CN105039499A - Preparation and application of fluorescence probe for detecting [gamma]-glutamyltransferase (GGT) in cancer cells - Google Patents
Preparation and application of fluorescence probe for detecting [gamma]-glutamyltransferase (GGT) in cancer cells Download PDFInfo
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- SHMVQAWJAQWUEO-UHFFFAOYSA-N CCC1=C(C2)CC=CC2CCC1 Chemical compound CCC1=C(C2)CC=CC2CCC1 SHMVQAWJAQWUEO-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to preparation and an application of a fluorescence probe for detecting [gamma]-glutamyltransferase (GGT) in cancer cells. By means of the character that the GGT can selectively catalytically-crack glutamic acid, a method of grafting glutathione (GSH) onto a fluorochrome is creatively designed in the invention to synthesize a series of novel fluorescence probes used for targetedly-detecting the GGT in the cancer cells. The probe can be reacted with over-expressed GGT in the cancer cells, in which an optical signal is significantly changed, thereby achieving detection of the GGT in the cancer cells. The fluorescence probe can be used for recognizing and detecting the cancer cells being rich in the GGT in organisms. The fluorescence probe has a specific selectivity on the GGT and is very high in sensitivity.
Description
[technical field]
The present invention relates to chemicobiology art field, specifically, is preparation method and the application thereof of γ-glutamine transferring enzyme fluorescent probe in the novel targeted detection cancer cells of a class.
[background technology]
γ-glutamine transferring enzyme (GGT) is extensively present in organism, it plays very important role in the metabolic processes of organism, the unbalance meeting of organism γ-glutamine transferring enzyme (GGT) content causes very many-sided disease, such as primary hepatocarcinoma, myocardial infarction, acute pancreatitis etc., γ-glutamine transferring enzyme (GGT) is as a common biomarker, it can form process LAN in the cytoplasmic membrane of tumour cell, but content in normal cell is also few, by can to the rapid detection of cancer to the tracing detection of biomarker, Diagnosis and Treat plays very important effect, and enzyme is as a kind of important biomarker, again because have biologic specificity, advantages such as quick catalysis reaction and there is more wide application prospect, therefore extremely important to γ-glutamine transferring enzyme (GGT) carries out specific recognition, by carrying out tracking monitor to γ-glutamine transferring enzyme (GGT), can to the clinical detection of cancer, Diagnosis and Treat plays very important effect.
Meanwhile, compared with the method for traditional detection γ-glutamine transferring enzyme (GGT), the method of fluoroscopic examination has been proved to be has very many superiority, comprise simple, detection limit is extremely low, easy to operate etc., most important one is also advantageous in that this method can carry out in good time analyzing and testing in vivo.
[summary of the invention]
The object of the invention is to overcome the deficiencies in the prior art, preparation method and the application thereof of γ-glutamine transferring enzyme fluorescent probe in the novel detection cancer cells of a class is provided.
The object of the invention is to be achieved through the following technical solutions:
First object of the present invention is to provide the fluorescent probe that a class target detects γ-glutamine transferring enzyme in cancer cells.
The present invention's second object is to provide the preparation method that a class target detects the fluorescent probe of γ-glutamine transferring enzyme in cancer cells.
The present invention's the 3rd object is to provide a class target and detects the fluorescent probe of γ-glutamine transferring enzyme in cancer cells in the application detecting γ-glutamine transferring enzyme in cancer cells.
The present invention's the 4th object is to provide the compound that a class target detects γ-glutamine transferring enzyme in cancer cells.
The present invention's the 5th object is to provide the preparation method that a class target detects the compound of γ-glutamine transferring enzyme in cancer cells.
The present invention's the 6th object is to provide a class target and detects the compound of γ-glutamine transferring enzyme in cancer cells in the application detecting γ-glutamine transferring enzyme in cancer cells.
γ-glutamine transferring enzyme fluorescent probe in a kind of detection cancer cells, it is characterized in that, described fluorescent probe is synthesized through chemical process by Novel BODIPY flourescent dye and gsh GSH.
The structure of described fluorescent probe is R-BODIPY-GSH, and the application mainly introduces wherein two M-BODIPY-GSH and C-BODIPY-GSH.
Wherein: R group is any group, reach by conversion R group the effect regulating photophysical property, can be used as decorating site simultaneously and make further research; Be specifically as follows electrophilic or to the group of electronics;
R substituent is carbonic ether group or methoxyl group.
Described BODIPY structure is the structural formula shown in following formula or like derivatives:
Target detects the fluorescent probe of γ-glutamine transferring enzyme GGT in cancer cells, and fluorescent probe is synthesized through chemical process by novel B ODIPY fluorescence dye and gsh (GSH).
Described fluorescent probe is the binding substances of novel B ODIPY fluorescence dye and gsh (GSH), and fluorescent probe has extraordinary water-soluble.
The action site of described fluorescent probe and γ-glutamine transferring enzyme (GGT) is grafting on the gsh on fluorescent probe (GSH), and gsh (GSH) is γ-glutamine transferring enzyme (GGT) acts on good substrate.
The reaction mechanism of described fluorescent probe and γ-glutamine transferring enzyme (GGT) is intramolecular nucleophilic substitution reaction.
The R group site of described fluorescent probe has extraordinary modifiability, not only can reach the effect regulating photophysical property, can be used as decorating site simultaneously and make further research.
Described target detects the fluorescent probe of γ-glutamine transferring enzyme (GGT) in cancer cells, and choosing described R substituent is methoxyl group.
Described target detects the fluorescent probe of γ-glutamine transferring enzyme (GGT) in cancer cells, and choosing described R substituent is carbonic ether group.
The present invention also provides target to detect the preparation method of the fluorescent probe of γ-glutamine transferring enzyme (GGT) in cancer cells, comprises the steps:
(1) take ethyl ethylacetoacetate as raw material through becoming ring, hydrolysis, decarboxylation, reduction obtains indolyl moiety;
(2) with Benzoyl chloride be raw material through acidylate, condensation, upper chlorine is obtained by reacting benzoyl pyrrole chlorine;
(3) indolyl moiety and benzoyl pyrrole chlorine react and generate novel B ODIPY;
(4) generate novel B ODIPY to be modified again by demethylation, then be obtained by reacting compound R-BODIPY-GSH with gsh GSH;
Wherein: R group is any group, reach by conversion R group the effect regulating photophysical property, can be used as decorating site simultaneously and make further research.Be specifically as follows electrophilic or to the group of electronics;
R substituent is carbonic ether group.
R substituent is methoxyl group.
The synthetic route that a kind of target detects the fluorescent probe of γ-glutamine transferring enzyme (GGT) in cancer cells is as follows:
(a, b) CH
2cl
2, BF
3oEt
2, TEA; (c) GSH; (d) BBr
3; (e, f) to nitro-ethyl carbonic acid benzene fat, GSH
The present invention be BODIPY through processes such as nucleophilic substitution, obtain to detect the fluorescent probe R-BODIPY-GSH of γ-glutamine transferring enzyme (GGT) in cancer cells by target.
Originality of the present invention is based on novel B ODIPY parent, utilize γ-glutamine transferring enzyme (GGT) can selectively catalytic pyrolysis L-glutamic acid, our creationary design has been invented and gsh (GSH) grafting has been reached to the method on fluorescence dye the object that target detects γ-glutamine transferring enzyme (GGT) in cancer cells, and gsh (GSH) is γ-glutamine transferring enzyme (GGT) when acting on good substrate, by the effect with γ-glutamine transferring enzyme (GGT), our discovery of result creates the change of beyond thought fluorescent signal in this process, thus reach the object detecting γ-glutamine transferring enzyme (GGT).
The present invention also provides a class target to detect the compound of γ-glutamine transferring enzyme (GGT) in cancer cells, and it is characterized in that, described compound is R-BODIPY-GSH, and its structural formula is:
Wherein: R group is any group, reach by conversion R group the effect regulating photophysical property, can be used as decorating site simultaneously and make further research.
Described R substituent is methoxyl group.
Described R substituent is carbonic ether group.
The present invention also provides a class target to detect the preparation method of the compound of γ-glutamine transferring enzyme (GGT) in cancer cells, comprises the steps:
(1) be that raw material obtains indolyl moiety through becoming ring, hydrolysis, decarboxylation, reduction with ethyl ethylacetoacetate;
(2) be that raw material is obtained by reacting benzoyl pyrrole chlorine through acidylate, condensation, upper chlorine with Benzoyl chloride;
(3) indolyl moiety described in and benzoyl pyrrole chlorine react and generate novel B ODIPY;
(4) the novel B ODIPY synthesized is modified by demethylation again, then acts on gsh (GSH) and obtain other compounds R-BODIPY-GSH;
Wherein: R group is any group, reach by conversion R group the effect regulating photophysical property, can be used as decorating site simultaneously and make further research.Be specifically as follows electrophilic or to the group of electronics;
R substituent is carbonic ether group.
R substituent is methoxyl group.
Detect a preparation method for γ in cancer cells-glutamine transferring enzyme fluorescent probe, comprise the steps:
(1) be that raw material obtains indolyl moiety through becoming ring, hydrolysis, decarboxylation, reduction with ethyl ethylacetoacetate;
(2) be that raw material is obtained by reacting benzoyl pyrrole chlorine through acidylate, condensation, fork boron with Benzoyl chloride;
(3) indolyl moiety described in and benzoyl pyrrole chlorine react and generate novel B ODIPY;
(4) described novel BODIPY obtains compound M-BODIPY-GSH through acting on gsh (GSH);
Wherein: R group is any group, reach by conversion R group the effect regulating photophysical property, can be used as decorating site simultaneously and make further research.
Described R substituent is methoxyl group.
Described R substituent is carbonic ether group.
Detect a preparation method for γ in cancer cells-glutamine transferring enzyme fluorescent probe, comprise the steps:
(1) be that raw material obtains indolyl moiety through becoming ring, hydrolysis, decarboxylation, reduction with ethyl ethylacetoacetate;
(2) be that raw material is obtained by reacting benzoyl pyrrole chlorine through acidylate, condensation, fork boron with Benzoyl chloride;
(3) indolyl moiety described in and benzoyl pyrrole chlorine react and generate novel B ODIPY;
(4) the novel B ODIPY synthesized is modified by demethylation again, then acts on gsh (GSH) and obtain Compound C-BODIPY-GSH;
Wherein: R group is any group, reach by conversion R group the effect regulating photophysical property, can be used as decorating site simultaneously and make further research.
Described R substituent is methoxyl group.
Described R substituent is carbonic ether group.
The synthetic route of described compound M-BODIPY-GSH and C-BODIPY-GSH is as follows:
(a, b) DCM, BF
3oEt
2, TEA; (c) GSH; (d) BBr
3; (e, f) to nitro-ethyl carbonic acid benzene fat, GSH
The present invention further provides a class target and detect the fluorescent probe of γ-glutamine transferring enzyme (GGT) in cancer cells in the application detecting γ-glutamine transferring enzyme (GGT) in cancer cells.
The present invention further provides a class target and detect the compound of γ-glutamine transferring enzyme (GGT) in cancer cells in the application detecting γ-glutamine transferring enzyme (GGT) in cancer cells.
A kind of target detects the preparation method of the fluorescent probe of γ-glutamine transferring enzyme (GGT) in cancer cells, and its concrete technology flow process is:
(1) synthesis of compound 1
In round-bottomed flask, adding 0.2mol2-ethyl ethylacetoacetate, take ethanol as solvent, and at 0 DEG C, churning time is 5 ~ 60min, for subsequent use; Getting 0.2mol m-anisidine joins in round-bottomed flask, then adds the water of 100ml and the concentrated hydrochloric acid of 50ml, stirs, adds the Sodium Nitrite of 0.22mol, stirs 15min; Then add the sodium acetate of 0.2mol, regulate pH, joined in above-mentioned 2-ethyl ethylacetoacetate and ethanolic soln subsequently, then add the potassium hydroxide of 0.18mol and the ice cube of 1mol, regulate between pH, be stirred to and react completely, extraction, dry, be spin-dried for obtain adopting dark liquid; At 70 DEG C, adopting dark liquid is instilled in the hydrogen chloride solution of ethanol, drip off backflow 2h; Ethanol is spun off, extraction after having reacted, dry, be spin-dried for; Product crosses silicagel column; The product obtained is dissolved in ethanol, adds potassium hydroxide, backflow, and reaction end spins off ethanol, adds dilute hydrochloric acid and has been adjusted to Precipitation, suction filtration, and washing is dried and obtained solid.Solid is dissolved in quinoline, adds copper powder decarboxylic reaction, the lower 240 DEG C of reaction 3h of helium protection; React rear cooling, adjusted pH, extraction, washing, dry, be spin-dried for; Product crosses silicagel column, obtains 0.62g brown solid and is compound 1;
(2) synthesis of compound 2
Get 02729mol benzoyl morpholine and 0.2729molPOCl
3be mixed to join in round-bottomed flask, stirring at room temperature evenly to all dissolving, is at room temperature reacted; Get pyrroles to be added dropwise in reaction system and to react; Question response is complete, by system as under condition of ice bath, drips excessive saturated Na wherein
2cO
3the aqueous solution, release to there is no bubble; Extraction, dry; Be spin-dried for, obtain white solid.Then 0.04molCuCl is taken
22H
2o is dissolved in CH
3in CN, add 0.02mol benzoyl pyrrole in system, heat up, question response is complete, and cooling, adds H
2sO
4solution, stirs, and extraction is dry, obtains compound 2.
(3) synthesis of compd B ODIPY
Taking 0.2433mmol compound 2 is dissolved in DCM, adds 0.97mmolPOCl wherein
3, stirring at room temperature; Above-mentioned system is added after 0.29mmol compound 1 is dissolved in DCM; Under rare gas element existence condition, reaction 2h; After having reacted, add saturated manganese hydrogen sodium regulating solution pH to neutral, extraction, washing, dry, be spin-dried for, intermediate be dissolved in appropriate DCM again, add 5mlEt
3n stirs 5min, adds 5mlBF under condition of ice bath
3et
2o, returns to room temperature, continues uniform stirring 3h, dichloromethane extraction, and washing merges organic phase, is spin-dried for after drying; Cross silicagel column, obtain atropurpureus solid BODIPY.
(4) synthesis of compound M-BODIPY-GSH
Getting 0.035mmolBODIPY is dissolved in acetonitrile, gets 0.35mmolGSH soluble in water simultaneously, adds above-mentioned system, reaction 5h, and react complete, vacuum is spin-dried for solvent, column chromatography, obtains product M-BODIPY-GSH;
(5) synthesis of compound 3
Get 0.1mmolBODIPY to be dissolved in dry DCM, under condition of ice bath, add 0.84mmolBBr
3reaction 24h, reacts complete and adds ethanol cancellation, then DCM extraction, and column chromatography obtains compound 3;
(6) synthesis of Compound C-BODIPY-GSH
Getting 0.2618mmol compound 3 is dissolved in dry acetonitrile, add 2mlTEA, then add nitro-ethyl carbonic acid benzene fat, reaction 1h, TLC detection reaction is complete, then 0.32mmolGSH is soluble in water, add in above-mentioned reaction system, reaction 5h, after reacting completely, vacuum is spin-dried for, and column chromatography obtains product C-BODIPY-GSH.
Compound R-the BODIPY-GSH detecting γ-glutamine transferring enzyme (GGT) in cancer cells for target provided by the invention, following structure:
Wherein: R group is any group, reach by conversion R group the effect regulating photophysical property, can be used as decorating site simultaneously and make further research.
Term:
Absorption (a.u.) is absorbance.
Fluoresenceintensity (a.u.) is fluorescence intensity.
BODIPY molecule is fluorine boron two pyrroles (BODIPY).
M-BODIPY-GSH is structure shown in figure;
C-BODIPY-GSH is structure shown in figure;
Compared with prior art, positively effect of the present invention is:
Provided by the inventionly detecting the fluorescent probe of γ-glutamine transferring enzyme (GGT) in cancer cells for target and can be used for detecting γ-glutamine transferring enzyme (GGT) in cancer cells, overcoming in prior art for detecting many defects of γ in cancer cells-glutamine transferring enzyme (GGT); Fluorescent probe of the present invention make use of creationary grafting gsh (GSH) on fluorescence dye and is used as the substrate of γ-glutamine transferring enzyme (GGT), not only substantially increase the water-soluble of fluorescence dye, solve the problem of traditional fluorescent probe poorly water-soluble, and gsh (GSH) is as the reaction substrate of γ-glutamine transferring enzyme (GGT), can improve the highly selective of probe; Fluorescent probe of the present invention can be applied to the detection of the cancer cells being rich in GGT.Fluorescent probe provided by the invention has specific selectivity to γ-glutamine transferring enzyme (GGT), has very high susceptibility.Its preparation method fairly simple easy to operate, environmental friendliness, with low cost, productive rate comparatively advantages of higher.
Target provided by the invention detects the compound R-BODIPY-GSH of γ-glutamine transferring enzyme (GGT) in cancer cells, can be used for detecting γ-glutamine transferring enzyme (GGT) in cancer cells, thus specific detection is rich in the detection of the cancer cells of GGT; Compound R-BODIPY-GSH is good, highly sensitive to γ-glutamine transferring enzyme (GGT) selectivity.
[accompanying drawing explanation]
Fig. 1 be compound M-BODIPY-GSH in PBS buffered soln (pH=7.4), when there is γ-glutamine transferring enzyme (GGT), the photophysical property change procedure of probe;
Fig. 1 (a) uv absorption spectra, Fig. 1 (b) fluorescence emission spectrogram;
Fig. 2 is the fluorescence interference variation diagram of compound M-BODIPY-GSH for other physiological environment;
Fig. 3 is the laser confocal imaging figure of compound M-BODIPY-GSH for different cell;
Fig. 4 is the hydrogen spectrum of compound M-BODIPY-GSH, and deuterated reagent is CD
3oD;
Fig. 5 is the high resolution mass spec figure HRMS (m/z) of compound M-BODIPY-GSH;
Fig. 6 is the hydrogen spectrum of Compound C-BODIPY-GSH, and deuterated reagent is CD
3oD;
Fig. 7 is the high resolution mass spec figure HRMS (m/z) of Compound C-BODIPY-GSH.
[embodiment]
In order to understand the present invention better, illustrate content of the present invention further below in conjunction with embodiment, but content of the present invention is not only confined to the following examples.
Embodiment 1: the synthesis of compound 1
In round-bottomed flask, add appropriate 2-ethyl ethylacetoacetate and ethanol, at 0 DEG C, stir for some time, for subsequent use.Getting m-anisidine joins in round-bottomed flask, then adds water and concentrated hydrochloric acid, stirs, adds Sodium Nitrite., stir 15min.Then add sodium acetate, regulate pH, joined subsequently in above-mentioned 2-ethyl ethylacetoacetate and ethanolic soln, then add potassium hydroxide and ice cube, regulate between pH, be stirred to and react completely, extraction, dry, be spin-dried for obtain adopting dark liquid.At 70 DEG C, adopting dark liquid is instilled in the hydrogen chloride solution of ethanol, drip off backflow 2h.Ethanol is spun off, extraction after having reacted, dry, be spin-dried for.Product crosses silicagel column,
The product obtained is dissolved in ethanol, adds potassium hydroxide, backflow, and reaction end spins off ethanol, adds dilute hydrochloric acid and has been adjusted to Precipitation, suction filtration, and washing is dried and obtained solid.Solid is dissolved in quinoline, adds copper powder, the lower 240 DEG C of reaction 3h of helium protection.React rear cooling, adjusted pH, extraction, washing, dry, be spin-dried for.Product crosses silicagel column, obtains 0.62g brown solid and is compound 1.
Embodiment 2: the synthesis of compound 2
Get benzoyl morpholine and POCl
3be mixed to join in round-bottomed flask, stirring at room temperature evenly to all dissolving, is at room temperature reacted; Get pyrroles to be added dropwise in reaction system and to react; Question response is complete, by system as under condition of ice bath, drips excessive saturated Na wherein
2cO
3the aqueous solution, release to there is no bubble; Extraction, dry; Be spin-dried for, obtain white solid.Then CuCl is taken
22H
2o is dissolved in CH
3in CN, in system, add benzoyl pyrrole, heat up, question response is complete, and cooling, adds H
2sO
4solution, stirs, and extraction is dry, obtains compound 2.
Embodiment 3: the synthesis of compd B ODIPY
Weigh Compound 2 is dissolved in DCM, adds POCl wherein
3, stirring at room temperature.Above-mentioned system is added after compound 1 is dissolved in a small amount of DCM.Under rare gas element existence condition, reaction 2h.After having reacted, add saturated manganese hydrogen sodium regulating solution pH to neutral, extraction, washing, dry, be spin-dried for, intermediate be dissolved in appropriate DCM again, add Et
3n stirs 5min, adds BF under condition of ice bath
3et
2o, returns to room temperature, continues uniform stirring 3h, dichloromethane extraction, and washing merges organic phase, is spin-dried for after drying.Cross silicagel column, obtain atropurpureus solid BODIPY.
Embodiment 4: the synthesis of compound M-BODIPY-GSH
Getting appropriate BODIPY is dissolved in acetonitrile, gets appropriate GSH soluble in water simultaneously, adds above-mentioned system, reaction 5h, and react complete, vacuum is spin-dried for solvent, column chromatography, obtains product M-BODIPY-GSH;
1hNMR (CD
3oD, 400MHz): δ 7.61-7.58 (m, 3H), 7.48-7.46 (m, 2H), 7.43-7.41 (d, 1H), 7.05 (s, 1H), 6.96-6.95 (d, 1H), 6.77-6.75 (d, 1H), 6.68-6.66 (dd, 1H), 4.57-4.53 (m, 1H), 3.94 (s, 3H), 3.76-3.73 (m, 2H), 3.59-3.57 (t, 1H), 3.07-3.02 (m, 2H), 2.62-2.57 (m, 2H), 2.17-2.15 (m, 2H), 1.77 (s, 3H);
13cNMR (CD
3oD, 100MHz): δ 193.38,189.75,188.86,186.59,182.92,179.29,175.34,173.09,166.71,156.96,147.92,134.30,132.84,130.66,130.23,127.34,117.62,115.06,108.88,95.17,64.34,60.76,54.56,53.76,44.80,30.80,30.36,23.79,13.02; HRMS (ESI) calcdforC
31h
32bF
2n
5naO
7s:690.1981, Found:690.1967 [M+Na]
+.
Embodiment 5: the synthesis of compound 3
Get appropriate BODIPY to be dissolved in dry DCM, under condition of ice bath, add appropriate BBr
3reaction 4h, reacts complete and adds ethanol cancellation, and then DCM extraction, column chromatography obtains compound 3
Embodiment 6: the synthesis of Compound C-BODIPY-GSH
Getting compound 3 is dissolved in dry acetonitrile, add appropriate TEA, then add nitro-ethyl carbonic acid benzene fat, reaction 1h, TLC detection reaction is complete, then appropriate GSH is dissolved in appropriate water, add in above-mentioned reaction system, reaction 5h, after reacting completely, vacuum is spin-dried for, and column chromatography obtains product C-BODIPY-GSH,
1hNMR (CD
3oD, 400MHz): δ 7.61-7.58 (m, 3H), 7.56-7.53 (d, 1H), 7.49-7.46 (m, 2H), 7.36 (s, 1H), 7.11-7.09 (d, 1H), 6.92-6.91 (d, 1H), 6.85-6.82 (dd, 1H), 4.54-4.51 (m, 1H), 4.35-4.29 (q, 2H), 3.85-3.80 (m, 2H), 3.65-3.62 (t, 1H), 2.87-2.82 (m, 2H), 2.61-2.50 (m, 2H), 2.20-2.05 (m, 2H), 1.78 (s, 3H), 1.40-1.36 (t, 3H),
13cNMR (CD
3oD, 100MHz): δ 200.66,181.05,176.26,175.46,175.32,174.61,174.21,173.91,172.41,171.54,169.94,153.52,152.07,130.65,129.93,123.30,123.05,122.21,114.75,113.76,79.86,59.65,57.20,54.04,52.91,43.01,33.04,27.78,26.78,14.53,11.96, HRMS (ESI) calcdforC
33h
35bF
2n
5o
9s:726.2217, Found:726.2221 [M+H]
+.
Effect example:
Referring to Fig. 1 compound M-BODIPY-GSH in PBS buffered soln (pH=7.4), when there is γ-glutamine transferring enzyme (GGT), the photophysical property change procedure of probe; Figure (a) uv absorption spectra, figure (b) fluorescence emission spectrogram.
Draw by figure: compound M-BODIPY-GSH is not when adding γ-glutamine transferring enzyme (GGT), its uv-absorbing wavelength in buffered soln, at 578nm, can produce faint fluorescence at 601nm place.And after adding γ-glutamine transferring enzyme (GGT), compound M-BODIPY-GSH and γ-glutamine transferring enzyme (GGT) reacts completely within 30min, there is larger blue shift in uv-absorbing, by original 578nm blue shift to 510nm, and produce isobestic point at 528nm place, as Fig. 1 a; Now not only there is blue shift in fluorescence, and at 582nm place, intensity also significantly strengthens (Fig. 1 b).This shows that M-BODIPY-GSH can carry out specific recognition to γ-glutamine transferring enzyme (GGT) and have very high susceptibility.
That compound M-BODIPY-GSH is for the fluorescence interference variation diagram under other physiological environment referring to Fig. 2.
From figure, we can find: M-BODIPY-GSH in PBS buffer solution system when containing 10% foetal calf serum, 10% human plasma, collagen hydrolysate enzyme, phosphoesterase, time under the condition such as peptase and saccharifying enzyme, it is very stable that our probe still shows, and optical property does not change substantially, this illustrates that our probe has very strong immunity from interference, and the specific recognition of molecule is very good.
Compound M-BODIPY-GSH for different cell and the laser confocal imaging figure adding different amount inhibitor referring to Fig. 3.
As can be seen from figure we: for the cell lacking γ-glutamine transferring enzyme (GGT), the yellow channels of cell imaging and red channel ratio r atio are about 0.4, and namely fluorescence still exists with the form of redness; And for the cancer cells of γ-glutamine transferring enzyme (GGT) process LAN, they are then about 1.4 at the ratio ratio of two passages, this explanation, after our probe and γ-glutamine transferring enzyme (GGT) act on, probe is main exists with the form of yellow fluorescence, and this illustrates that the probe that we design obtains good application in bio-imaging.
Referring to Fig. 4,5,6,7 is the phenogram of M-BODIPY-GSH and C-BODIPY-GSH.
The present invention's fluorescent probe required for protection the experiment proved that the function all had described in similar above-mentioned effect example, i.e. the fluorescent probe of γ in detection cancer cells provided by the invention-glutamine transferring enzyme (GGT) and compound R-BODIPY-GSH; Wherein compound R-BODIPY-GSH:
Wherein: R group is any group, reach by conversion R group the effect regulating photophysical property, can be used as decorating site simultaneously and make further research.
Provided by the inventionly detecting the fluorescent probe of γ-glutamine transferring enzyme (GGT) in cancer cells for target and can be used for detecting γ-glutamine transferring enzyme (GGT) in cancer cells, overcoming in prior art for detecting many defects of γ in cancer cells-glutamine transferring enzyme (GGT); Fluorescent probe of the present invention make use of creationary grafting gsh (GSH) on fluorescence dye and is used as the substrate of γ-glutamine transferring enzyme (GGT), not only substantially increase the water-soluble of fluorescence dye, solve the problem of traditional fluorescent probe poorly water-soluble, and gsh (GSH) is as the reaction substrate of γ-glutamine transferring enzyme (GGT), there is in physiology and pathology very many advantages; Fluorescent probe of the present invention can be applied to target in organism and detect γ-glutamine transferring enzyme (GGT) in cancer cells.Fluorescent probe provided by the invention has specific selectivity to γ-glutamine transferring enzyme (GGT), has very high susceptibility.Its preparation method fairly simple easy to operate, environmental friendliness, with low cost, productive rate comparatively advantages of higher.
Target provided by the invention detects the compound R-BODIPY-GSH of γ-glutamine transferring enzyme (GGT) in cancer cells, can be used for detecting γ-glutamine transferring enzyme (GGT) in cancer cells; Compound R-BODIPY-GSH is good, highly sensitive to γ-glutamine transferring enzyme (GGT) selectivity.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, without departing from the inventive concept of the premise; can also make some improvements and modifications, these improvements and modifications also should be considered within the scope of protection of the present invention.
Claims (10)
1. detect γ-glutamine transferring enzyme fluorescent probe in cancer cells, it is characterized in that, described fluorescent probe is synthesized through chemical process by Novel BODIPY flourescent dye and gsh GSH.
2. as claimed in claim 1 a kind of detect γ-glutamine transferring enzyme fluorescent probe in cancer cells, it is characterized in that, described fluorescent probe be R-BODIPY-GSH, structural formula is:
3. γ-glutamine transferring enzyme fluorescent probe in a kind of detection cancer cells as claimed in claim 1, it is characterized in that, described fluorescent probe is the binding substances of novel B ODIPY fluorescence dye and gsh GSH.
4. γ-glutamine transferring enzyme fluorescent probe in a kind of detection cancer cells as claimed in claim 1, it is characterized in that, the action site of described fluorescent probe and γ-glutamine transferring enzyme GGT is grafting on the gsh GSH on fluorescent probe, and gsh GSH is γ-glutamine transferring enzyme GGT acts on good substrate;
The reaction mechanism of described fluorescent probe and γ-glutamine transferring enzyme (GGT) is intramolecular nucleophilic substitution reaction.
5. a kind of preparation method detecting γ in cancer cells-glutamine transferring enzyme fluorescent probe as claimed in claim 1, is characterized in that, comprise the steps:
(1) be that raw material obtains indolyl moiety through becoming ring, hydrolysis, decarboxylation, reduction with ethyl ethylacetoacetate;
(2) be that raw material is obtained by reacting benzoyl pyrrole chlorine through acidylate, condensation, fork boron with Benzoyl chloride;
(3) indolyl moiety described in and benzoyl pyrrole chlorine react and generate novel B ODIPY;
(4) described novel BODIPY obtains compound M-BODIPY-GSH through acting on gsh (GSH);
6. a kind of preparation method detecting γ in cancer cells-glutamine transferring enzyme fluorescent probe as claimed in claim 1, is characterized in that, comprise the steps:
(1) be that raw material obtains indolyl moiety through becoming ring, hydrolysis, decarboxylation, reduction with ethyl ethylacetoacetate;
(2) be that raw material is obtained by reacting benzoyl pyrrole chlorine through acidylate, condensation, fork boron with Benzoyl chloride;
(3) indolyl moiety described in and benzoyl pyrrole chlorine react and generate novel B ODIPY;
(4) the novel B ODIPY synthesized is modified by demethylation again, then acts on gsh (GSH) and obtain Compound C-BODIPY-GSH;
7. a kind of preparation method detecting γ in cancer cells-glutamine transferring enzyme fluorescent probe as claimed in claim 1, is characterized in that, comprise the steps:
(1) be that raw material obtains indolyl moiety through becoming ring, hydrolysis, decarboxylation, reduction with ethyl ethylacetoacetate;
(2) be that raw material is obtained by reacting benzoyl pyrrole chlorine through acidylate, condensation, fork boron with Benzoyl chloride;
(3) indolyl moiety described in and benzoyl pyrrole chlorine react and generate novel B ODIPY;
(4) the novel B ODIPY synthesized is modified by demethylation again, then acts on gsh (GSH) and obtain other compounds R-BODIPY-GSH;
8. detect a preparation method for γ in cancer cells-glutamine transferring enzyme fluorescent probe, it is characterized in that, its concrete technology flow process is:
(1) synthesis of compound 1
In round-bottomed flask, adding 0.2mol2-ethyl ethylacetoacetate, take ethanol as solvent, and at 0 DEG C, churning time is 5 ~ 60min, for subsequent use; Getting 0.2mol m-anisidine joins in round-bottomed flask, then adds the water of 100ml and the concentrated hydrochloric acid of 50ml, stirs, adds the Sodium Nitrite of 0.22mol, stirs 15min; Then add the sodium acetate of 0.2mol, regulate pH, joined in above-mentioned 2-ethyl ethylacetoacetate and ethanolic soln subsequently, then add the potassium hydroxide of 0.18mol and the ice cube of 1mol, regulate between pH, be stirred to and react completely, extraction, dry, be spin-dried for obtain adopting dark liquid; At 70 DEG C, adopting dark liquid is instilled in the hydrogen chloride solution of ethanol, drip off backflow 2h; Ethanol is spun off, extraction after having reacted, dry, be spin-dried for; Product crosses silicagel column; The product obtained is dissolved in ethanol, adds potassium hydroxide, backflow, and reaction end spins off ethanol, adds dilute hydrochloric acid and has been adjusted to Precipitation, suction filtration, and washing is dried and obtained solid.Solid is dissolved in quinoline, adds copper powder decarboxylic reaction, the lower 240 DEG C of reaction 3h of helium protection; React rear cooling, adjusted pH, extraction, washing, dry, be spin-dried for; Product crosses silicagel column, obtains 0.62g brown solid and is compound 1;
(2) synthesis of compound 2
Get 02729mol benzoyl morpholine and 0.2729molPOCl
3be mixed to join in round-bottomed flask, stirring at room temperature evenly to all dissolving, is at room temperature reacted; Get pyrroles to be added dropwise in reaction system and to react; Question response is complete, by system as under condition of ice bath, drips excessive saturated Na wherein
2cO
3the aqueous solution, release to there is no bubble; Extraction, dry; Be spin-dried for, obtain white solid.Then 0.04molCuCl is taken
22H
2o is dissolved in CH
3in CN, add 0.02mol benzoyl pyrrole in system, heat up, question response is complete, and cooling, adds H
2sO
4solution, stirs, and extraction is dry, obtains compound 2.
(3) synthesis of compd B ODIPY
Taking 0.2433mmol compound 2 is dissolved in DCM, adds 0.97mmolPOCl wherein
3, stirring at room temperature; Above-mentioned system is added after 0.29mmol compound 1 is dissolved in DCM; Under rare gas element existence condition, reaction 2h; After having reacted, add saturated manganese hydrogen sodium regulating solution pH to neutral, extraction, washing, dry, be spin-dried for, intermediate be dissolved in appropriate DCM again, add 5mlEt
3n stirs 5min, adds 5mlBF under condition of ice bath
3et
2o, returns to room temperature, continues uniform stirring 3h, dichloromethane extraction, and washing merges organic phase, is spin-dried for after drying; Cross silicagel column, obtain atropurpureus solid BODIPY.
(4) synthesis of compound M-BODIPY-GSH
Getting 0.035mmolBODIPY is dissolved in acetonitrile, gets 0.35mmolGSH soluble in water simultaneously, adds above-mentioned system, reaction 5h, and react complete, vacuum is spin-dried for solvent, column chromatography, obtains product M-BODIPY-GSH;
(5) synthesis of compound 3
Get 0.1mmolBODIPY to be dissolved in dry DCM, under condition of ice bath, add 0.84mmolBBr
3reaction 24h, reacts complete and adds ethanol cancellation, then DCM extraction, and column chromatography obtains compound 3;
(6) synthesis of Compound C-BODIPY-GSH
Getting 0.2618mmol compound 3 is dissolved in dry acetonitrile, add 2mlTEA, then add nitro-ethyl carbonic acid benzene fat, reaction 1h, TLC detection reaction is complete, then 0.32mmolGSH is soluble in water, add in above-mentioned reaction system, reaction 5h, after reacting completely, vacuum is spin-dried for, and column chromatography obtains product C-BODIPY-GSH.
9. one kind is detected γ in cancer cells-glutamine transferring enzyme fluorescent probe in the application detecting γ-glutamine transferring enzyme GGT in cancer cells.
10. the compound detecting γ in cancer cells-glutamine transferring enzyme GGT is in the application detecting γ-glutamine transferring enzyme GGT in cancer cells.
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| CN110862818A (en) * | 2019-11-14 | 2020-03-06 | 南通大学 | Gamma-glutamyl transpeptidase near-infrared fluorescent probe, preparation method and application thereof |
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| CN110862818A (en) * | 2019-11-14 | 2020-03-06 | 南通大学 | Gamma-glutamyl transpeptidase near-infrared fluorescent probe, preparation method and application thereof |
| CN110862818B (en) * | 2019-11-14 | 2022-03-18 | 南通大学 | Gamma-glutamyl transpeptidase near-infrared fluorescent probe, preparation method and application thereof |
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