivate 8h under condition.Before adding probe, cell PBS damping fluid (pH 7.4) is washed once, adds the 1pM probe culture medium solution prepared in advance and contains FBS, at 37 DEG C, cultivate 2h, 1h, 0.5h.Before taking pictures, PBS damping fluid (pH 7.4) washes three times, and removing does not enter the probe of cell.
The chemical synthesis process of embodiment 6 5-nitro acenaphthenequinone
The nitric acid of 65% of 3.46mL is mixed with the 35mL vitriol oil, and under ice bath, above-mentioned mixed solution instillation has been dissolved in the 300mL vitriol oil of 9.11g acenaphthenequinone, normal-temperature reaction is poured into after three hours in frozen water, filters; Sodium bicarbonate aqueous solution washing with 15% to neutral, and then is washed by massive laundering, with recrystallized from acetonitrile, obtains yellow needles solid.
1H NMR(400MHz,CDCl
3)δ9.18(d,J＝8.7Hz,1H)，8.77(d,J＝7.7Hz,1H)，8.34(d,J＝7.1Hz,1H)，8.25(d,J＝7.7Hz,1H)，8.18(dd,J＝8.7,7.1Hz,1H)。
Claims (7)
1. a specificity fluorescent probe for nitroreductase, is characterized in that: the nitryl group in this probe substrate can by nitroreductase specific metabolic for amino, and its structural formula is such as formula shown in (1), and this substrate is 5-nitro acenaphthenequinone.
2. the application of the specificity fluorescent probe of nitroreductase described in a claim 1, it is characterized in that: adopt above-mentioned formula (1) compound as the specific substrate of nitroreductase, carried out the activity of nitroreductase in the different living things system of quantitative assay by the production rate of the substrate elimination factor in the detection by quantitative unit time or its product.
3. the application of the specificity fluorescent probe of nitroreductase according to claim 2, is characterized in that: described incubated in vitro reaction conditions is: concentration of substrate is between 1/10 ~ 10K
mbetween; Incubation system pH is between 5.5 ~ 10.5; Temperature of reaction is between 20 ~ 60 DEG C.
4. the application of the specificity fluorescent probe of nitroreductase according to claim 2, is characterized in that: the production rate of described substrate elimination factor or product is lower than 20%.
5. the application of the specificity fluorescent probe of nitroreductase according to claim 2, is characterized in that: this probe substrate does not have fluorescence, and product possesses fluorescence, and fluorimetric detector can be adopted to realize quick, the Sensitive Detection of Substrate fluorescence change.
6. the application of the specificity fluorescent probe of nitroreductase according to claim 2, is characterized in that: this probe substrate also can be used for the rapid screening of nitroreduction enzyme inhibitors and the quantitative evaluation of rejection ability.
7. the application of the specificity fluorescent probe of nitroreductase according to claim 2, it is characterized in that: described fluorescent probe meta-bolites can be excited by two-photon laser, this wavelength is as excitation light source, biological sample background fluorescence is faint, is applicable to detecting thiophenol content in cell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107286925A (en) * | 2016-03-31 | 2017-10-24 | 中国科学院大连化学物理研究所 | A kind of fluorescence probe for detecting nitroreductase and its application |
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| JPS4811005B1 (en) * | 1970-08-11 | 1973-04-10 | ||
| JPH0811005B2 (en) * | 1987-01-07 | 1996-02-07 | 井関農機株式会社 | Inverting device for pot type nursery equipment |
| CN103242306A (en) * | 2013-05-13 | 2013-08-14 | 中国科学院化学研究所 | Bacteria nitroreductase detection kit and special-purpose fluorescence probe thereof |
-
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- 2014-12-29 CN CN201410836056.0A patent/CN104592984A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS4811005B1 (en) * | 1970-08-11 | 1973-04-10 | ||
| JPH0811005B2 (en) * | 1987-01-07 | 1996-02-07 | 井関農機株式会社 | Inverting device for pot type nursery equipment |
| CN103242306A (en) * | 2013-05-13 | 2013-08-14 | 中国科学院化学研究所 | Bacteria nitroreductase detection kit and special-purpose fluorescence probe thereof |
Non-Patent Citations (2)
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| WEIPING ZHU ET AL.: "Novel nitroheterocyclic hypoxic markers for solid tumor: Synthesis and biological evaluation", 《BIOORGANIC & MEDICINAL CHEMISTRY》 * |
| 高灵芝等: "新型苊并吡嗪类高灵敏度极性探针", 《中国科技论文》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107286925A (en) * | 2016-03-31 | 2017-10-24 | 中国科学院大连化学物理研究所 | A kind of fluorescence probe for detecting nitroreductase and its application |
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