CN104673278A - Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe - Google Patents
Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe Download PDFInfo
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Abstract
The invention discloses a fluorescence probe for detecting glutathione and a preparation method and a use method of the fluorescence probe. A classical ICT system is constructed by utilizing 1,8-naphthalimides, a benzene sulfoxide part is introduced at 4-position, and the ICT effect of probe molecules is adjusted and controlled by utilizing a classical ICT system constructed by 1,8-naphthalimide and introducing benzene sulfoxide part at the fourth position. Under the condition that no glutathione exists, the probe molecules do not emit fluorescence light because of strong electron-withdrawing effect of 4-position benzene sulfoxide; under the condition that glutathione exists, the benzene sulfoxide part can be replaced by the glutathione, and thus intramolecular electron transfers from a 4-position sulphur atom (from the glutathione) to the 1,8-naphthalimides, and the probe molecules emit hyperfluorescence because of the ICT effect. According to the invention, the detection of the intracellular glutathione can be realized, and the fluorescence probe has the advantages of convenient operation, low cost, sensitive response, easy promotion and application and the like.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of naphthalimide-benzene sulfoxide derivant as the use of gsh fluorescence probe material and preparation method thereof and using method.
Background technology
The tri-peptide molecule that gsh (Glutathione, GSH) is made up of L-glutamic acid, halfcystine and glycine, be in human body content maximum containing mercaptoamino-acid.In life entity, gsh plays an important role to maintaining cellular redox balance, the metabolism of allogenic material, Intracellular signals conversion and generegulation etc.The exception of glutathion inside cell concentration will cause a series of physiological maladies, such as white corpuscle disappearance, psoriatic, liver injury, cancer and acquired immune deficiency syndrome (AIDS) etc. are (see L.A.Herzenberg, S.C.De Rosa, J.G.Dubs etc., Glutathione deficiency is associated with impaired survival in HIV disease, Proc.Natl.Acad.Sci.USA., 1997,94:1967-1972).Therefore, quantitatively, the content of monitoring bio body glutathion inside is biological chemistry and chemicobiological study hotspot always in real time.
Fluorescence detection due to its outstanding detection sensitivity and selectivity, and can realize the extensive concern real-time, the on-line checkingi of biological sample being subject to investigator.Naphthalimide fluorescent molecule has the particular advantages such as good light stability, high molar extinction coefficient and quantum yield because of it and becomes one of most important fluorescent parent of the method, is widely used in the fluoroscopic examination of multiple testing molecule.
That at present to have developed designs for the small-molecule fluorescent probe detecting gsh mainly reacts based on the specific chemical between sulfydryl and 2,4-dinitrobenzene sulfonamido or 2,4-dinitrobenzene semi-annular jade pendant acyl ester group.When under the condition that there is gsh, in probe molecule 2,4-dinitrobenzene sulfonamido or 2,4-dinitrobenzene semi-annular jade pendant acyl ester group can be replaced by the sulfydryl in gsh, former 2,4-dinitrobenzene sulfonamido or 2,4-dinitrobenzene semi-annular jade pendant acyl ester group are left away and cause the photoluminescent property of probe molecule to change, thus realize setting the specificity of gsh.
But, the gsh probe great majority reported are subject to the same amino acid containing sulfydryl in organism, as: the interference of halfcystine (Cysteine, Cys) and homocysteine (Homocysteine, Hcy) is (see J.Bouffard, Y.Kim, T.M.Swager etc., A Highly Selective Fluorescent Probe for Thiol Bioimaging, Org.Lett., 2008,10:37-40; X.-D.Jiang, J.Zhang, X.Shao etc., A selective fluorescent turn-on NIR probe for cysteine, Org.Biomol.Chem., 2012,10:1966-1968), be difficult to realize the specific detection that it carries out gsh in the organism of complexity.Therefore need a kind of novelty, there is good biological stability and can realize outstanding specificly-response for organism glutathion inside detect fluorescent probe.
Summary of the invention
In order to overcome above-mentioned defect of the prior art, the present invention aim to provide a kind of from naphthalimide and benzene sulfoxide for fluorescent probe detecting gsh and preparation method thereof and using method.
Core of the present invention is to utilize 1,8-naphthalimide to construct classical ICT system, and introduces benzene sulfoxide moiety in 4-position, the ICT effect of regulation and control probe molecule.When without under the existence of gsh, the strong sucting electronic effect due to 4-position benzene sulfoxide causes probe molecule unstressed configuration to be launched; And when under the condition that there is gsh, benzene sulfoxide moiety can be replaced by gsh, and then the cyclic voltammetry method occurred from 4-position sulphur atom (from gsh) to 1,8-naphthalimide, and this ICT effect makes probe molecule launch hyperfluorescenceZeng Yongminggaoyingguang.By such scheme, obtain the fluorescence response of height gsh specific " co " type, substantially increase selectivity and the sensitivity of detection.
First, in order to achieve the above object, the invention provides one such as formula the naphthalimide shown in (I)-benzene sulfoxide derivant.
In formula (I), n is the atomicity of 0 ~ 18; R
1for hydrogen, or methyl, or ethyl, or sec.-propyl, or fluorine; R
2for methyl, or hydroxyl, or carboxyl, or sulfonic group, or (triphenyl) phosphorus base, or in appoint
what is a kind of.
Shown in formula (I), compound is specially compound (Nap-G) shown in formula (II),
The preparation method of above-mentioned probe comprises the following steps:
Step one: under an inert atmosphere, shown in formula (III), bromo-1, the 8-naphthalic anhydride of 4-and aminocompound are obtained by reacting bromo-1, the 8-naphthalimide of 4-replaced shown in formula (IV) in alcohol.
In formula (IV), n is the atomicity of 0 ~ 18, R
2for methyl, or hydroxyl, or carboxyl, or sulfonic group, or (triphenyl) phosphorus base, or
in any one.
Step 2: under an inert atmosphere, in the presence of a base, shown in formula (IV), shown in compound and formula (V), compound is obtained by reacting 4-thiophenol base-1, the 8-naphthalimide replaced shown in formula (VI) in alcohol.
In formula (VI), n is the atomicity of 0 ~ 18; R
1for hydrogen, or methyl, or ethyl, or sec.-propyl, or fluorine; R
2for methyl, or hydroxyl, or carboxyl, or sulfonic group, or (triphenyl) phosphorus base, or
in any one.
Step 3: under an inert atmosphere, compound and metachloroperbenzoic acid shown in formula (VI) react in organic solvent and obtain compound shown in formula (I).
Described in step one, aminocompound is specially n-Butyl Amine 99; Shown in formula (III), the mol ratio of bromo-1, the 8-naphthalic anhydride of 4-and described aminocompound is 1 ~ 50:1; Described alcohol is methyl alcohol, ethanol, n-propyl alcohol, Virahol, propyl carbinol or ethylene glycol monomethyl ether; Described temperature of reaction is 50 ~ 120 degree, and the reaction times is 1 ~ 20 hour;
As preferably, shown in formula (III), the mol ratio of bromo-1, the 8-naphthalic anhydride of 4-and described aminocompound is 10:1; Described alcohol is ethanol; Described temperature of reaction is 80 degree; Reaction times is 5 hours.
Above-mentioned preparation method, alcohol described in step 2 is methyl alcohol, ethanol, propyl alcohol, butanols, ethylene glycol monomethyl ether or diethylene glycol monomethyl ether; Shown in formula (IV), shown in bromo-1, the 8-naphthalimide of 4-and formula (V), the mol ratio of thiophenol compound is 1 ~ 10:1;
Described alkali is organic bases or mineral alkali;
Described organic bases is in triethylamine, pyridine or diisopropyl ethyl amine; Described mineral alkali is salt of wormwood, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or saleratus;
Temperature of reaction is 0 ~ 140 degree; Reaction times is 1 ~ 48 hour.
As preferably, the ethylene glycol monomethyl ether of alcohol described in step 2; Shown in formula (IV), shown in bromo-1, the 8-naphthalimide of 4-and formula (V), the mol ratio of thiophenol compound is 5:1; Temperature of reaction is for being 100 degree; 24 hours reaction times.
The mol ratio of 4-thiophenol base-1, the 8-naphthalimide replaced shown in metachloroperbenzoic acid described in step (3) and formula (VI) is 0.5 ~ 1.5:1;
Described organic solvent is chloroform, methylene dichloride, acetonitrile, DMF, DMSO or 1,2-ethylene dichloride;
Temperature of reaction is 0 ~ 100 degree; Reaction times is 1 ~ 24 hour.
As preferably, the mol ratio 1:1 of 4-thiophenol base-1, the 8-naphthalimide replaced shown in metachloroperbenzoic acid and formula (VI); Described organic solvent is methylene dichloride; Temperature of reaction is 25 degree; Reaction times is 5 hours.
A kind of using method detecting the fluorescent probe of gsh; The method specifically comprises the following steps:
Step 1: the shown compound of formula (I) adding same concentrations in the buffering salt of different concns gsh, configures the standardized solution containing compound shown in formula (I) of at least 3 kinds of different glutathione contents.
Shown in buffered soln to be any one in phosphate buffer soln, Tris-HCl buffered soln, HEPES buffered soln, boric acid-sodium borate buffered soln, specifically to be phosphate buffer soln;
Shown in the pH value of standardized solution be 6 ~ 11, specifically being 7.2;
Shown in shown standardized solution Chinese style (I), the concentration of compound is 1nM ~ 10 μM;
The content of shown standardized solution GSH-PX activity is 0.1nM ~ 1mM;
Step 2: the fluorescence emission spectrum measuring described standardized solution respectively, excitation wavelength is 366nm, take glutathione concentrations as X-coordinate, with I
482for ordinate zou, Criterion curve.
I
482represent that described standardized solution is the fluorescence emission peak intensity level at 482nm place at wavelength;
Step 3: add compound shown in formula (I) in testing sample, control its concentration equal with the concentration of compound described standardized solution Chinese style (I) Suo Shi; Measuring it is fluorescence emission spectrum under the exciting light of 366nm in excitation wavelength, namely calculates the glutathione content of testing sample according to typical curve.
Above-mentioned steps: 2 or step 3 in fluorescence intensity detect on luminoscope.
The present invention has following features:
1) fluorescent probe provided by the invention is white solid, and the molecular characterization of naphthalimide and sulfoxide ensure that structure and the optical stability of probe.
2) fluorescent probe provided by the invention, its solution is to the concentration sensitive of gsh, and along with the increase of glutathione concentrations, the fluorescence observing its aqueous solution under ultraviolet lamp becomes bright green from colourless.
3) fluorescent probe provided by the invention, its emission wavelength is 482nm, belongs to " co " type fluorescence response, and when greatly can eliminate detection, background difference is on the impact of result, improves the sensitivity detected.
4) fluorescent probe provided by the invention has outstanding response susceptibility to gsh, commonly coexists containing the amino acid of sulfydryl, as halfcystine, homocysteine etc. are noiseless to detection.
5) fluorescent probe provided by the invention is linear to glutathione concentrations, for the accurate measurement of gsh.
" co " type gsh probe of naphthalimide dyestuff provided by the invention and test kit thereof have good response to glutathione solution, the detection to glutathion inside cell can be realized, have easy and simple to handle, with low cost, respond sensitive, be easy to the advantages such as promotion and application.
Accompanying drawing explanation
Fig. 1 is the synthetic route of fluorescent probe Nap-G prepared by embodiment 1.
Fig. 2 is that the Nap-G test kit of embodiment 6 preparation is to the color response figure of the gsh aqueous solution.
Fig. 3 is that the Nap-G test kit of embodiment 6 preparation is to the fluorescence response figure of the different gsh aqueous solution.
Fig. 4 is the ratio I of the fluorescent emission intensity of Nap-G test kit under wavelength 482nm prepared by embodiment 6
482with glutathione concentrations relation curve.
Fig. 5 is that the Nap-G test kit prepared of embodiment 6 is to the fluorescence response figure of common coexisting ion or biological micromolecule.
Fig. 6 is that the Nap-G test kit of embodiment 6 preparation is to the fluorescence imaging figure of glutathion inside cell; Wherein, (a) be do not add Nap-G before cell fluorescence image; B () is for adding the cell fluorescence image after Nap-G; C () is for adding cell fluorescence image after Nap-G and gsh.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, obtain all from commercial channels.
As shown in Figure 1, the preparation of embodiment 1, fluorescent probe Nap-G
Step is a): under an inert atmosphere, joined in 100mL dehydrated alcohol by bromo-for 5.00g 4-1,8-naphthalic anhydride, reinject 2.5mL n-Butyl Amine 99, at the temperature of 50 DEG C, and back flow reaction 6 hours.After reacting completely, placing spends the night has needle crystal to separate out, and filter, cold washing with alcohol three times, obtains bromo-1, the 8-naphthalimide 4.20g of intermediate N normal-butyl-4-(productive rate is 80%).
Step b): under an inert atmosphere, bromo-1, the 8-naphthalimide of 1.00g N-normal-butyl-4-and 1.87g are added in 50mL three-necked bottle to methylbenzene phenyl-sulfhydrate, inject 20mL ethylene glycol monomethyl ether and 2.1mL triethylamine, at the temperature of 0 DEG C, back flow reaction 5 hours.After reacting completely, poured into by reaction solution in 200mL frozen water, have a large amount of solid to separate out, filter, washing, vacuum-drying obtains, N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide 0.967g (productive rate 85%), yellow solid powder.
1H NMR(400MHz,CDCl
3)δ8.63(d,J＝7.9Hz,2H),8.32(d,J＝7.9Hz,1H),7.77(t,J＝7.9Hz,1H),7.45(d,J＝7.9Hz,2H),7.28(d,J＝7.9Hz,2H),7.16(d,J＝7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J＝15.2,7.6Hz,3H),1.44(dq,J＝14.8,7.3Hz,3H),0.97(t,J＝7.3Hz,4H)。
Step c): under an inert atmosphere, 0.9g N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide is dissolved in 20mL methylene dichloride, in system, adds 0.45g metachloroperbenzoic acid in three batches, room temperature reaction 1 hour.After question response is complete, is spin-dried for reaction solution, obtains final product Nap-G 0.6g (productive rate 62%) through column chromatographic isolation and purification, white solid.
1H NMR(400MHz,CDCl
3)δ8.65(d,J＝7.7Hz,1H),8.51(d,J＝7.3Hz,1H),8.40(dd,J＝8.1,2.7Hz,2H),7.68(t,J＝7.9Hz,1H),7.49(d,J＝8.1Hz,2H),7.13(d,J＝8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J＝7.4Hz,3H)；
13C NMR(100MHz,CDCl
3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82；HRMS(ESI-TOF):m/z 391.1245[M+H]
+,calc’d.391.1242.
The preparation of embodiment 2, fluorescent probe Nap-G
Step is a): under an inert atmosphere, joined in 100mL anhydrous methanol by bromo-for 5.00g 4-1,8-naphthalic anhydride, reinject 5.0mL n-Butyl Amine 99, at the temperature of 80 DEG C, and back flow reaction 1 hour.After reacting completely, placing spends the night has needle crystal to separate out, and filter, cold washing with alcohol three times, obtains bromo-1, the 8-naphthalimide 4.40g of intermediate N normal-butyl-4-(productive rate is 84%).
Step b): under an inert atmosphere, bromo-1, the 8-naphthalimide of 1.00g N-normal-butyl-4-and 3.94g are added in 50mL three-necked bottle to methylbenzene phenyl-sulfhydrate, injects 20mL butanols and 2.5mL diisopropyl ethyl amine, at the temperature of 100 DEG C, back flow reaction 1 hour.After reacting completely, poured into by reaction solution in 200mL frozen water, have a large amount of solid to separate out, filter, washing, vacuum-drying obtains, N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide 1.0g (productive rate 88%), yellow solid powder.
1H NMR(400MHz,CDCl
3)δ8.63(d,J＝7.9Hz,2H),8.32(d,J＝7.9Hz,1H),7.77(t,J＝7.9Hz,1H),7.45(d,J＝7.9Hz,2H),7.28(d,J＝7.9Hz,2H),7.16(d,J＝7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J＝15.2,7.6Hz,3H),1.44(dq,J＝14.8,7.3Hz,3H),0.97(t,J＝7.3Hz,4H)。
Step c): under an inert atmosphere, 0.9g N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide is dissolved in 20mL chloroform, in system, adds 0.45g metachloroperbenzoic acid in three batches, at the temperature of 0 DEG C, react 5 hours.After question response is complete, is spin-dried for reaction solution, obtains final product Nap-G 0.56g (productive rate 57%) through column chromatographic isolation and purification, white solid.
1H NMR(400MHz,CDCl
3)δ8.65(d,J＝7.7Hz,1H),8.51(d,J＝7.3Hz,1H),8.40(dd,J＝8.1,2.7Hz,2H),7.68(t,J＝7.9Hz,1H),7.49(d,J＝8.1Hz,2H),7.13(d,J＝8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J＝7.4Hz,3H)；
13C NMR(100MHz,CDCl
3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82；HRMS(ESI-TOF):m/z 391.1245[M+H]
+,calc’d.391.1242.
The preparation of embodiment 3, fluorescent probe Nap-G
Step is a): under an inert atmosphere, joined in the anhydrous n-propyl alcohol of 100mL by bromo-for 5.00g 4-1,8-naphthalic anhydride, reinject 7.5mL n-Butyl Amine 99, at the temperature of 120 DEG C, and back flow reaction 5 hours.After reacting completely, placing spends the night has needle crystal to separate out, and filter, cold washing with alcohol three times, obtains bromo-1, the 8-naphthalimide 4.60g of intermediate N normal-butyl-4-(productive rate is 87%).
Step b): under an inert atmosphere, bromo-1, the 8-naphthalimide of 1.00g N-normal-butyl-4-and 0.38g are added in 50mL three-necked bottle to methylbenzene phenyl-sulfhydrate, injects 20mL diethylene glycol monomethyl ether and 2.9g salt of wormwood, at the temperature of 140 DEG C, back flow reaction 48 hours.After reacting completely, poured into by reaction solution in 200mL frozen water, have a large amount of solid to separate out, filter, washing, vacuum-drying obtains, N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide 0.67g (productive rate 59%), yellow solid powder.
1H NMR(400MHz,CDCl
3)δ8.63(d,J＝7.9Hz,2H),8.32(d,J＝7.9Hz,1H),7.77(t,J＝7.9Hz,1H),7.45(d,J＝7.9Hz,2H),7.28(d,J＝7.9Hz,2H),7.16(d,J＝7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J＝15.2,7.6Hz,3H),1.44(dq,J＝14.8,7.3Hz,3H),0.97(t,J＝7.3Hz,4H)。
Step c): under an inert atmosphere, 0.9g N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide is dissolved in 20mL 1,2-ethylene dichloride, in system, adds 0.23g metachloroperbenzoic acid in three batches, reaction 24 hours under 0 degree.After question response is complete, is spin-dried for reaction solution, obtains final product Nap-G 0.32g (productive rate 34%) through column chromatographic isolation and purification, white solid.
1H NMR(400MHz,CDCl
3)δ8.65(d,J＝7.7Hz,1H),8.51(d,J＝7.3Hz,1H),8.40(dd,J＝8.1,2.7Hz,2H),7.68(t,J＝7.9Hz,1H),7.49(d,J＝8.1Hz,2H),7.13(d,J＝8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J＝7.4Hz,3H)；
13C NMR(100MHz,CDCl
3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82；HRMS(ESI-TOF):m/z 391.1245[M+H]
+,calc’d.391.1242.
The preparation of embodiment 4, fluorescent probe Nap-G
Step is a): under an inert atmosphere, joined in 100mL anhydrous normal butyl alcohol by bromo-for 5.00g 4-1,8-naphthalic anhydride, reinject 10.0mL n-Butyl Amine 99, at the temperature of 50 DEG C, and back flow reaction 20 hours.After reacting completely, placing spends the night has needle crystal to separate out, and filter, cold washing with alcohol three times, obtains bromo-1, the 8-naphthalimide 4.84g of intermediate N normal-butyl-4-(productive rate is 88%).
Step b): under an inert atmosphere, bromo-1, the 8-naphthalimide of 1.00g N-normal-butyl-4-and 2.87g are added in 50mL three-necked bottle to methylbenzene phenyl-sulfhydrate, inject 20mL ethanol and 3.5g potassium hydroxide, at the temperature of 100 DEG C, back flow reaction 12 hours.After reacting completely, poured into by reaction solution in 200mL frozen water, have a large amount of solid to separate out, filter, washing, vacuum-drying obtains, N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide 0.84g (productive rate 74%), yellow solid powder.
1H NMR(400MHz,CDCl
3)δ8.63(d,J＝7.9Hz,2H),8.32(d,J＝7.9Hz,1H),7.77(t,J＝7.9Hz,1H),7.45(d,J＝7.9Hz,2H),7.28(d,J＝7.9Hz,2H),7.16(d,J＝7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J＝15.2,7.6Hz,3H),1.44(dq,J＝14.8,7.3Hz,3H),0.97(t,J＝7.3Hz,4H)。
Step c): under an inert atmosphere, 0.9g N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide is dissolved in 20mL DMSO, in system, adds 0.30g metachloroperbenzoic acid in three batches, in 100 degree of lower reactions 1 hour.After question response is complete, is spin-dried for reaction solution, obtains final product Nap-G 0.23g (productive rate 24%) through column chromatographic isolation and purification, white solid.
1H NMR(400MHz,CDCl
3)δ8.65(d,J＝7.7Hz,1H),8.51(d,J＝7.3Hz,1H),8.40(dd,J＝8.1,2.7Hz,2H),7.68(t,J＝7.9Hz,1H),7.49(d,J＝8.1Hz,2H),7.13(d,J＝8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J＝7.4Hz,3H)；
13C NMR(100MHz,CDCl
3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82；HRMS(ESI-TOF):m/z 391.1245[M+H]
+,calc’d.391.1242.
The preparation of embodiment 5, fluorescent probe Nap-G
Step is a): under an inert atmosphere, joined in the anhydrous ethylene glycol monomethyl ether of 100mL by bromo-for 5.00g 4-1,8-naphthalic anhydride, reinject 0.5mL n-Butyl Amine 99, at the temperature of 80 DEG C, and back flow reaction 20 hours.After reacting completely, placing spends the night has needle crystal to separate out, and filter, cold washing with alcohol three times, obtains bromo-1, the 8-naphthalimide 3.2g of intermediate N normal-butyl-4-(productive rate is 60%).
Step b): under an inert atmosphere, bromo-1, the 8-naphthalimide of 1.00g N-normal-butyl-4-and 2.15g are added in 50mL three-necked bottle to methylbenzene phenyl-sulfhydrate, inject 20mL propyl alcohol and 2.0mL pyridine, at the temperature of 50 DEG C, back flow reaction 8 hours.After reacting completely, poured into by reaction solution in 200mL frozen water, have a large amount of solid to separate out, filter, washing, vacuum-drying obtains, N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide 0.76g (productive rate 67%), yellow solid powder.
1H NMR(400MHz,CDCl
3)δ8.63(d,J＝7.9Hz,2H),8.32(d,J＝7.9Hz,1H),7.77(t,J＝7.9Hz,1H),7.45(d,J＝7.9Hz,2H),7.28(d,J＝7.9Hz,2H),7.16(d,J＝7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70(dt,J＝15.2,7.6Hz,3H),1.44(dq,J＝14.8,7.3Hz,3H),0.97(t,J＝7.3Hz,4H)。
Step c): under an inert atmosphere, 0.9g N-normal-butyl-4-(to methylphenyl-sulfanyl)-1,8-naphthalimide is dissolved in 20mL acetonitrile, in system, adds 0.45g metachloroperbenzoic acid in three batches, room temperature reaction 2 hours.After question response is complete, is spin-dried for reaction solution, obtains final product Nap-G 0.55g (productive rate 57%) through column chromatographic isolation and purification, white solid.
1H NMR(400MHz,CDCl
3)δ8.65(d,J＝7.7Hz,1H),8.51(d,J＝7.3Hz,1H),8.40(dd,J＝8.1,2.7Hz,2H),7.68(t,J＝7.9Hz,1H),7.49(d,J＝8.1Hz,2H),7.13(d,J＝8.0Hz,2H),4.16-3.96(m,2H),1.68-1.53(m,2H),1.42-1.28(m,2H),0.88(t,J＝7.4Hz,3H)；
13C NMR(100MHz,CDCl
3)δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82；HRMS(ESI-TOF):m/z 391.1245[M+H]
+,calc’d.391.1242.
The spectral quality of compound shown in embodiment 6, formula (I)
Take 3.9mg Nap-G, be dissolved in 10mL DMSO, be made into mother liquor (1mM), namely obtain Nap-G test kit.This mother liquor of 100 μ L is added drop-wise in the phosphate buffered saline buffer of different concns gsh, and with corresponding phosphate buffered saline buffer constant volume to 10mL.Measure its fluorescence emission spectrum.Fluorescence emission spectrum excites with 366nm when measuring, and the intensity of emission peak is I
482; The slit width excited and launch is respectively 5/5.
Fig. 2 is the color response figure of Nap-G test kit to the gsh aqueous solution.Known by Fig. 2, after adding the gsh aqueous solution, the colour-change being observed visually solution is not obvious, but the fluorescence of solution becomes the bright green fluorescence after adding from unstressed configuration when not adding gsh.Prove that test kit of the present invention has developing response intuitively to gsh.
Fig. 3 is the fluorescence response figure of Nap-G test kit to the different gsh aqueous solution.Known by Fig. 3, along with the increase of glutathione concentrations, wavelength is that the fluorescence intensity of the emission peak at 482nm place increases gradually, proves that test kit of the present invention has sensitive " co " type response to gsh.
Fig. 4 is the fluorescent emission intensity I of Nap-G test kit under wavelength 482nm
482with glutathione concentrations relation curve.Known by Fig. 4, along with the increase of aqueous solution Glutathione peptide concentration, fluorescent emission intensity I
482increase gradually.In the scope that glutathione concentrations is 0 ~ 1mM, the fluorescent emission intensity I of emission peak
482with the linear relationship (R that the concentration of aqueous solution GSH-PX activity is good
2=0.995).Prove that test kit of the present invention is to carry out Measurement accuracy to gsh.
Fig. 5 is Nap-G test kit to the fluorescence response figure of common coexisting ion or biological micromolecule.Known by Fig. 5, the common positively charged ion that coexists, negatively charged ion, biological micromolecule add the fluorescent emission intensity I that can not make solution
482change.Prove that test kit of the present invention has outstanding selectivity to gsh.
The mensuration of embodiment 7, glutathion inside cell content
1) at 37 degree and 5% (v/v) CO
2under condition, with the DMEM culture medium culturing HeLa cell of the Streptomycin sulphate containing 10% (v/v) FBS (foetal calf serum), 100U/mL penicillin, 100 μ g/mL.Cell uses PBS buffer solution for cleaning before using.
2) in HeLa cell, add PBS (pH 7.4), then add Nap-G (10 μMs) and hatch 30min, after washing three times with PBS, carry out confocal fluorescent imaging, wherein excitation wavelength is 366nm, and collection wave band is 400-650nm.Then, in above-mentioned HeLa cell, add the phosphate buffered saline(PBS) of GSH (1mM) again, after continuing to hatch 30min, laser confocal microscope carries out imaging, wherein excitation wavelength is 366nm, and collection wave band is 400-650nm.
Known by Fig. 6, the cell being loaded with Nap-G presented fluorescence "off" state before not adding gsh, and unstressed configuration is launched; And after add gsh, cell presents fluorescence "open" state, transmitting green fluorescence, show that Nap-G is with permeate through cell membranes well, and with in cell with gsh generation specificly-response.Prove that test kit of the present invention to detect gsh in cell.
Finally it should be noted that above-described embodiment is only enumerated with Nap-G compound for fluorescent reagent, all the other fluorescent reagents due to structure and properties close, its concentration, experiment excite band selection not list one by one, but it is not intended to limit the present invention.Any those skilled in the art, without departing from the spirit and scope of the present invention, should to make various changes and modifications.
Claims (7)
1. detect a fluorescent probe for gsh, it is characterized in that: this probe is compound shown in formula (I),
In formula (I), n is the atomicity of 0 ~ 18; R
1for hydrogen, or methyl, or ethyl, or sec.-propyl, or fluorine; R
2for methyl, or hydroxyl, or carboxyl, or sulfonic group, or (triphenyl) phosphorus base, or
in any one.
2. a kind of fluorescent probe detecting gsh according to claim 1, is characterized in that: this probe structure formula such as formula (II),
3. a kind of preparation method detecting the fluorescent probe of gsh described in claim 1, it is characterized in that, the method comprises the following steps:
Step one: under an inert atmosphere, shown in formula (III), bromo-1, the 8-naphthalic anhydride of 4-and aminocompound are obtained by reacting bromo-1, the 8-naphthalimide of 4-replaced shown in formula (IV) in alcohol;
In formula (IV), n is the atomicity of 0 ~ 18, R
2for methyl, or hydroxyl, or carboxyl, or sulfonic group, or (triphenyl) phosphorus base, or
in any one;
Step 2: under an inert atmosphere, in the presence of a base, shown in formula (IV), shown in compound and formula (V), compound is obtained by reacting 4-thiophenol base-1, the 8-naphthalimide replaced shown in formula (VI) in alcohol;
In formula (VI), n is the atomicity of 0 ~ 18; R
1for hydrogen, or methyl, or ethyl, or sec.-propyl, or fluorine; R
2for methyl, or hydroxyl, or carboxyl, or sulfonic group, or (triphenyl) phosphorus base, or
in any one;
Step 3: under an inert atmosphere, compound and metachloroperbenzoic acid shown in formula (VI) react in organic solvent and obtain compound shown in formula (I).
4. a kind of preparation method detecting the fluorescent probe of gsh according to claim 3, is characterized in that:
Described in step one, aminocompound is specially n-Butyl Amine 99; Shown in formula (III), the mol ratio of bromo-1, the 8-naphthalic anhydride of 4-and described aminocompound is 1 ~ 50:1; Described alcohol is methyl alcohol, ethanol, n-propyl alcohol, Virahol, propyl carbinol or ethylene glycol monomethyl ether; Described temperature of reaction is 50 ~ 120 degree, and the reaction times is 1 ~ 20 hour;
Alcohol described in step 2 is methyl alcohol, ethanol, propyl alcohol, butanols, ethylene glycol monomethyl ether or diethylene glycol monomethyl ether; Shown in formula (IV), shown in bromo-1, the 8-naphthalimide of 4-and formula (V), the mol ratio of thiophenol compound is 1 ~ 10:1; Described alkali is organic bases or mineral alkali; Described organic bases is in triethylamine, pyridine or diisopropyl ethyl amine; Described mineral alkali is salt of wormwood, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or saleratus; Temperature of reaction is 0 ~ 140 degree; Reaction times is 1 ~ 48 hour;
The mol ratio of 4-thiophenol base-1, the 8-naphthalimide replaced shown in metachloroperbenzoic acid described in step (3) and formula (VI) is 0.5 ~ 1.5:1; Described organic solvent is chloroform, methylene dichloride, acetonitrile, DMF, DMSO or 1,2-ethylene dichloride; Temperature of reaction is 0 ~ 100 degree; Reaction times is 1 ~ 24 hour.
5. a kind of preparation method detecting the fluorescent probe of gsh according to claim 3 or 4, is characterized in that:
In step one, shown in formula (III), the mol ratio of bromo-1, the 8-naphthalic anhydride of 4-and described aminocompound is 10:1; Described alcohol is ethanol; Described temperature of reaction is 80 degree; Reaction times is 5 hours;
The ethylene glycol monomethyl ether of alcohol described in step 2; Shown in formula (IV), shown in bromo-1, the 8-naphthalimide of 4-and formula (V), the mol ratio of thiophenol compound is 5:1; Temperature of reaction is for being 100 degree; 24 hours reaction times;
In step 3, the mol ratio 1:1 of 4-thiophenol base-1, the 8-naphthalimide replaced shown in metachloroperbenzoic acid and formula (VI); Described organic solvent is methylene dichloride; Temperature of reaction is 25 degree; Reaction times is 5 hours.
6. one kind is detected the using method of the fluorescent probe of gsh; It is characterized in that, the method specifically comprises the following steps:
Step 1: the shown compound of formula (I) adding same concentrations in the buffered soln of different concns gsh, configures the standardized solution containing compound shown in formula (I) of at least 3 kinds of different glutathione contents;
Shown in buffered soln to be any one in phosphate buffer soln, Tris-HCl buffered soln, HEPES buffered soln, boric acid-sodium borate buffered soln,
Shown in the pH value of standardized solution be 6 ~ 11, specifically being 7.2;
Shown in shown standardized solution Chinese style (I), the concentration of compound is 1nM ~ 10 μM;
The content of shown standardized solution GSH-PX activity is 0.1nM ~ 1mM;
Step 2: the fluorescence emission spectrum measuring described standardized solution respectively, excitation wavelength is 366nm, take glutathione concentrations as X-coordinate, with I
482for ordinate zou, Criterion curve;
I
482represent that described standardized solution is the fluorescence emission peak intensity level at 482nm place at wavelength;
Step 3: add compound shown in formula (I) in testing sample, control its concentration equal with the concentration of compound described standardized solution Chinese style (I) Suo Shi; Measuring it is fluorescence emission spectrum under the exciting light of 366nm in excitation wavelength, namely calculates the glutathione content of testing sample according to typical curve;
Above-mentioned steps: 2 or step 3 in fluorescence intensity detect on luminoscope.
7. a kind of using method detecting the fluorescent probe of gsh according to claim 6; It is characterized in that: the buffered soln in step 1 is phosphate buffer soln.
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