CN106995424A - It is a kind of to be used to detect enhanced fluorescence probe of carboxy-lesterase 1 and preparation method and application - Google Patents
It is a kind of to be used to detect enhanced fluorescence probe of carboxy-lesterase 1 and preparation method and application Download PDFInfo
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- 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
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- 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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- 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"
- G01N2021/6432—Quenching
Abstract
The invention discloses a kind of enhanced fluorescence probe for being used to detect carboxy-lesterase 1 and preparation method and application, belong to fluorescence probe material technical field.The fluorescence probe is the acetic acid esters of 3 bromomethyl, 2 oxo 2H chromenes 7, and preparation method comprises the following steps：（1）By the acetic acid esters of 3 methyl, 2 oxo 2H chromenes 7, N NBSs and azodiisobutyronitrile are dissolved in carbon tetrachloride, obtain mixed liquor；（2）Mixed liquor is heated to backflow, room temperature is cooled to after reaction, reaction product obtains the acetic acid esters of 3 bromomethyl, 2 oxo 2H chromenes 7 through isolating and purifying.The fluorescence probe can realize the Fluorescence Increasing type detection to CES1, and with effectively directly perceived, it is easy to the advantage of observation, and stability is high, is avoided that the interference of external condition, improves the precision of detection.It can be widely used in the qualitative and quantitative analysis of CES1 in the samples such as environment, chemistry.
Description
Technical field
The invention belongs to fluorescence probe material technical field, and in particular to one kind is used to detect carboxy-lesterase 1 (CES1)
Enhanced fluorescence probe and preparation method and application.
Background technology
Carboxy-lesterase (Carboxylesterase, CES) is a kind of polyprotein, with a variety of esters chemical combination of catalyzing hydrolysis
Alcohol, carboxylic acid and hydrone are discharged after the ability of thing, hydrolysis.The spy of a variety of esters substrates can be catalyzed based on carboxy-lesterase
Property, carboxy-lesterase extensive application in terms of the practical applications such as industrial production, organic synthesis, active agent of medicament.Carboxy-lesterase
It is broadly divided into two hypotypes：Carboxy-lesterase 1 (CES1) and carboxy-lesterase 2 (CES2), both have obvious poor on catalytic performance
It is different：CES1 can preference catalyzing hydrolysis can obtain the carboxyl of small fragment (be free of benzene ring structure) and the hydroxyl of large fragment
Esters substrate, and CES2 can preference catalyzing hydrolysis can obtain the carboxyl and small fragment of large fragment (contain benzene ring structure)
The esters substrate of hydroxy compounds.Therefore applications of the CES1 and CES2 in terms of industrial production, organic synthesis also has
Marked difference.Then a kind of new method for being capable of selective enumeration method CES1 or CES2 is developed as current industrial production, organic
The study hotspot in the fields such as synthesis.
A variety of analysis methods for being used to detect CES existing at present, such as chemoluminescence method, high performance liquid chromatography (HPLC) with
And fluorescence detection etc..
Chemoluminescence method mainly has two kinds of liquid chemiluminescence method and biochemiluminescence method.Liquid chemiluminescence method refers to
Under the catalytic action of biology enzyme, carboxy-lesterase and oxygen generate the matrix of hydrogen peroxide, then are reacted life with alkaline solution
Into the sub- ketone of N- methyl of excitation state, maximum emission wavelength 470nm light is produced, so that indirect determination enzyme content；Biochemistry is sent out
Light method is combined chemiluminescent high sensitivity with enzyme reaction, continuous luminescent radiation is produced, so that indirect determination enzyme content.So
And chemoluminescence method is selectively poor, luminescence system is relatively fewer.
HPLC methods are used to detect carboxy-lesterase, and cushioning liquid is selected first and makees solvent, and sets flow velocity and column temperature.Elution
Afterwards, change linear gradient, then elute a period of time.Subsequent chromatographic column is rinsed with eluent, after flushing is finished, and is continued with beginning bar
Part is eluted.After each composition is separated in post, detected into detector, so that the analysis to sample is realized, but HPLC
Method is very cumbersome for the detection process of carboxy-lesterase, and is easily disturbed.
In recent years, all circles are enjoyed to pay close attention to as the fluorescence detection of new detection means, what fluorescence detection was used sets
Standby simple, response is fast, with excellent sensitivity and the features such as can realize the real-time detection to material.In addition, for glimmering
The structure of the compound of light detection is mostly fairly simple, it is easy to which design meets the demand of a variety of detection samples with improving.To sum up
Described, fluorescence detection is quite suitable for the analysis detection to carboxy-lesterase.But the invention at this stage for detecting CES2 is special
Profit is more, such as Chinese patent (application number：201310587411.0, application number CN201410513296.7, application number
CN201410836704.2, CN201510224354.9 etc.).Detect that CES1 report is relatively fewer, document report has synthesized one
Detection CES1 of the kind based on luciferin fluorescence probe DME (Chem.Commun., 2016,52,3183-3186), but worm
The expensive of fluorescein, synthesis are complicated, and probe steady is poor, it is necessary to preserved in a low temperature of subzero 20 degrees Celsius, because
This is not suitable for prolonged preservation, is unfavorable for practical application.Chinese patent (application number CN201610119590.9) is prepared for one
(S) -4,5- dihydros -2- (benzothiazole -2- bases) 4-thiazolecarboxylic acid ester derivant (abbreviation DBT-R) is planted, as CES1's
Detection probe；Probe is hydrolyzed into substrate (S) -4,5- dihydros -2- (benzothiazole -2- bases) thiophene of luciferase after being reacted with CES1
Azoles -4- carboxylic acid compounds (abbreviation DBTC), produce bioluminescence with luciferase reaction using DBTC and are detected.But it is such a
The preparation method of probe is cumbersome, need to can just show result by 2 secondary responses；Luciferase is easily inactivated, and testing result easily goes out
Now than larger deviation；PH buffer solutions value is limited to 6.5 used in follow-up test, does not probe into the knot of wider array of acid-base value scope
Really, using limited；The Cymag of severe toxicity has been used in preparation process, has been unfavorable for large-scale industrial production.In summary, at present
It is badly in need of a kind of degree of accuracy height of development, strong interference immunity, prepares the carboxy-lesterase 1 that simple and toxicity is low, have a wide range of application in this area
Detection method.
The content of the invention
In place of shortcoming and deficiency in order to solve currently available technology, primary and foremost purpose of the invention is that providing one kind is used for
Detect the enhanced fluorescence probe of carboxy-lesterase 1 (CES1).The fluorescence probe is catalyzed end ester group using cumarin as parent in CES1
Under conditions of generation hydrolysis, the Fluorescence Increasing inspection to CES1 is realized by cyclic voltammetry method effect (ICT effects)
Survey.
Another object of the present invention is to provide a kind of simple method for preparing of above-mentioned enhanced fluorescence probe.
It is still another object of the present invention to provide the practical application of above-mentioned enhanced fluorescence probe.The probe is in carboxylate
Application in the detection of enzyme 1.
The object of the invention is achieved through the following technical solutions.
It is a kind of to be used to detecting the enhanced fluorescence probe of carboxy-lesterase 1, the fluorescence probe be 3- bromomethyl -2- oxos -
2H- chromene -7- acetic acid esters, chemical structural formula is as follows：
Prepare a kind of above-described method for being used to detect the enhanced fluorescence probe of carboxy-lesterase 1, including following step
Suddenly：
(1) by 3- methyl -2- oxo -2H- chromene -7- acetic acid esters, N-bromosuccinimide (NBS) and the isobutyl of azo two
Nitrile (AIBN) is dissolved in carbon tetrachloride, obtains mixed liquor；
(2) mixed liquor is heated to backflow, room temperature is cooled to after reaction, reaction product obtains 3- bromine first through isolating and purifying
Base -2- oxo -2H- chromene -7- acetic acid esters.
It is preferred that, step (1) 3- methyl -2- oxos -2H- chromenes -7- acetic acid esters and N-bromosuccinimide
Mol ratio is 1:(0.9-1).
It is preferred that, the consumption of step (1) described azodiisobutyronitrile is 3- methyl -2- oxo -2H- chromene -7- acetic acid esters
Mole 0.1%.
It is preferred that, the temperature of step (2) described reaction is 80 DEG C -85 DEG C.
It is preferred that, the time of step (2) described reaction is 4-5 hours.
It is preferred that, the step of being isolated and purified described in step (2) is：By reaction product suction filtration and organic phase is collected, then rotated
Evaporating organic solvent, the purifying of gained solid by silica gel chromatography post.
Application of the above-described enhanced fluorescence probe in detection carboxy-lesterase 1.
It is preferred that, the enhanced fluorescence probe carries out qualitative and quantitative analysis to carboxy-lesterase 1.
Relative to prior art, the invention has the advantages that：
(1) fluorescent probe compounds of the invention, by introducing bromine atoms, have adjusted the electronics effect of 3- bit substituents
Should, therefore detection of the probe compound to carboxy-lesterase 1 (CES1) has excellent anti-interference, common ion, protein
It can not be reacted Deng interfering material with the ester group hydrolyses in probe, therefore will not cause the enhancing of fluorescence, so that without interference with
Probe shows that probe molecule has excellent detection specificity for CES1 detection.
(2) fluorescent probe compounds of the invention still maintain good stability in different pH cushioning liquid,
Test effect is not also influenceed by pH, expands application of the probe under different pH values environment.
(3) probe compound in the present invention is by the ester group as recognition group and the fluorogen tonka-bean with photoluminescent property
Element is constituted；Under conditions of in the absence of CES1, the fluorescence of cumarin is quenched in probe compound, and fluorescence signal weakens, and is visited
The electronics effect of bromine atoms on hydrolysis, 3- substituents just can rapidly occur only under conditions of it there is CES1 for pin compound
It should may advantageously facilitate after the reaction of CES1 and ester group, reaction that electrophilic ester group is transformed into the hydroxyl of supplied for electronic in molecular structure,
7- hydroxyl -3- bromomethyl -2H- pyran-2-ones are obtained, has triggered the electro transfer between intramolecular different groups, result in
The generation (ICT effects) of Intramolecular electron transfer effect, has promoted the fluorescence signal of cumarin parent to recover.Therefore this probe energy
The fluoroscopic examination to CES1 is realized, and along with the increase of CES1 concentration, produced blue-fluorescence also gradually strengthens.It is this
The detection pattern of Fluorescence Increasing type has can be effectively directly perceived, it is easy to the advantage of observation, and this probe improves existing probe
Detection precision and accuracy.In certain concentration range, there is good linear relationship in fluorescence intensity with CES1 concentration,
Available for quantitative detection.
(4) to construct a kind of brand-new accuracy high, sensitive for the detection architecture of the enhanced fluorescence probe obtained by the present invention
The high detection CES1 of degree method, it is easy to use, be conducive to its popularization and application.
(5) product yield of the synthesis and preparation process of probe molecule of the present invention is high, and preparation method is simple and with low cost.
Brief description of the drawings
Fig. 1 is the synthetic route chart of enhanced fluorescence probe of the invention.
Fig. 2 is that the nuclear-magnetism of enhanced fluorescence probe 3- bromomethyls -2- oxos -2H- chromene -7- acetic acid esters in embodiment 1 is total to
Shake hydrogen spectrogram.
Fig. 3 is the mass spectrogram of enhanced fluorescence probe 3- bromomethyls -2- oxos -2H- chromene -7- acetic acid esters in embodiment 1.
Fig. 4 be the present invention fluorescence probe in the PBS cushioning liquid that pH is 7.4 in the presence of carboxy-lesterase 1 is whether there is
Fluorescence spectra.
The fluorescence probe that Fig. 5 is the present invention coexists in the PBS cushioning liquid that pH is 7.4 with various concentrations carboxy-lesterase 1
When fluorescence spectra.
Fig. 6 deposits the fluorescence light for being in or be not in carboxy-lesterase 1 for the fluorescence probe of the present invention in different PBS values cushioning liquid
Spectrogram.
Fig. 7 is the fluorescence spectra of the specific test of the fluorescence probe of the present invention.
Fig. 8 detects test chart for the anti-interference of the fluorescence probe of the present invention.
Embodiment
For a better understanding of the present invention, further details of elaboration is made to the present invention with reference to embodiment and accompanying drawing,
But the implementation of the present invention is not limited to this.
The synthetic route chart of the enhanced fluorescence probe of the present invention is as shown in Figure 1.
Embodiment 1：The preparation technology flow 1 of probe compound 3- bromomethyl -2- oxo -2H- chromene -7- acetic acid esters
By 1090mg 3- methyl -2- oxo -2H- chromene -7- acetic acid esters (5mmol), 890mg N-bromosuccinimides
(5mmol) and 0.82mg azodiisobutyronitriles are dissolved in 30mL carbon tetrachloride, and mixed liquor is heated into backflow, controlling reaction temperature
Reacted for 80 DEG C, reaction stops reaction after 4 hours, is cooled to room temperature, suction filtration is carried out to mixed solution, collection has machine filter
Liquid, rotary evaporation removes organic solvent, and (eluent is petrol ether/ethyl acetate, V/V to the purifying of gained solid by silica gel chromatography post
=4:1), obtain white solid 1115mg (yield is 75.1%).The product is characterized by proton nmr spectra, as a result
As shown in Fig. 2 characterize data is as follows：1H NMR(600MHz,CDCl3):δ 7.85 (s, 1H), 7.51 (d, J=8.5Hz, 1H),
(s, the 3H) of 7.14 (d, J=2.1Hz, 1H), 7.08 (dd, J=8.5,2.1Hz, 1H), 4.42 (s, 2H), 2.35 is wherein
After f correspondence bromination reactions at 4.42ppm, 2 H of residue proton peak after a H is replaced by bromine on methyl.A at 2.35ppm
The proton peak of 1 H in e correspondence double bonds at the proton peak of 3 H on the methyl of correspondence connection ester group, 7.85ppm, 7.08-
B at 7.51ppm, c, d are the proton peak of 3 H on phenyl ring.In addition, having carried out secondary proof by high-resolution mass spectrum, as a result
As shown in figure 3, appraising datum is as follows：HR-MS(ESI):calcd for C12H9BrO4([M+H]+)296.9757,found:
296.9756.By nuclear-magnetism and mass spectrographic analysis can determine synthesized product for target product 3- bromomethyl -2- oxos -
2H- chromene -7- acetic acid esters.
Embodiment 2：The preparation technology flow 2 of probe compound 3- bromomethyl -2- oxo -2H- chromene -7- acetic acid esters
By 545mg 3- methyl -2- oxo -2H- chromene -7- acetic acid esters (2.5mmol), 400mg N- bromos succinyl is sub-
Amine (2.25mmol) and 0.41mg azodiisobutyronitriles are dissolved in 15mL carbon tetrachloride, and mixed liquor is heated into backflow, and control is anti-
Temperature is answered to be reacted for 83 DEG C, reaction stops reaction after 4.5 hours, is cooled to room temperature, and suction filtration is carried out to mixed solution, collects
Organic filtrate, rotary evaporation removes organic solvent, and (eluent is petroleum ether/acetic acid second to the purifying of gained solid by silica gel chromatography post
Ester, V/V=4:1) white solid 487mg (yield 72.9%), is obtained.The present embodiment middle probe 3- bromomethyl -2- oxos -2H-
It is identical that chromene -7- acetate compounds, which are characterized with the result in embodiment 1,.
Embodiment 3：The preparation technology flow 3 of probe compound 3- bromomethyl -2- oxo -2H- chromene -7- acetic acid esters
By 1635mg 3- methyl -2- oxo -2H- chromene -7- acetic acid esters (7.5mmol), 1270mg N- bromo succinyls
Imines (7.125mmol) and 1.23mg azodiisobutyronitriles are dissolved in 50mL carbon tetrachloride, and mixed liquor is heated into backflow, control
Reaction temperature is 85 DEG C and reacted that reaction stops reaction after 5 hours, is cooled to room temperature, and suction filtration is carried out to mixed solution, collects
Organic filtrate, rotary evaporation removes organic solvent, and (eluent is petroleum ether/acetic acid second to the purifying of gained solid by silica gel chromatography post
Ester, V/V=4:1) white solid 1508mg (yield 71.3%), is obtained.The present embodiment middle probe 3- bromomethyl -2- oxos -2H-
The sign of chromene -7- acetate compounds is identical with the result in embodiment 1.
The performance test of gained probe compound 3- bromomethyl -2- oxo -2H- chromene -7- acetic acid esters of the invention：
Performance test is carried out to fluorescence probe 3- bromomethyl -2- oxo -2H- chromene -7- acetic acid esters prepared by embodiment 1,
Test result is as shown in Fig. 4-Fig. 8
(1) photoluminescent property of probe compound 3- bromomethyls -2- oxos -2H- chromene -7- acetic acid esters：Use PBS cushioning liquid
(pH=7.4) blank control sample and test specimens are prepared, blank sample middle probe compound concentration is 5 μM, is added without carboxy-lesterase；Survey
Sample middle probe compound concentration is 5 μM, and CES1 concentration is 100U/L.Under wavelength 405nm burst of ultraviolel light irradiation, its is glimmering
Light emission spectrum figure is as shown in Figure 4.Blank sample fluorescence intensity is close to zero；But what the probe molecule in test specimens existed in CES1
Under the conditions of, the hydroxyl that electrophilic ester group in hydrolysis, molecular structure is transformed into supplied for electronic occurs for ester group, obtains 7- hydroxyls
Base -3- bromomethyl -2H- pyran-2-ones, have triggered the electro transfer between intramolecular different groups, result in intramolecular electricity
The generation (ICT effects) of lotus transfer effect, has promoted the fluorescence signal of cumarin parent to recover, therefore hydrolysis of ester group is into after hydroxyl
Probe molecule launches very significant blue-fluorescence in 462nm or so.The probe prepared from result above, the present invention
Compound 3- bromomethyl -2- oxo -2H- chromene -7- acetic acid esters can be used in environmental sample, biological sample and chemical example
CES1 carries out qualitative detection analysis.
(2) it is right in probe compound 3- bromomethyls -2- oxos -2H- chromene -7- acetic acid esters PBS cushioning liquid (pH=7.4)
Various concentrations CES1 fluorescence response test
It is respectively 1,2,5,10,20,30,40,50,80,100,150,200U/L to prepare CES1 concentration, and probe compound is dense
Degree is a series of 5 μM of PBS cushioning liquid (pH=7.4).Burst of ultraviolel of the every group of test specimens in wavelength 405nm is determined respectively
Fluorescent emission spectrogram under light, test result is as shown in Figure 5.With the increase of CES1 concentration, blue-fluorescence intensity gradually strengthens,
Therefore the Fluorescence Increasing type detection to CES1 can be realized, this detection pattern is intuitively effective, can improve the accuracy of monitoring.By the above
As a result visible, probe compound 3- bromomethyl -2- oxo -2H- chromene -7- acetic acid esters prepared by the present invention can be used for environment sample
CES1 carries out quantitative detecting analysis in product, biological sample and chemical example.
(3) stability test of the probe compound in different pH buffer solutions
A series of different test specimens of pH value are prepared, wherein probe compound concentration is 5 μM, and pH value is respectively 3,4,5,
6,7,8,9 PBS cushioning liquid, determines fluorescence of each group in the case of there is CES1 (5 μM of concentration) or in the absence of CES1, paints
The point and line chart of fluorescence intensity at the corresponding 462nm of each group pH value (pH 3-9) processed, as a result as shown in Figure 6.Test result shows,
Probe in the present invention has good stability for pH, and test effect will not also be disturbed by pH, and this expands probe and existed
Application under different pH values environment.
(4) the specific detection test of probe compound 3- bromomethyls -2- oxos -2H- chromene -7- acetic acid esters
Compound concentration is 50mM Hcy, GSH (glutathione), Cys (cysteine), Lys, Phe (phenylpropyl alcohol ammonia respectively
Acid), Arg (arginine), DTT (dithiothreitol (DTT)), Gly (glycine) test specimens PBS cushioning liquid (pH=7.4), concentration is
100mM Na+、K+、Ca2+、Mg2+、Gly、Cys、DTT、H2O2、H3BO3Test specimens PBS cushioning liquid (pH=7.4), concentration is
100U/L CES1 and CES2 PBS cushioning liquid (pH=7.4).The concentration of probe compound is 5 μM, determines the glimmering of each group
Light, draws the block diagram of the fluorescence intensity at the corresponding 462nm of interfering material, as a result (1.Na as shown in Figure 7+, 2.K+, 3.Ca2 +, 4.Mg2+, 5.Gly, 6.Cys, 7.DTT, 8.H2O2, 9.H3BO3, 10.Hcy, 11.GSH, 12.Cys, 13.Lys, 14.Phe,
15.Arg, 16.DTT, 17.Gly, 18.CES2,19.CES1).Test experiments show, except the experimental group that CES1 is present, other
The fluorescence of interfering material experimental group is all very faint, illustrates that detection of the probe compound of the present invention to CES1 has specific
Advantage.
(5) the anti-interference detection test of probe compound 3- bromomethyls -2- oxos -2H- chromene -7- acetic acid esters
The test specimens PBS cushioning liquid (pH=7.4) that other disturbance materials coexist with CES1, wherein Na are prepared respectively+、K+、Ca2+、Mg2+、Gly、Cys、DTT、H2O2、H3BO3Concentration be 100mM, Hcy, GSH, Cys, Lys, Phe, Arg, DTT,
Gly concentration is 50mM, and CES2 concentration is 100U/L.The concentration of each group probe compound is 5 μM, and CES1 concentration is
100U/L.Fluorometric investigation is carried out to each group, fluorescence is strong at corresponding 462nm in the case that drafting disturbance thing coexists with CES1
The block diagram of degree, as a result as shown in Figure 8 (1.Na++ CES1,2.K++ CES1,3.Ca2++ CES1,4.Mg2++ CES1,5.Gly+
CES1,6.Cys+CES1,7.DTT+CES1,8.H2O2+ CES1,9.H3BO3+ CES1,10.Hcy+CES1,11.GSH+CES1,
12.Cys+CES1,13.Lys+CES1,14.Phe+CES1,15.Arg+CES1,16.DTT+CES1,17.Gly+CES1,
18.CES2+CES1,19.CES1).As a result show, the detection of the presence of other materials without interference with probe compound to CES1,
This illustrates that the probe of the present invention has excellent anti-interference, CES1 detection can be realized good accuracy with it is sensitive
Degree.
The present invention is with enhanced fluorescent probe molecule 3- bromomethyls -2- oxo -2H- chromene -7- acetic acid esters to carboxy-lesterase 1
(CES1) detected, recognition group of the probe compound using ester group as CES1, the bromination cumarin with photoluminescent property
Molecule is used as fluorescence signal group.Under conditions of in the absence of CES1, probe compound does not have any fluorescence signal, probe
Hydrolysis can quickly occur under conditions of it there is CES1 for compound, and molecular structure is transformed into supplied for electronic by electrophilic ester group
Hydroxyl, obtain 7- hydroxyl -3- bromomethyl -2H- pyran-2-ones, this process has triggered the electricity between intramolecular different groups
Son transfer, result in the generation (ICT effects) of Intramolecular electron transfer effect, has promoted the fluorescence signal of bromination cumarin parent
Enhancing.Under the irradiation of wavelength 405nm uv excitation light, the probe molecule after hydrolysis of ester group launches bright in wavelength 462nm
Aobvious blue-fluorescence.Therefore the enhanced fluoroscopic examination to CES1 can be realized, this detection pattern have can it is effectively directly perceived,
It is easy to the advantage of observation, and this probe improves the precision and accuracy of the detection of existing probe.Test result shows：The invention
In probe compound good stability can be kept under conditions of different pH, expand probe under different pH values environment
Application；Probe compound has fine specific detection effect to CES1, will not be reacted with other materials, even in
Under conditions of other materials are present, also without interference with detection of the probe for CES1, illustrate that probe of the invention has excellent
Anti-interference property.Material toxicity simultaneously used in this preparation method is low, and yield is high, is conducive to production and application.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
1. a kind of enhanced fluorescence probe for being used to detect carboxy-lesterase 1, it is characterised in that the fluorescence probe is 3- bromine first
Base -2- oxo -2H- chromene -7- acetic acid esters, chemical structural formula is as follows：
2. preparing a kind of method for being used to detect the enhanced fluorescence probe of carboxy-lesterase 1 described in claim 1, its feature exists
In comprising the following steps：
(1) by 3- methyl -2- oxo -2H- chromene -7- acetic acid esters, N-bromosuccinimide and azodiisobutyronitrile are dissolved in four
In chlorination carbon, mixed liquor is obtained；
(2) mixed liquor is heated to backflow, room temperature is cooled to after reaction, reaction product obtains 3- bromomethyls -2- through isolating and purifying
Oxo -2H- chromene -7- acetic acid esters.
3. a kind of preparation method for being used to detect the enhanced fluorescence probe of carboxy-lesterase 1 according to claim 2, it is special
Levy and be, the mol ratio of step (1) 3- methyl -2- oxos -2H- chromenes -7- acetic acid esters and N-bromosuccinimide is
1:(0.9-1)。
4. a kind of preparation method for being used to detect the enhanced fluorescence probe of carboxy-lesterase 1 according to claim 2, it is special
Levy and be, the consumption of step (1) described azodiisobutyronitrile is 3- methyl -2- oxo -2H- chromene -7- acetic acid esters moles
0.1%.
5. a kind of preparation method for being used to detect the enhanced fluorescence probe of carboxy-lesterase 1 according to claim 2, it is special
Levy and be, the temperature of step (2) described reaction is 80 DEG C -85 DEG C.
6. a kind of preparation method for being used to detect the enhanced fluorescence probe of carboxy-lesterase 1 according to claim 2, it is special
Levy and be, the time of step (2) described reaction is 4-5 hours.
7. a kind of preparation method for being used to detect the enhanced fluorescence probe of carboxy-lesterase 1 according to claim 2, it is special
Levy and be, the step of being isolated and purified described in step (2) is：By reaction product suction filtration and organic phase is collected, then rotary evaporation is removed
Organic solvent, the purifying of gained solid by silica gel chromatography post.
8. application of the enhanced fluorescence probe in detection carboxy-lesterase 1 described in claim 1.
9. application according to claim 8, it is characterised in that the enhanced fluorescence probe is determined carboxy-lesterase 1
Property and quantitative analysis.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108164450A (en) * | 2018-02-08 | 2018-06-15 | 赣南师范大学 | A kind of carboxy-lesterase fluorescence probe and its preparation method and application |
| CN112812141A (en) * | 2019-11-18 | 2021-05-18 | 华东理工大学 | Preparation method of 3-fluoromethyl-substituted coumarin compound and fluorescent probe |
| CN115819374A (en) * | 2022-11-18 | 2023-03-21 | 遵义医科大学 | Near-infrared carboxylesterase small-molecule fluorescent probe and preparation method and application thereof |
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| US6133460A (en) * | 1997-11-20 | 2000-10-17 | Cerus Corporation | Psoralens for pathogen inactivation |
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| US6133460A (en) * | 1997-11-20 | 2000-10-17 | Cerus Corporation | Psoralens for pathogen inactivation |
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| IAN CUMMINS,ET AL: "Identification of a carboxylesterase expressed in protoplasts using fluorescence-activated cell sorting", 《PLANT BIOTECHNOLOGY JOURNAL》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108164450A (en) * | 2018-02-08 | 2018-06-15 | 赣南师范大学 | A kind of carboxy-lesterase fluorescence probe and its preparation method and application |
| CN108164450B (en) * | 2018-02-08 | 2019-08-16 | 赣南师范大学 | A kind of carboxy-lesterase fluorescence probe and its preparation method and application |
| CN112812141A (en) * | 2019-11-18 | 2021-05-18 | 华东理工大学 | Preparation method of 3-fluoromethyl-substituted coumarin compound and fluorescent probe |
| CN115819374A (en) * | 2022-11-18 | 2023-03-21 | 遵义医科大学 | Near-infrared carboxylesterase small-molecule fluorescent probe and preparation method and application thereof |
| CN115819374B (en) * | 2022-11-18 | 2024-01-05 | 遵义医科大学 | Near-infrared carboxylesterase small-molecule fluorescent probe and preparation method and application thereof |
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