KR101757729B1 - Probe based on nitrobenzothiadiazole structure for selectively detect cysteine and homocysteine - Google Patents

Probe based on nitrobenzothiadiazole structure for selectively detect cysteine and homocysteine Download PDF

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KR101757729B1
KR101757729B1 KR1020150042512A KR20150042512A KR101757729B1 KR 101757729 B1 KR101757729 B1 KR 101757729B1 KR 1020150042512 A KR1020150042512 A KR 1020150042512A KR 20150042512 A KR20150042512 A KR 20150042512A KR 101757729 B1 KR101757729 B1 KR 101757729B1
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cysteine
homocysteine
cys
hcy
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KR20160116179A (en
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윤주영
윤준
이다영
김경미
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이화여자대학교 산학협력단
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/14—Thiadiazoles; Hydrogenated thiadiazoles condensed with carbocyclic rings or ring systems
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    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Abstract

The present invention relates to a nitrobenzothiadiazole structure-based probe for selectively detecting cysteine and homocysteine, wherein the nitrobenzothiadiazole structure-based probe according to the present invention maintains its structure even in an intracellular environment; (Cys, Cysteine) and homocysteine (Hcy, Homocysteine) among the amino acids including the thiol group (R-SH) such as cysteine, homocysteine and glutathione in the neutral condition (pH 7.4) Changing; Cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) are simultaneously detected in a biological sample by reacting selectively with cysteine (Cys, Cysteine) in a slightly acidic condition (pH 6.0) And can be usefully used for detection or selective detection.

Description

(Probe based on nitrobenzothiadiazole structure for selectively detecting cysteine or homocysteine)

The present invention relates to probes based on nitrobenzothiadiazole structures for selectively detecting cysteine or homocysteine.

Amino acids containing thiol groups (R-SH) maintain an equilibrium between reduced free thiol and oxidized disulfide forms and play an important role in maintaining oxidation-reduction homeostasis in vivo And is a component of numerous peptides to be performed (Non-Patent Document 1).

In Non-Patent Document 2, abnormal levels of amino acids including in-vivo thyroids (R-SH) may be caused by the occurrence of certain diseases such as liver damage, cancer, AIDS, osteoporosis, Alzheimer's disease, inflammatory bowel disease, And the like. That is, if the level of the biothiol, which is an amino acid containing the in-vivo thiol group (R-SH), is evaluated, early diagnosis will be possible for some related diseases.

Accordingly, various fluorescent probes for detecting amino acids including a thiol group (R-SH) selectively among various amino acids in vivo have been developed. Specifically, Non-Patent Document 3 discloses a probe that a thiol group undergoes a unique nucleophilic addition and substitution reaction. However, in the case of the above probe, an amino acid including a thiol group such as cysteine (Cys, cysteine), homocysteine (Hcy, Homocysteine), glutathione (GSH, Glutathione) among bio- thiol which is an amino acid including a thiol group And the respective amino acids can not be selectively detected.

Cysteine and homocysteine in the biothiol are related to cellular functions such as intracellular redox activity, xenobiotic metabolism, intracellular signal transduction and gene regulation. This is especially important.

Several fluorescent probes based on organic dyes have been developed to selectively detect cysteine (Cys, cysteine) or homocysteine (Hcy, Homocysteine). For example, Non-Patent Document 4 discloses a probe that can be used for selectively detecting cysteine (Cys, cysteine) in the above-described biothyrol. In addition, Non-Patent Document 5 discloses a probe based on a BODIPY structure that can be used for selectively detecting homocysteine (Hcy, homocysteine) in the above biosyield.

However, the probes can be used for detecting only cysteine (Cys, Cysteine) independently (Non-Patent Document 4) or for detecting homocysteine (Hcy, Homocysteine) (Cys, cysteine) and homocysteine (Hcy, Homocysteine) can not be simultaneously detected or selectively detected.

The inventors of the present invention have been studying to develop a probe for simultaneously detecting or selectively detecting cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) in a biological sample with a single probe, The thiadiazole structure-based probe retains its structure even in the intracellular environment; (Cys, Cysteine) and homocysteine (Hcy, Homocysteine) among the amino acids including the thiol group (R-SH) such as cysteine, homocysteine and glutathione in the neutral condition (pH 7.4) Changing; Cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) are simultaneously detected in a biological sample by reacting selectively with cysteine (Cys, Cysteine) in a slightly acidic condition (pH 6.0) The present invention has been completed based on this finding.

Wood, Z. A .; Schroder, E .; Robin Harris, J .; Poole, L. B. Trends Biochem. Sci. 2003, 28, 32. Herzenberg, L. A .; De Rosa, S. C .; Dubs, J. G .; Roederer, M .; Anderson, M. T .; Ela, S. W .; Deresinski S. C .; Herzenberg, L. A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1967. Yin, L.-L .; Chen, Z.-Z .; Tong, L.-L .; Xu, K.-H .; Tang, B. Chin. J. Analy. Chem. 2009, 37, 1073. RSC Adv., 2014, 4, 29340-29343. Chem. Commun., 2014, 50, 13668-13671.

It is an object of the present invention to provide a compound based on a nitrobenzothiadiazole structure for selectively sensing cysteine or homocysteine.

Another object of the present invention is to provide a process for producing the above compound.

It is a further object of the present invention to provide a chemical sensor for the detection of cysteine or homocysteine comprising said compound.

It is another object of the present invention to provide a method for detecting cysteine or homocysteine using the chemical sensor.

In order to achieve the above object, the present invention provides a compound represented by the following general formula (1).

[Chemical Formula 1]

Figure 112015029794135-pat00001

In Formula 1,

R 1 , R 2 and R 3 are independently selected from the group consisting of -H, -OH, -CN, -NO 2 , halogen, C 1-10 linear or branched alkyl, C 1-10 linear or branched alkoxy or -NR 4 R 5 ,

R 4 and R 5 are independently -H, C 1-10 straight or branched alkyl or C 1-10 straight or branched alkoxy.

The present invention also relates to a process for producing a compound represented by the formula (1)

A step of reacting a compound represented by the formula (2) with a compound represented by the formula (3) to prepare a compound represented by the formula (1) (step 1).

[Reaction Scheme 1]

Figure 112015029794135-pat00002

In the above Reaction Scheme 1,

R 1 , R 2 and R 3 are independently as defined in the above formula (1).

Furthermore, the present invention provides a chemical sensor for detecting cysteine or homocysteine comprising the above compound.

Further, the present invention provides a method for detecting a chemical substance, comprising the steps of: (1) adding the chemical sensor to a sample;

Irradiating the sample prepared in the step 1 with a light source (step 2); And

(Step 3) of measuring a change in absorption or fluorescence characteristic of the cysteine or homocysteine, either singly or in combination.

The nitrobenzothiadiazole structure-based probe according to the present invention maintains its structure even in an intracellular environment; (Cys, Cysteine) and homocysteine (Hcy, Homocysteine) among the amino acids including the thiol group (R-SH) such as cysteine, homocysteine and glutathione in the neutral condition (pH 7.4) Changing; Cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) are simultaneously detected in a biological sample by reacting selectively with cysteine (Cys, Cysteine) in a slightly acidic condition (pH 6.0) And can be usefully used for detection or selective detection.

FIG. 1 (A) is a graph showing changes in the absorption spectrum observed when the compound prepared in Example 1 was reacted with cysteine (Cys, cysteine), homocysteine (Hcy, homocysteine) and glutathione (GSH, Glutathione) Observed image.
FIG. 1 (B) is a graph showing fluorescence emission spectra obtained by reacting the compound prepared in Example 1 with cysteine (Cys, cysteine), homocysteine (Hcy), and glutathione (GSH, Glutathione) And an illustration is an image showing a sample in which fluorescence emission of each sample is observed.
FIG. 2 is a graph showing the relationship between the compound prepared in Example 1 and cysteine (Cys, Cysteine), homocysteine (Hcy), glutathione (GSH), glutathione, Alanine, Ala, arginine Asparagine, Asp, Asp, Dithiothreitol, Dtt, Glutamine, Gln, Glutamic acid, Glu, Glycine, Gly, Histidine, His, Isoleucine, Ile, Lysine, Lys, Methionine, Met, Phenylalanine, Phe, Proline, Pro, Serine, Ser), Tryptophan (Trp), Tyrosine (Tyr), and Valine (Val) were reacted to observe the relative fluorescence intensity of 533 nm wavelength band.
FIG. 3 is an image obtained by reacting the compound prepared in Example 1 with various concentrations of cysteine (Cys, Cysteine) and observing changes in normalized fluorescence responses.
4 is an image obtained by reacting the compound prepared in Example 1 with various concentrations of homocysteine (Hcy, Homocysteine) and observing changes in normalized fluorescence responses.
FIG. 5 is an image of fluorescence emission (533 nm wavelength region) generated by reacting the compound prepared in Example 1 and cysteine (Cys, Cysteine) with time.
FIG. 6 is an image of fluorescence emission (533 nm wavelength region) generated by reacting the compound prepared in Example 1 with homocysteine (Hcy, Homocysteine) observed over time.
FIG. 7A is a graph showing changes in absorption spectrum observed when the compound prepared in Example 1, cysteine (Cys, cysteine), homocysteine (Hcy, homocysteine) and glutathione (GSH, Glutathione) Observed image.
FIG. 7 (B) shows fluorescence emission spectra obtained by reacting the compound prepared in Example 1 with cysteine (Cys, cysteine), homocysteine (Hcy, homocysteine) and glutathione (GSH, Glutathione) And an illustration is an image showing a sample in which fluorescence emission of each sample is observed.
FIG. 8 is a graph showing the effect of the compound prepared in Example 1 and cysteine (Cys, cysteine), homocysteine (Hcy), glutathione (GSH), glutathione, Alanine, Ala, arginine Asparagine, Asp, Asp, Dithiothreitol, Dtt, Glutamine, Gln, Glutamic acid, Glu, Glycine, Gly, Histidine, His, Isoleucine, Ile, Lysine, Lys, Methionine, Met, Ornithine, Orn, Phenylalanine, Phe, Serine, , Ser), Threonine (Thr), Tryptophan (Trp), and Valine (Val) were reacted to observe the relative fluorescence intensity of 533 nm wavelength band.
FIG. 9 is an image showing a change in fluorescence emission (533 nm wavelength region) caused by reaction between the compound prepared in Example 1 and cysteine (Cys, Cysteine) with time.
10 is an image showing the change in fluorescence emission (533 nm wavelength region) caused by reaction of the compound prepared in Example 1 with homocysteine (Hcy, Homocysteine) over time.
FIG. 11 is a graph showing changes observed by titrating the compound prepared in Example 1 with cysteine (Cys, Cysteine) at a concentration of 0-500 μM through a fluorescence emission spectrum (RF-5301 / PC fluorescence spectrophotometer, Shimadzu) It is an image.
FIG. 12 is a graph showing changes observed by titrating and reacting the compound prepared in Example 1 with homocysteine (Hcy, Homocysteine) at a concentration of 0-500 μM through a fluorescence emission spectrum (RF-5301 / PC fluorescence spectrophotometer, Shimadzu) It is an image.
13 (a) is a fluorescence image and differential interference channel observed by HeLa cells cultured together with the compound prepared in Example 1 using a confocal laser scanning microscope under neutral conditions.
Observing the HeLa cells incubated with the compound prepared in were pretreated with ethyl maleimide (N -ethylmaleimide, NEM), Example 1 - Fig. 13 (b) is N, using the confocal laser scanning microscope at neutral conditions Fluorescence image and differential interference channel.
Were incubated with a solution of the compound prepared in Example 1 was pretreated with ethyl maleimide (N -ethylmaleimide, NEM), the addition of cysteine, and - Fig. 13 (c) is N, using the confocal laser scanning microscope at neutral conditions HeLa cells are fluorescent image and differential interference channel.
Were incubated with a solution of the compound prepared in Example 1 was pretreated with ethyl maleimide (N -ethylmaleimide, NEM), the addition of homocysteine, and - Fig. 13 (d) is N, using the confocal laser scanning microscope at neutral conditions HeLa cells are fluorescent image and differential interference channel.
Were incubated with a solution of the compound prepared in were pretreated with ethyl maleimide (N -ethylmaleimide, NEM), in Example 1 the addition of glutathione, and - Fig. 13 (e) is N, using the confocal laser scanning microscope at neutral conditions HeLa cells are fluorescent image and differential interference channel.
Fig. 14 (a) is a fluorescence image and differential interference channel observing HeLa cells cultured together with the compound prepared in Example 1 using a confocal laser scanning microscope under slightly acidic conditions. Fig.
Observing the HeLa cells incubated with the compound prepared in were pretreated with ethyl maleimide (N -ethylmaleimide, NEM), Example 1 - Fig. 14 (b), using the confocal laser scanning microscope in a weakly acidic condition N Fluorescence image and differential interference channel.
Were incubated with a solution of the compound prepared in Example 1 was pretreated with ethyl maleimide (N -ethylmaleimide, NEM), the addition of cysteine, and - Fig. 14 (c), using the confocal laser scanning microscope in a weakly acidic condition N HeLa cells are fluorescent image and differential interference channel.
Were incubated with a solution of the compound prepared in Example 1 was pretreated with ethyl maleimide (N -ethylmaleimide, NEM), the addition of homocysteine, and - Fig. 14 (d), using the confocal laser scanning microscope in a weakly acidic condition N HeLa cells are fluorescent image and differential interference channel.
15 is an image for evaluating cytotoxicity of the compound prepared in Example 1. Fig.

Hereinafter, the present invention will be described in detail.

The present invention provides a compound represented by the following formula (1).

[Chemical Formula 1]

Figure 112015029794135-pat00003

In Formula 1,

R 1 , R 2 and R 3 are independently selected from the group consisting of -H, -OH, -CN, -NO 2 , halogen, C 1-10 linear or branched alkyl, C 1-10 linear or branched alkoxy or -NR 4 R 5 ,

R 4 and R 5 are independently -H, C 1-10 straight or branched alkyl or C 1-10 straight or branched alkoxy.

Preferably,

R 1 , R 2 and R 3 are independently -H, halogen, C 1-10 linear or branched alkyl, C 1-10 straight or branched alkoxy or -NR 4 R 5 ,

R 4 and R 5 are independently -H or C 1-10 straight-chain or branched alkyl.

More preferably,

R 1 , R 2 and R 3 are independently -H or -NR 4 R 5 ,

Wherein R < 4 > and R < 5 > are independently -H.

Preferable examples of the compound represented by the formula (1) according to the present invention include the following compounds.

(1) 4- (7-Nitrobenzo [c] [1,2,5] thiadiazol-4-ylthio) benzene amine.

The compound can be used for selectively detecting cysteine or homocysteine among various amino acids present in a cell.

The present invention also relates to a process for producing a compound represented by the formula (1)

A step of reacting a compound represented by the formula (2) with a compound represented by the formula (3) to prepare a compound represented by the formula (1) (step 1).

[Reaction Scheme 1]

Figure 112015029794135-pat00004

In the above Reaction Scheme 1,

R 1 , R 2 and R 3 are independently as defined in the above formula (1).

Hereinafter, the process for preparing the compound represented by the formula (1) according to the present invention will be described in detail.

In the process for preparing the compound represented by the formula (1) according to the present invention, the above step 1 is a step of reacting the compound represented by the formula (2) with the compound represented by the formula (3) to prepare the compound represented by the formula Specifically, the compound represented by the formula (1) is dissolved in a solvent and the compound represented by the formula (1) is added to the triethylamine mixture and stirred to prepare the compound represented by the formula (1).

At this time, as the reaction solvent, tetrahydrofuran; Dioxane; Ether solvents including ethyl ether, 1,2-dimethoxyethane and the like; Lower alcohols including methanol, ethanol, propanol and butanol; But are not limited to, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonitrile, acetonazenesulfonate, toluene sulfonate, chlorobenzene sulfonate, Sulfonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate, naphthalene-2-sulphonate , Mandelate and the like can be used, and it is preferable to use tetrahydrofuran.

The reaction temperature is preferably between 0 ° C and the boiling point of the solvent. The reaction time is not particularly limited, but it is preferably 0.5-10 hours.

Furthermore, the present invention provides a chemical sensor for detecting cysteine or homocysteine comprising the above compound. Here, the chemical sensor is characterized by detecting cysteine or homocysteine in a biological sample.

Further, the present invention provides a method for detecting a chemical substance, comprising the steps of: (1) adding the chemical sensor to a sample;

Irradiating the sample prepared in the step 1 with a light source (step 2); And

(Step 3) of measuring a change in absorption or fluorescence characteristic of the cysteine or homocysteine, either singly or in combination.

Hereinafter, the method of detecting cysteine or homocysteine according to the present invention will be described step by step.

In the method for detecting cysteine or homocysteine according to the present invention, step 1 is a step of adding the chemical sensor to a sample. Here, the sample is preferably a biological sample, and most preferably a HeLa cell.

In the method for detecting cysteine or homocysteine according to the present invention, step 2 is a step of irradiating a sample prepared in step 1 with a light source. Here, the light source is preferably irradiated using a fluorescence spectrophotometer or UV absorption spectroscopy.

In the method for detecting cysteine or homocysteine according to the present invention, step 3 is a step of measuring changes in absorbance or fluorescence characteristics singly or in combination. Specifically, a phenyl derivative containing the sulfur atom of the compound represented by the formula (1) (

Figure 112015029794135-pat00005
Cysteine or homocysteine is separated from the cysteine or homocysteine by the action of a cysteine by itself or by mixing a change in the absorption or fluorescence characteristic caused by the change of the - Or homocysteine can be detected.

The nitrobenzothiadiazole structure-based compound according to the present invention maintains its structure even in the intracellular environment and selectively reacts with exogenous or endogenous cysteine or homocysteine to change its absorption or fluorescence spectrum The following series of experiments were conducted to prove this.

First, the experiment was conducted to evaluate the selectivity for cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) under neutral conditions. As a result, the compound prepared in Example 1 showed cysteine (Cys, Cysteine (Cys, Cysteine) and homocysteine (Hcy, Homocysteine) in homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione) 1, and FIG. 2 of Experimental Example 2).

Experiments were conducted to evaluate the changes in normalized fluorescence responses depending on the concentration of cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) under neutral conditions. As a result, cysteine (Cys, cysteine) or homocysteine Hcy, Homocysteine) was increased, the degree of luminescence of normalized fluorescence responses was increased (see FIGS. 3 and 4 of Experimental Example 3).

Further, experiments were carried out to evaluate the stability of fluorescence over time of fluorescence emission by cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) under neutral conditions. As a result, the compound prepared in Example 1 and cysteine , Fluorescence emission (533 nm wavelength region) generated by the reaction of cysteine or homocysteine (Hcy, Homocysteine) was converged and increased with respect to a specific fluorescence intensity over time, 4 and Fig. 5 and Fig. 6).

As a result of conducting experiments to evaluate the selectivity to cysteine (Cys, cysteine) under a slightly acidic condition, the compound prepared in Example 1 was found to have cysteine (Cys, cysteine), homocysteine (Hcy, Homocysteine and glutathione (GSH, Glutathione) were observed to react with cysteine (Cys, Cysteine) to show changes in absorption or fluorescence emission spectra (FIG. 7 of Experimental Example 5 and FIG. 8 of Experimental Example 6).

Furthermore, experiments were carried out to evaluate the stability of fluorescence emission of cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) over time in a slightly acidic condition. As a result, the compound prepared in Example 1 and cysteine , Fluorescence emission (533 nm wavelength region) generated by the reaction of cysteine or homocysteine (Hcy, Homocysteine) was converged and increased with respect to a specific fluorescence intensity over time, 9 and 10 of Fig. 7).

Cysteine (Cys), homocysteine (Hcy) and homocysteine (Hcy) were measured in the neutral condition to evaluate the absorption or fluorescence spectrum change according to the concentrations of cysteine and homocysteine. And the degree of fluorescence emission of the 535 nm wavelength band increases as the concentration of the dye increases. (See FIGS. 11 and 12 of Experimental Example 8).

Further, the present inventors conducted experiments to evaluate the detection ability of intracellular cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) under neutral conditions. As a result, (R-SH) contained in the cytoplasm, selectively reacts with cysteine and homocysteine to generate green fluorescence, and thus it can be used for selectively detecting cysteine and homocysteine 13 of Experimental Example 9).

Further, as a result of conducting experiments to evaluate intracellular cysteine (Cys, cysteine) detection ability under weakly acidic conditions, the compound prepared in Example 1 infiltrated into HeLa cells in a slightly acidic condition (pH 7.4) (R-SH), it was confirmed that green fluorescence was generated selectively by reacting with cysteine selectively among the amino acids including R-SH (R-SH).

Furthermore, when the compound prepared in Example 1 was incubated with HeLa cells at 0 to 3 μM, the survival rate of the cells was found to be about 85 % (See Fig. 15 of Experimental Example 11).

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the present invention is not limited thereto.

< Example  1 > 4- (7- Nitrobenzo [c] [1,2,5] thiadiazole -4- Ithio ) Benzene amine  Produce

Figure 112015029794135-pat00006

To a solution of 4-aminothiophenol (125 mg, 1.0 mmol) and triethylamine (303 mg, 3.0 mmol) in dry THF (20 mL) was added 4-chloro- 5] thiadiazole (215 mg, 1.0 mm) was added. The mixture was refluxed for 4 hours under a nitrogen atmosphere. After cooling to room temperature, the solvent was removed, washed with water and extracted with dichloromethane. When the crude product was formed, it was purified by column chromatography (eluent: hexane / dichloromethane 1: 1) to give the title compound (yield: 93%).

1 H NMR (300 MHz, CDCl 3) δ 6.66 (d, 1H), 6.83 (d, 2H), 7.22 (d, 2H), 8.24 (d, 1H), 4.10 (NH 2);

13 C NMR (75 MHz, CDCl 3) δ 149.36, 148.34, 145.12, 142.55, 137.22, 130.83, 120. 82, 116.43, 112.31; And

FAB-MS m / z = XXXX [M + H] +, calcd for C 12 H 8 N 4 O 2 S 2 = 304.35.

< Experimental Example  1 > Cysteine in neutral condition ( Cys , Cysteine ) And homocysteine Hcy , Homocysteine) 1

Among the cysteine (Cys, Cysteine), homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione) in the biothiol which is an amino acid containing thiol group (R-SH) Cysteine) and homocysteine (Hcy, Homocysteine) under neutral (pH 7.4) conditions.

10 μM of the compound prepared in Example 1 and 100 μM of each of HEPES (4- ((4-hydroxyphenyl) -1,3-dioxolan-2-ylmethyl) glycine, Cys, Cysteine, Hcy, Homocysteine and Glutathione (UVIKON 933 Double Beam UV / VIS Spectrometer) at 25 ° C, and the pH of the solution was measured by the same method as in Example 1, Fluorescence emission spectra (RF-5301 / PC fluorescence spectrophotometer, Shimadzu) were observed. The results are shown in Figs. 1 (A) and 1 (B), respectively.

FIG. 1 (A) is a graph showing changes in the absorption spectrum observed when the compound prepared in Example 1 was reacted with cysteine (Cys, cysteine), homocysteine (Hcy, homocysteine) and glutathione (GSH, Glutathione) Observed image.

FIG. 1 (B) is a graph showing fluorescence emission spectra obtained by reacting the compound prepared in Example 1 with cysteine (Cys, cysteine), homocysteine (Hcy), and glutathione (GSH, Glutathione) And an illustration is an image showing a sample in which fluorescence emission of each sample is observed.

As shown in FIG. 1 (A), the compound prepared in Example 1 reacted with cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) at a neutral (pH 7.4) While glutathione (GSH, Glutathione) showed similar absorption to the untreated group.

1 (B), the compound prepared in Example 1 reacted with cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) under neutral (pH 7.4) , Whereas glutathione (GSH, Glutathione) showed similar fluorescence emission to the untreated group.

Therefore, the compound prepared in Example 1 was found to have cysteine (Cys, cysteine), homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione) Can be usefully used for selectively detecting the signal.

< Experimental Example  2 > Cysteine in neutral condition ( Cys , Cysteine ) And homocysteine Hcy , Homocysteine) 2

Cysteine (Cys, Cysteine), homocysteine and glutathione (GSH, Glutathione) as well as various kinds of amino acids used in Experimental Example 1, And homocysteine (Hcy, Homocysteine), the following experiment was conducted.

In addition to cysteine (Cys, cysteine), homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione), alanine (Ala), arginine (Arg), asparagine, Asn, aspartic acid , Asp, dithiothreitol (Dtt), glutamine, Gln, glutamic acid, Glu, glycine, Gly, histidine, His, isoleucine, , Lysine, Lys, Methionine, Met, Phenylalanine, Phe, Proline, Pro, Serine, Ser, Tryptophan, Trp, Tyrosine, The relative fluorescence intensity at 533 nm was evaluated in the same manner as in Experimental Example 1, except that Valine (Val) was further used. The results are shown in FIG.

FIG. 2 is a graph showing the relationship between the compound prepared in Example 1 and cysteine (Cys, Cysteine), homocysteine (Hcy), glutathione (GSH), glutathione, Alanine, Ala, arginine Asparagine, Asp, Asp, Dithiothreitol, Dtt, Glutamine, Gln, Glutamic acid, Glu, Glycine, Gly, Histidine, His, Isoleucine, Ile, Lysine, Lys, Methionine, Met, Phenylalanine, Phe, Proline, Pro, Serine, Ser), Tryptophan (Trp), Tyrosine (Tyr), and Valine (Val) were reacted to observe the relative fluorescence intensity of 533 nm wavelength band.

As shown in FIG. 2, the compound prepared in Example 1 reacted with cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) under neutral (pH 7.4) conditions and was found to exhibit strong fluorescence emission at a wavelength of 533 nm On the other hand, fluorescence emission was not observed for the remaining amino acids.

Therefore, the compound prepared in Example 1 can be used to selectively detect only cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) among various amino acids under neutral (pH 7.4) conditions.

< Experimental Example  3> Cysteine in neutral condition ( Cys , Cysteine ) And homocysteine Hcy , Homocysteine) in the presence or absence of normalized fluorescence responses

The concentration of cysteine (Cys, Cysteine) and the concentration of the compound prepared in Example 1 at constant concentration (1 μM) in neutral conditions and concentrated in DMSO-HEPES (0.01 M, pH 7.4) (1:99, v / v) The changes in normalized fluorescence responses observed by reacting homocysteine (Hcy, Homocysteine) were evaluated by fluorescence emission spectra (RF-5301 / PC fluorescence spectrophotometer, Shimadzu) Respectively.

FIG. 3 is an image obtained by reacting the compound prepared in Example 1 with cysteine (Cys, Cysteine) at various concentrations and observing changes in normalized fluorescence responses.

4 is an image obtained by reacting the compound prepared in Example 1 with various concentrations of homocysteine (Hcy, Homocysteine) and observing changes in normalized fluorescence responses.

As shown in FIG. 3, as the concentration of cysteine (Cys, Cysteine) was increased, the degree of luminescence of normalized fluorescence responses was increased. Here, it was confirmed that the lower limit of detection of cysteine (Cys, cysteine) was 9.93056 × 10 -7 M.

Also, as shown in FIG. 4, as the concentration of homocysteine (Hcy, Homocysteine) was increased, the degree of luminescence of normalized fluorescence responses was increased. Here, it was confirmed that the lower limit of detection of homocysteine (Hcy, Homocysteine) was 5.91741 × 10 -7 M.

< Experimental Example  4> In the neutral condition, cysteine ( Cys , Cysteine ) And homocysteine Hcy , Homocysteine) on the stability of fluorescence emission over time

(Cys, Cysteine) or homocysteine (DMSO) concentrated at a constant concentration (10 μM) in the neutral condition and the compound prepared in Example 1 and concentrated in DMSO-HEPES (0.01 M, pH 7.4) (RF-5301 / PC fluorescence spectrophotometer, Shimadzu) in order to evaluate whether the fluorescence emission generated by the reaction of Hcy and Homocysteine stably stays over time, Are shown in Fig. 5 and Fig. 6, respectively.

FIG. 5 is an image of fluorescence emission (533 nm wavelength region) generated by reacting the compound prepared in Example 1 and cysteine (Cys, Cysteine) with time.

FIG. 6 is an image of fluorescence emission (533 nm wavelength region) generated by reacting the compound prepared in Example 1 with homocysteine (Hcy, Homocysteine) observed over time.

As shown in FIG. 5, fluorescence emission (533 nm wavelength region) generated by reacting the compound prepared in Example 1 with cysteine (Cys, Cysteine) converges to a specific fluorescence intensity with time, The luminescence holding stability was high.

Further, as shown in FIG. 6, fluorescence emission (533 nm wavelength region) generated by reacting the compound prepared in Example 1 with homocysteine (Hcy, Homocysteine) converges and increases with respect to a specific fluorescence intensity over time And the fluorescence emission retention stability was high.

< Experimental Example  5> Under slightly acidic conditions, cysteine ( Cys , Cysteine ) 1

Among the cysteine (Cys, Cysteine), homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione) in the biothiol which is an amino acid containing thiol group (R-SH) Cysteine (pH 6.0), the following experiment was carried out.

10 μM of the compound prepared in Example 1 and 100 μM of cysteine (Cys, Cysteine), homocysteine (Hcy, Homocysteine) and glutathione (GSH) were dissolved in citric acid containing 1% dimethylsulfoxide (DMSO) The fluorescence emission spectrum (RF-5301 / PC fluorescence spectrophotometer) was measured at 25 ° C using a UV absorption spectrometer (UVIKON 933 Double Beam UV / VIS Spectrometer) and added to -Na 2 HPO 4 (0.01 M, pH = 6.0) , Shimadzu) were observed. The results are shown in Figs. 7 (A) and 7 (B), respectively.

FIG. 7A is a graph showing changes in absorption spectrum observed when the compound prepared in Example 1, cysteine (Cys, cysteine), homocysteine (Hcy, homocysteine) and glutathione (GSH, Glutathione) Observed image.

FIG. 7 (B) shows fluorescence emission spectra obtained by reacting the compound prepared in Example 1 with cysteine (Cys, cysteine), homocysteine (Hcy, homocysteine) and glutathione (GSH, Glutathione) And an illustration is an image showing a sample in which fluorescence emission of each sample is observed.

As shown in FIG. 7 (A), the compound prepared in Example 1 reacted with cysteine (Cys) at a slightly acidic (pH 6.0) condition and showed strong absorption at a wavelength of 475 nm, while homocysteine , Homocysteine) and glutathione (GSH, Glutathione) showed similar absorption to the untreated group.

As shown in FIG. 7 (B), the compound prepared in Example 1 reacted with cysteine (Cys, Cysteine) at a slightly acidic (pH 6.0) condition and was found to exhibit strong fluorescence emission at a wavelength of 535 nm, Homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione) showed similar fluorescence emission to the untreated group.

Therefore, the compound prepared in Example 1 selectively detects only cysteine (Cys, Cysteine) among cysteine (Cys, cysteine), homocysteine (Hcy) and glutathione (GSH) It can be useful.

< Experimental Example  6> Under slightly acidic conditions, cysteine ( Cys , Cysteine ) Selectivity evaluation 2

Cysteine (Cys, Cysteine), homocysteine and glutathione (GSH, Glutathione), as well as various kinds of amino acids used in Experimental Example 5, The following experiments were carried out in order to evaluate the selectivity with respect to.

In addition to cysteine (Cys, cysteine), homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione), alanine (Ala), arginine (Arg), asparagine, Asn, aspartic acid , Asp, dithiothreitol (Dtt), glutamine, Gln, glutamic acid, Glu, glycine, Gly, histidine, His, isoleucine, , Lysine, Lys, Methionine, Met, Ornithine, Orn, Phenylalanine, Phe, Serine, Threonine, Thr, Tryptophan, And Valine (Val) were further used in the same manner as in Experimental Example 1, and the relative fluorescence intensity at 533 nm was evaluated. The results are shown in FIG.

FIG. 8 is a graph showing the effect of the compound prepared in Example 1 and cysteine (Cys, cysteine), homocysteine (Hcy), glutathione (GSH), glutathione, Alanine, Ala, arginine Asparagine, Asp, Asp, Dithiothreitol, Dtt, Glutamine, Gln, Glutamic acid, Glu, Glycine, Gly, Histidine, His, Isoleucine, Ile, Lysine, Lys, Methionine, Met, Ornithine, Orn, Phenylalanine, Phe, Serine, , Ser), Threonine (Thr), Tryptophan (Trp), and Valine (Val) were reacted to observe the relative fluorescence intensity of 533 nm wavelength band.

As shown in FIG. 8, it was found that the compound prepared in Example 1 reacted with cysteine (Cys) only at a slightly acidic (pH 6.0) condition and showed strong fluorescence emission at a wavelength of 533 nm, The fluorescence emission was not observed.

Therefore, the compound prepared in Example 1 can be used to selectively detect only cysteine (Cys, cysteine) among various amino acids under a slightly acidic condition (pH 6.0).

< Experimental Example  7> Cysteine in weakly acidic conditions Cys , Cysteine ) And homocysteine Hcy , Homocysteine) on the stability of fluorescence emission over time

The concentration of the compound prepared in Example 1 and the citric acid -Na 2 HPO 4 (0.01 M, pH = 6.0) containing 1% dimethylsulfoxide (DMSO) at a constant concentration (10 μM) (RF-5301 / PC fluorescence spectrophotometer, Shimadzu) was used in order to evaluate whether fluorescence emission caused by the reaction of 100 μM cysteine (Cys, cysteine) or homocysteine (Hcy, Homocysteine) ). The results are shown in Figs. 9 and 10, respectively.

FIG. 9 is an image showing a change in fluorescence emission (533 nm wavelength region) caused by reaction between the compound prepared in Example 1 and cysteine (Cys, Cysteine) with time.

10 is an image showing the change in fluorescence emission (533 nm wavelength region) caused by reaction of the compound prepared in Example 1 with homocysteine (Hcy, Homocysteine) over time.

As shown in FIG. 9, fluorescence emission (533 nm wavelength region) generated by reacting the compound prepared in Example 1 with cysteine (Cys, Cysteine) converges to a specific fluorescence intensity with time, The luminescence holding stability was high.

10, fluorescence emission (533 nm wavelength region) generated by reacting the compound prepared in Example 1 with homocysteine (Hcy, Homocysteine) was observed when the cysteine (Cys, Cysteine) was used over time And the fluorescence emission stability was higher than that of the control.

< Experimental Example  8 > Cysteine in neutral condition ( Cys , Cysteine ) And homocysteine Hcy , Homocysteine) concentration or fluorescence spectra

(Cys, Cysteine) or homocysteine (Hcy) in concentrated condition (10 μM) in the neutral condition and the compound prepared in Example 1 and concentrated in DMSO-HEPES (0.01 M, pH 7.4) , Homocysteine) was titrated to 0-500 μM, and the changes observed were evaluated by fluorescence emission spectra (RF-5301 / PC fluorescence spectrophotometer, Shimadzu), and the results are shown in FIGS. 11 and 12, respectively .

FIG. 11 is a graph showing changes observed by titrating the compound prepared in Example 1 with cysteine (Cys, Cysteine) at a concentration of 0-500 μM through a fluorescence emission spectrum (RF-5301 / PC fluorescence spectrophotometer, Shimadzu) It is an image.

FIG. 12 is a graph showing changes observed by titrating and reacting the compound prepared in Example 1 with homocysteine (Hcy, Homocysteine) at a concentration of 0-500 μM through a fluorescence emission spectrum (RF-5301 / PC fluorescence spectrophotometer, Shimadzu) It is an image.

As shown in FIG. 11, the fluorescence intensity of the 535 nm wavelength region increases with an increase in the appropriate concentration of cysteine (Cys, Cysteine).

Also, as shown in FIG. 12, the fluorescence emission intensity at 535 nm wavelength region increased with increasing the proper concentration of homocysteine (Hcy, Homocysteine).

Therefore, it was confirmed that the fluorescence intensity induced by the reaction of the compound prepared in Example 1 with cysteine (Cys, cysteine) or homocysteine (Hcy, Homocysteine) was concentration-dependent.

< Experimental Example  9> In the neutral condition, intracellular cysteine Cys , Cysteine ) And homocysteine (Hcy, Homocysteine ) Detection ability evaluation

Among the cysteine (Cys, Cysteine), homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione) in the biothiol which is an amino acid containing a thiol group (R-SH) In order to evaluate the selectivity of cysteine (Cys, cysteine) and homocysteine (Hcy, Homocysteine) in cells, the following experiment was conducted.

10% heat-inactive fetal calf serum (Fetal Bovine Serum, FBS), 100 U / mL penicillin and 100 U / mL streptomycin is added to the DMEM (Dulbecco Modified Eagle Medium, pH 7.4) within 37 ℃ 5% CO 2 environment HeLa cells (human epithelial adenocarcinoma cells, Korea Cell Line Bank) were cultured and prepared. N, known as thiol-blocker-ethyl maleimide (N -ethylmaleimide, NEM) without pre-processing or the 1mM, for 30 minutes, thiol group (R-SH) in the amino acid cysteine, homocysteine, glutathione, each comprising an added 300μM after, DMEM were seeded (seed) of the cells in each dish in culture medium (per dish) 3 × 10 5 cells to a density 35-mm glass bottom dish (glass bottomed dishes). After 24 hours, 3 mu M of the compound prepared in Example 1 was added and cultured together at 37 DEG C for 30 minutes. Then, images were imaged using a confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). The results are shown in Fig.

13 (a) is a fluorescence image and differential interference channel observed by HeLa cells cultured together with the compound prepared in Example 1 using a confocal laser scanning microscope under neutral conditions.

Observing the HeLa cells incubated with the compound prepared in were pretreated with ethyl maleimide (N -ethylmaleimide, NEM), Example 1 - Fig. 13 (b) is N, using the confocal laser scanning microscope at neutral conditions Fluorescence image and differential interference channel.

Were incubated with a solution of the compound prepared in Example 1 was pretreated with ethyl maleimide (N -ethylmaleimide, NEM), the addition of cysteine, and - Fig. 13 (c) is N, using the confocal laser scanning microscope at neutral conditions HeLa cells are fluorescent image and differential interference channel.

Were incubated with a solution of the compound prepared in Example 1 was pretreated with ethyl maleimide (N -ethylmaleimide, NEM), the addition of homocysteine, and - Fig. 13 (d) is N, using the confocal laser scanning microscope at neutral conditions HeLa cells are fluorescent image and differential interference channel.

Were incubated with a solution of the compound prepared in were pretreated with ethyl maleimide (N -ethylmaleimide, NEM), in Example 1 the addition of glutathione, and - Fig. 13 (e) is N, using the confocal laser scanning microscope at neutral conditions HeLa cells are fluorescent image and differential interference channel.

As shown in Fig. 13 (a), it was confirmed that green fluorescence appeared in HeLa cells cultured together with the compound prepared in Example 1. From the result, the compound prepared in Example 1 showed stable cytoplasmic structure of HeLa cells And it reacts with endogenous cysteine and homocysteine and shows fluorescence emission.

On the other hand, Figure 13 (b) as shown in, N - Confirm ethyl maleimide (N -ethylmaleimide, NEM) is the green fluorescence of the compound prepared in the pre-treatment and then Example 1, in a HeLa cell cultures does not appear with the From this, it can be seen that NEM blocks the endogenous cysteine and homocysteine as a thiol blocker, and fluorescence emission does not appear because no amino acid can be reacted with the compound prepared in Example 1.

In addition, as shown in FIG. 13 (c), N - ethyl maleimide (N -ethylmaleimide, NEM) after a pre-treatment, green fluorescence in HeLa cells incubated with the compound prepared in Example 1 was added to cysteine, and And that the compound prepared in Example 1 stably maintains the cytoplasmic structure of HeLa cells and reacts with exogenous cysteine to show fluorescence emission.

Furthermore, as shown in FIG. 13 (d), N - ethyl maleimide (N -ethylmaleimide, NEM) after a pre-treatment, green fluorescence in HeLa cells incubated with the compound prepared in Example 1 was added to homocysteine, and And that the compound prepared in Example 1 maintains the intracellular structure of HeLa cells stably and reacts with extrinsic homocysteine to show fluorescence emission.

In addition, as shown in FIG. 13 (e), N - ethyl maleimide (N -ethylmaleimide, NEM) after a pre-treatment, green fluorescence in HeLa cells incubated with the compound prepared in Example 1 was added to glutathione, and , Indicating that the compound prepared in Example 1 had no activity against glutathione.

Therefore, the compound prepared in Example 1 penetrates into HeLa cells under neutral (pH 7.4) conditions and selectively reacts with cysteine and homocysteine among amino acids including a thiol group (R-SH) present in cytoplasm, Therefore, it can be usefully used for selectively detecting cysteine and homocysteine.

< Experimental Example  10 > Cysteine in a slightly acidic condition Cys , Cysteine ) Detection ability evaluation

Among the cysteine (Cys, Cysteine), homocysteine (Hcy, Homocysteine) and glutathione (GSH, Glutathione) in the biothiol which is an amino acid containing a thiol group (R-SH) To evaluate the selectivity for cysteine (Cys, Cysteine) in cells, the following experiment was conducted.

HBSS (Hanks' balanced salt solution) in the medium, Experimental Example 9 HeLa cells prepared from N, known as thiol blocking agent in (a human epithelial carcinoma cell, Korea Cell Line Bank) - ethyl maleimide (N -ethylmaleimide, NEM) 1 mM of cysteine or homocysteine, which is an amino acid containing a thiol group (R-SH), was added in a concentration of 300 μM and then dissolved in citric acid-Na 2 HPO 4 buffer (pH 6.0) 3 [mu] M of the compound prepared in Example 1 was added and cultured together at 37 DEG C for 30 minutes. Then, images were imaged using a confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). The results are shown in Fig.

Fig. 14 (a) is a fluorescence image and differential interference channel observing HeLa cells cultured together with the compound prepared in Example 1 using a confocal laser scanning microscope under slightly acidic conditions. Fig.

Observing the HeLa cells incubated with the compound prepared in were pretreated with ethyl maleimide (N -ethylmaleimide, NEM), Example 1 - Fig. 14 (b), using the confocal laser scanning microscope in a weakly acidic condition N Fluorescence image and differential interference channel.

Were incubated with a solution of the compound prepared in Example 1 was pretreated with ethyl maleimide (N -ethylmaleimide, NEM), the addition of cysteine, and - Fig. 14 (c), using the confocal laser scanning microscope in a weakly acidic condition N HeLa cells are fluorescent image and differential interference channel.

Were incubated with a solution of the compound prepared in Example 1 was pretreated with ethyl maleimide (N -ethylmaleimide, NEM), the addition of homocysteine, and - Fig. 14 (d), using the confocal laser scanning microscope in a weakly acidic condition N HeLa cells are fluorescent image and differential interference channel.

As shown in Fig. 14 (a), it was confirmed that green fluorescence appeared in HeLa cells cultured together with the compound prepared in Example 1. From this, the compound prepared in Example 1 showed stable cytoplasmic structure of HeLa cells And it reacts with endogenous cysteine to show fluorescence emission.

On the other hand, Figure 14 (b) as shown in, N - ethyl maleimide confirmed that the green fluorescence does not appear in the HeLa cells incubated with the compound prepared in (N -ethylmaleimide, NEM) after a pre-treatment, Example 1 From this, it can be seen that NEM blocks the endogenous cysteine as a thiol blocker and fluorescence emission does not appear because there is no amino acid that the compound prepared in Example 1 can react to.

In addition, as shown in FIG. 14 (c), N - ethyl maleimide (N -ethylmaleimide, NEM) after a pre-treatment, green fluorescence in HeLa cells incubated with the compound prepared in Example 1 was added to cysteine, and And that the compound prepared in Example 1 stably maintains the cytoplasmic structure of HeLa cells and reacts with exogenous cysteine to show fluorescence emission.

Furthermore, as shown in FIG. 14 (d), N - ethyl maleimide (N -ethylmaleimide, NEM) after a pre-treatment, green fluorescence in HeLa cells incubated with the compound prepared in Example 1 was added to homocysteine, and . From this, it can be seen that the compound prepared in Example 1 is not active against homocysteine under a slightly acidic condition (pH 6.0).

Therefore, the compound prepared in Example 1 penetrates into HeLa cells under a weakly acidic condition (pH 7.4) and selectively reacts with cysteine among amino acids including a thiol group (R-SH) present in the cytoplasm to generate green fluorescence Therefore, it can be usefully used for selectively detecting cysteine.

< Experimental Example  11> Assessment of cytotoxicity

In order to evaluate the cytotoxicity of the compound prepared in Example 1, the following experiment was conducted.

HeLa cells (human epithelial adenocarcinoma cells, Korea Cell Line Bank) prepared in Experimental Example 9 were seeded in a 96-well plate. After culturing for one day, the cells were cultured together with the compound (0-3 μM) prepared in Example 1 at 37 ° C. for 24 hours. After washing with Dulbecco's Phosphate Buffered Saline (DPBS), 0.5 mg / mL of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma) The resulting formazan was dissolved in 0.1 mL DMSO and read at OD 650 nm using a Spectramax Microwell plate reader. The results are shown in Fig.

15 is an image for evaluating cytotoxicity of the compound prepared in Example 1. Fig.

As shown in FIG. 15, when the compound prepared in Example 1 was incubated with HeLa cells at 0 to 3 μM, the survival rate of the cells was found to be about 85% or more.

Claims (10)

delete delete delete delete As shown in Scheme 1 below,
Reacting a compound represented by formula (2) with a compound represented by formula (3) to prepare a compound represented by formula (1) (step 1).
Preparation of 4- (7-nitrobenzo [c] [1,2,5] thiadiazol-4-ylthio) benzenamine represented by the formula (1)
[Reaction Scheme 1]
Figure 112017032801613-pat00026
.
A chemical sensor for detecting cysteine or homocysteine comprising a compound represented by the following formula (1):
[Chemical Formula 1]
Figure 112017004235602-pat00025

(In the formula 1,
R 1 , R 2 and R 3 are independently selected from the group consisting of -H, -OH, -CN, -NO 2 , halogen, C 1-10 linear or branched alkyl, C 1-10 linear or branched alkoxy or -NR 4 R 5 ,
Wherein R 4 and R 5 are independently -H, C 1-10 linear or branched alkyl or C 1-10 straight or branched alkoxy.
The method according to claim 6,
Wherein the chemical sensor detects cysteine or homocysteine in a biological sample.
Adding the chemical sensor of claim 6 to the sample (step 1);
Irradiating the sample prepared in the step 1 with a light source (step 2); And
(Step 3) of measuring a change in absorption or fluorescence characteristic, either singly or in combination.
9. The method of claim 8,
Wherein the detection method detects cysteine or homocysteine in a biological sample.
9. The method of claim 8,
The above-mentioned detection method can be carried out by using a phenyl derivative containing the sulfur atom of the compound represented by the general formula (1)
Figure 112017004235602-pat00009
) Is separated by reacting with cysteine or homocysteine, and the absorption or fluorescence characteristic change caused by the change of the--conjugation system occurring thereon is measured singly or in combination .
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