CN105906534A - 2,4-Dinitrobenzene sulfonamide derivative and application thereof in mercaptan detection - Google Patents

2,4-Dinitrobenzene sulfonamide derivative and application thereof in mercaptan detection Download PDF

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CN105906534A
CN105906534A CN201610255278.2A CN201610255278A CN105906534A CN 105906534 A CN105906534 A CN 105906534A CN 201610255278 A CN201610255278 A CN 201610255278A CN 105906534 A CN105906534 A CN 105906534A
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dspdf
cys
detection
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阴彩霞
张伟杰
霍方俊
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Shanxi University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C311/21—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
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    • 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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Abstract

The invention provides a 2,4-dinitrobenzene sulfonamide derivative and an application thereof in mercaptan detection. The 2,4-dinitrobenzene sulfonamide derivative is (E)-N-(4-(4-diphenylamino)styryl)phenyl)-2,4-dinitrobenzene sulfonamide, and is called DSPDF for short. The DSPDF is used in the mercaptan detection as a detection agent, and a mercapto group fractures an amide bond in an HEPES buffer solution with the pH value of 7.4 to generate an amino group in order to realize fluorescence enhancement and realize biological mercaptan detection. A detection method has the advantages of high sensitivity and selectivity in biological mercaptan, cheap detection reagent, simple, sensitive and rapid detection process, and accurate detection result.

Description

2,4-dinitro benzene sulfamide derivative and the application in detection mercaptan thereof
Technical field
The present invention relates to biological thiol detection technique, particularly belong to a kind of with 2,4-dinitrophenyl chloride derivant, and by it As fluorometric reagent, quick, the method for quantitative fluorescence detection biological thiol.
Background technology
In organism, the change of thiol levels is closely related with a lot of diseases, such as psoriasis, cancer and acquired immune deficiency syndrome (AIDS).Internal shortage half Cystine (Cys) can cause a lot of disease, and as children growth is slow, skin injury, hair fades, liver edema.In _ blood The homocysteine (Hcy) of high concentration is the independent risk factor of coronary heart disease and cerebral infarction.Glutathion (GSH) be intracellular Rich nonprotein mercaptan, plays an important role in the oxidoreduction dynamic equilibrium maintaining cell, and the water of glutathion Put down also to have with numerous disease and cancer and directly contact.Therefore, in detection by quantitative living things system the concentration of mercaptan at biochemistry Significant with in clinical chemistry.Fluoroscopic examination have highly sensitive, detection limit is low, it is possible in monomer or intracellular reality Now visualize tracking, thus become a kind of important means of thiol class substance in detection organism at present.Therefore, business is used in development Industry can obtain, cheap reagent, and high, the highly sensitive biological thiol quantitative detecting method of easy to operate, selectivity is particularly important.
Summary of the invention:
The invention aims to overcome problem present in the detection of prior art biological thiol, it is provided that a kind of system is cheap, behaviour Make reagent and the method for fluoroscopic examination biological thiol convenient, that selectivity is high, highly sensitive.
The one 2 that the present invention provides, the derivant of 4-dinitro benzene sulfonamide, it is (E)-N-(4-(4-(diphenylamino) styryl) phenyl)-2,4-dinitrobenzenesulfonamide (it is called for short: DSPDF), in Literary fame (E)-N-(4-(4-(diphenyl amino) styryl) phenyl)-2,4-dinitro benzene sulfonamide.Its structural formula is:
The synthetic method of DSPDF, step is:
By 2,4-dinitrophenyl chloride and (E)-4-(4-aminostyryl)-N, N-diphenyl amino are 11 complete in molar ratio It is dissolved in CH2Cl2In, gradually drip triethylamine, control temperature and react 8 hours under the conditions of the ice-water bath of 0 DEG C～5 DEG C, reacted Cheng Hou, sucking filtration, washing, it is dried, mistake post obtains yellow-brown solid and is (E)-N-(4-(4-(diphenyl amino) benzene second Thiazolinyl) phenyl)-2,4-dinitro benzene sulfonamide.
The derivant of described 2,4-dinitro benzene sulfonamide can be used as detecting the reagent of biological thiol.
A kind of method detecting mercaptan that the present invention provides, is based on 2, and the derivant of 4-dinitro benzene sulfonamide, is 7.4 at pH HEPES solution detects the content of mercaptan quantitatively.This detection method, shows high susceptiveness and selectivity to mercaptan, inspection Survey process is easy, sensitive, quick, and testing result is accurate.Concrete detecting step is:
(1), preparation pH=7.4, concentration are the HEPES buffer solution of 10mM, and prepare 2mM DSPDF with DMSO DMSO solution.
(2), the DMSO solution of HEPES buffer solution and DSPDF is added to clean fluorescence cuvette by 1:1 by volume In, Fluorescence spectrophotometer detects, along with treating the addition of test sample, the fluorescence intensity of 440nm gradually strengthens；
(3), with the Cys solution of distilled water preparation 2mM, the HEPES buffer solution of 2mL and the DMSO solution of DSPDF are added In fluorescence cuvette, be gradually added into the volume of Cys solution be 6,12,18,24,30,36,42,48,54,60,66, 72,78 μ L, measure in fluorescence spectrophotometer simultaneously corresponding fluorescence intensity F of 440nm be 428.6,712.2,997.5,1292, 1612,1920,2219,2494,2726,2947,3218,3431,3614, with Cys concentration as abscissa, with relatively Fluorescence intensity F0-F is vertical coordinate drafting figure, F0133.7, obtain the working curve of Cys concentration；Equation of linear regression is: F0-F The unit of=0.9049+45.1972c, c is 10-6mol/L；
(4), when measuring sample solution, the fluorescence intensity that record is substituted into equation of linear regression, Cys can be tried to achieve, concentration.
The experiment proved that, other analyte not interference system mensuration to biological thiol.
Compared with prior art, the invention have the advantages that and effect: 1, reagent is readily synthesized and inexpensively, detection system cost Cheap；2, the detection method of the present invention, shows high susceptiveness and selectivity to biological thiol；3, detection means is simple, Have only to can realize by fluorescence spectrophotometer.
Accompanying drawing illustrates:
The nucleus magnetic hydrogen spectrum of the DSPDF of Fig. 1 a embodiment 1 preparation
The nuclear-magnetism carbon spectrum of the DSPDF of Fig. 1 b embodiment 1 preparation
The mass spectrum of the DSPDF of Fig. 1 c embodiment 1 preparation
Fig. 2 embodiment 2DSPDF and the fluorescent emission figure of Cys effect
Fig. 3 embodiment 3DSPDF and the fluorescence block diagram of various analytes
Fig. 4 embodiment 4 working curve
Fig. 5 embodiment 5 cell imaging figure
Detailed description of the invention:
Embodiment 1
0.543g 2,4-dinitrophenyl chloride and 0.598 (E)-4-(4-aminostyryl)-N, N-diphenyl amino by mole It is dissolved completely in CH than 112Cl2In, gradually dropping triethylamine 0.362g, control temperature anti-under the conditions of the ice-water bath of 0 DEG C～5 DEG C Answer 8 hours, after having reacted, sucking filtration, washing, it is dried, mistake post obtains yellow-brown solid and is (E)-N-(4-(4-(two Phenyl amino) styryl) phenyl)-2,4-dinitro benzene sulfonamide.
1H NMR (DMSO-d6,600MHz): δ (ppm): 11.12 (s, 1H), 8.90 (s, 1H), 8.61 (d, J=8.0Hz, 1H), 8.23 (d, J=8.5Hz, 1H), 7.48 (dd, J=20.4,6.9Hz, 4H), 7.31 (d, J=6.7Hz, 4H), 7.16 7.00 (m, 10H), 6.94 (d, J=7.0Hz, 2H) (Fig. 1 a).13C NMR(DMSO-d6,150MHz):δ(ppm):130.06(s),128.05 (s), 127.73 (s), 124.62 (s), 123.76 (s), 123.42 (s), 121.80 (s), 40.48 (d, J=18.3Hz), 40.28 (s), 40.14 (s), 40.00 (s), 39.86 (s), 39.72 (s), 39.59 (s) (Fig. 1 b) .ESI MS m/z:[probe+H]+Calcd.For C32H25N4O6S 592.14, Found 591.67 (Fig. 1 c).
Embodiment 2
Preparation pH=7.0, concentration are the HEPES buffer solution of 10mM, and prepare the solution of 2mM DSPDF with DMSO； The HEPES buffer solution of 2mL and the DMSO solution of 10 μ L DSPDF are added in clean fluorescence cuvette, take Cys Solution, be gradually added in this cuvette with microsyringe, sample-adding limit, limit is detected, along with Cys on Fluorescence spectrophotometer Addition, at 440nm, fluorescence intensity gradually strengthens.Fluorescent emission figure is shown in Fig. 2.
Embodiment 3
Preparation pH=7.4, concentration are the HEPES buffer solution of 10mM, and the DSPDF with DMSO preparation 2mM is molten Liquid；In 22 fluorescence cuvette, each HEPES-DMSO (1:1, pH 7.4) solution adding 2mL and 10 μ L's The DMSO solution of DSPDF, then be separately added into 8 molar equivalent Cys, Hcy, GSH, and 50 molar equivalents is various Analyte: Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, IIe, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, VaL detects on Fluorescence spectrophotometer, draw 440nm relative intensity of fluorescence corresponding to different analyte block diagram (see Fig. 3).Mercaptan makes the fluorescence intensity of DSPDF be remarkably reinforced, and other analyte does not the most cause DSPDF fluorescence intensity Change.
The experiment proved that, other analyte not interference system mensuration to mercaptan.
Embodiment 4
Preparation pH=7.4, concentration are the HEPES buffer solution of 10mM, and with the DSPDF solution of DMSO preparation 2mM, use The Cys solution of distilled water preparation 2mM；HEPES-DMSO (1:1, pH 7.4) solution and the DSPDF of 10 μ L of 2mL DMSO solution be added in fluorescence cuvette, add respectively the volume of Cys solution be 6,12,18,24,30,36,42, 48, during 54,60,66,72,78 μ L, Fluorescence spectrophotometer measures fluorescence intensity F corresponding to 440nm be 428.6, 712.2,997.5,1292,1612,1920,2219,2494,2726,2947,3218,3431,3614.Dense with Cys Degree is abscissa, with relative intensity of fluorescence F-F0Figure, F is drawn for vertical coordinate0133.7, obtain the working curve of Cys concentration；Line Property regression equation is: F0The unit of-F=0.9049+45.1972c, c is 10-6mol/L；(see Fig. 4).
Embodiment 5
Probe DMSO solution with DMSO preparation 2mM；Probe DMSO solution is added in hepatoma carcinoma cell culture fluid, Making its concentration is 20 μMs, with hepatocellular carcinoma H22 at 37 DEG C, reacts 30min, and system is at fluorescence co-focusing imager Under present blue-fluorescence；The mercaptan scavenger (NEM) of 20 μMs is joined hepatoma carcinoma cell culture fluid, adds 20 μMs of spies The DMSO solution reaction 30min of pin, system does not has fluorescent emission under fluorescence co-focusing imager；Then external source is added Cys so that its concentration is 20 μMs, with hepatocellular carcinoma H22 at 37 DEG C, then reacts 30min, and system is in fluorescence copolymerization Strong blue-fluorescence is shown under burnt imager；The probe being i.e. introduced into cell and the Cys effect subsequently entering cell, make Its Fluorescence Increasing.Fig. 5 is image A (display blueness after hepatocellular carcinoma H22 and probe effect under fluorescence co-focusing imager Fluorescence).Hepatocellular carcinoma H22 adds probe imaging figure B (not showing fluorescence) after first acting on mercaptan scavenger (NEM). Hepatocellular carcinoma H22 first act on mercaptan scavenger (NEM) after external source again Cys effect after cell imaging figure C (show Blue-fluorescence).

Claims (4)

1. one kind 2,4-dinitro benzene sulfamide derivative, referred to as DSPDF, it is characterised in that structural formula is:
2. one 2 as claimed in claim 1, the synthetic method of 4-dinitro benzene sulfamide derivative, it is characterised in that step is:
By 2,4-dinitrophenyl chloride and (E)-4-(4-aminostyryl)-N, N-diphenyl amino are 11 complete in molar ratio It is dissolved in CH2Cl2In, gradually drip triethylamine, control temperature and react 8 hours under the conditions of the ice-water bath of 0 DEG C～5 DEG C, reacted Cheng Hou, sucking filtration, washing, it is dried, mistake post obtains yellow-brown solid and is (E)-N-(4-(4-(diphenyl amino) benzene second Thiazolinyl) phenyl)-2,4-dinitro benzene sulfonamide.
3. 2,4-dinitro benzene sulfamide derivative as claimed in claim 1 is used as the reagent of detection mercaptan.
4. the method detecting mercaptan: it is characterized in that, step is:
(1), preparation pH=7.4, concentration are the HEPES buffer solution of 10mM, and prepare 2mM DSPDF with DMSO DMSO solution.
(2), the DMSO solution of HEPES buffer solution and DSPDF is added to clean fluorescence cuvette by 1:1 by volume In, Fluorescence spectrophotometer detects, along with treating the addition of test sample, the fluorescence intensity of 440nm gradually strengthens；
(3), with the Cys solution of distilled water preparation 2mM, molten to the HEPES buffer solution of 2mL and the DMSO of DSPDF Liquid is added in fluorescence cuvette, be gradually added into the volume of Cys solution be 6,12,18,24,30,36,42,48,54,60, 66,72,78uL, corresponding fluorescence intensity F simultaneously measuring 440nm in fluorescence spectrophotometer is, 428.6,712.2,997.5, 1292,1612,1920,2219,2494,2726,2947,3218,3431,3614, with Cys concentration as abscissa, With relative intensity of fluorescence F0-F is vertical coordinate drafting figure, F0133.7, obtain the working curve of Cys concentration；Equation of linear regression is: F0The unit of-F=0.9049+45.1972c, c is 10-6mol/L；
(4), when measuring sample solution, the fluorescence intensity that record is substituted into equation of linear regression, the concentration of Cys can be tried to achieve.
CN201610255278.2A 2016-04-22 2016-04-22 2,4-Dinitrobenzene sulfonamide derivative and application thereof in mercaptan detection Pending CN105906534A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106770080A (en) * 2016-11-15 2017-05-31 山西大学 A kind of Pd2+Detection reagent and its synthetic method and application
CN106905199A (en) * 2017-03-15 2017-06-30 赣南师范大学 A kind of synthesis and application of the fluorometric reagent for being used for selective enumeration method cysteine based on aggregation-induced emission principle

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103575727A (en) * 2013-10-18 2014-02-12 山西大学 Reagent and method for detecting thiol

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103575727A (en) * 2013-10-18 2014-02-12 山西大学 Reagent and method for detecting thiol

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MINGJIE WEI,ET AL.: "A new turn-on fluorescent probe for selective detection of glutathione and cysteine in living cells", 《CHEM. COMMUN》 *
WEIJIE ZHANG,ET AL.: "A turn-on fluorescent probe based on 2,4-dinitrosulfonyl functional group and its application for bioimaging", 《SENSORS AND ACTUATORS B: CHEMICAL》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106770080A (en) * 2016-11-15 2017-05-31 山西大学 A kind of Pd2+Detection reagent and its synthetic method and application
CN106905199A (en) * 2017-03-15 2017-06-30 赣南师范大学 A kind of synthesis and application of the fluorometric reagent for being used for selective enumeration method cysteine based on aggregation-induced emission principle
CN106905199B (en) * 2017-03-15 2018-04-20 赣南师范大学 A kind of synthesis and application of the fluorometric reagent for being used for selective enumeration method cysteine based on aggregation-induced emission principle

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