KR102114159B1 - Multi-fucntional compound for glutathione sensing and Method for manufacturing it - Google Patents

Multi-fucntional compound for glutathione sensing and Method for manufacturing it Download PDF

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KR102114159B1
KR102114159B1 KR1020180080183A KR20180080183A KR102114159B1 KR 102114159 B1 KR102114159 B1 KR 102114159B1 KR 1020180080183 A KR1020180080183 A KR 1020180080183A KR 20180080183 A KR20180080183 A KR 20180080183A KR 102114159 B1 KR102114159 B1 KR 102114159B1
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디라제무라레
홍성철
하크 마무눌
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이준석
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Abstract

본 발명은 글루타티온 검출을 위한 화합물 및 이의 제조방법에 대한 것으로, 더욱 상세하게는 하기 화학식 1 또는 화학식 2로 표시되는 화합물을 제공함으로써, 세포 내 고침투성을 가지고 여러 바이오 티올에 대하여 글루타티온에 높은 선택성을 가져 글루타티온을 효과적으로 검출할 수 있다.

Figure 112018067993524-pat00024
Figure 112018067993524-pat00025

[화학식 1] [화학식 2]The present invention relates to a compound for detecting glutathione and a method for preparing the same, and more specifically, by providing a compound represented by the following Chemical Formula 1 or Chemical Formula 2, it has high permeability in cells and has high selectivity to glutathione against several biothiols. It can effectively detect glutathione.
Figure 112018067993524-pat00024
Figure 112018067993524-pat00025

[Formula 1] [Formula 2]

Description

글루타티온 검출을 위한 다기능성 화합물 및 이의 제조방법{Multi-fucntional compound for glutathione sensing and Method for manufacturing it}Multi-fucntional compound for glutathione sensing and method for manufacturing it

본원발명은 글루타티온 검출을 위한 화합물 및 그 제조방법에 대한 것이다.The present invention relates to a compound for detecting glutathione and a method for manufacturing the same.

글루타티온(GSH)은 세포 내에서 가장 풍부한 환원제로, 활성산소종 및 암세포 등의 과다한 물질대사에 따른 산화작용을 막아주는 역할을 수행한다. 따라서, 다양한 종류의 암을 포함하여 다양한 종류의 표지자로 사용되고 있다. 종래 글루타티온 검출 방법은 하기 특허문헌처럼 GSH의 티올(thiol)기가 친핵반응을 일으킬 수 있는 작용기를 형광유기분자에 유도체화하는 방식이 널리 사용되고 있다.Glutathione (GSH) is the most abundant reducing agent in the cell, and serves to prevent oxidation due to excessive metabolism of free radicals and cancer cells. Therefore, it is used as a marker of various types, including various types of cancer. In the conventional glutathione detection method, a method in which a thiol group of GSH is derivatized to a fluorescent organic molecule is widely used as in the following patent document.

<특허문헌><Patent Document>

특허공개공보 제10-2013-0043922호(2013. 05. 02. 공개) "티올 선택성을 갖는 화학정량 형광 표지자, 그 제조방법 및 이를 이용한 생체내 티올 영상화 방법"Patent Publication No. 10-2013-0043922 (published on 05. 02. 2013) "Chemical quantitative fluorescent marker with thiol selectivity, manufacturing method thereof, and in vivo thiol imaging method"

하지만, 생체 분자 내에는 티올 그룹을 가진 물질이 많기 때문에, 상기와 같은 종래 글루타티온 검출 방법을 이용하여, 글루타티온을 선택적으로 검출하는 것에 어려움이 있다.However, since there are many substances having a thiol group in the biomolecule, it is difficult to selectively detect glutathione using the conventional glutathione detection method as described above.

본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로,The present invention was made to solve the above problems,

본원발명은 세포 내 고침투성을 가지고 여러 바이오 티올에 대하여 글루타티온에 높은 선택성을 가져 글루타티온을 효과적으로 검출할 수 있는 화합물 및 그 제조방법을 제공하는데 그 목적이 있다.An object of the present invention is to provide a compound capable of effectively detecting glutathione having high permeability in cells and having high selectivity to glutathione for various biothiols, and a method for manufacturing the same.

본 발명은 앞서 본 목적을 달성하기 위하여 다음과 같은 구성을 가진 실시예에 의해 구현된다.The present invention is implemented by an embodiment having the following configuration in order to achieve the above object.

본 발명의 일 실시예에 따르면, 본 발명에 따른 화합물은 하기 화학식 1로 표시된다.According to an embodiment of the present invention, the compound according to the present invention is represented by Formula 1 below.

Figure 112018067993524-pat00001
Figure 112018067993524-pat00001

[화학식 1]        [Formula 1]

본 발명의 다른 실시예에 따르면, 본 발명에 따른 화합물은 하기 화학식 2로 표시된다.According to another embodiment of the present invention, the compound according to the present invention is represented by Formula 2 below.

Figure 112018067993524-pat00002
Figure 112018067993524-pat00002

[화학식 2]       [Formula 2]

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 화학식 1 또는 2로 표시되는 화합물은 글루타티온을 검출하기 위한 용도로 사용되는 것을 특징으로 한다.According to another embodiment of the present invention, the compound represented by Chemical Formula 1 or 2 according to the present invention is characterized in that it is used for the purpose of detecting glutathione.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 화학식 1로 표시되는 화합물은 녹색 형광을 발생시키는 것을 특징으로 한다.According to another embodiment of the present invention, the compound represented by Chemical Formula 1 according to the present invention is characterized in that it generates green fluorescence.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 화학식 2로 표시되는 화합물은 적색 형광을 발생시키는 것을 특징으로 한다.According to another embodiment of the present invention, the compound represented by Chemical Formula 2 according to the present invention is characterized in that it generates red fluorescence.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 글루타티온을 검출하는 방법은 화학식 1로 표시되는 화합물을 이용한다.According to another embodiment of the present invention, the method for detecting glutathione according to the present invention uses a compound represented by Chemical Formula 1.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 글루타티온을 검출하는 방법은 화학식 2로 표시되는 화합물을 이용한다.According to another embodiment of the present invention, the method for detecting glutathione according to the present invention uses a compound represented by Chemical Formula 2.

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 글루타티온 검출용 화합물의 제조방법은 하기 화학식 6으로 표시되는 화합물과 염화메틸렌, 2,4-디메틸피롤, 염화포스포릴, 보론트리플루오라이드에터레이트 및 디이소프로필에틸아민을 반응시켜 하기 화학식 1로 표시되는 화합물을 형성하는 것을 특징으로 한다.According to another embodiment of the present invention, a method for preparing a compound for detecting glutathione according to the present invention includes a compound represented by the following Chemical Formula 6, methylene chloride, 2,4-dimethylpyrrole, phosphoryl chloride, and boron trifluoride ether. It is characterized by reacting the rate and diisopropylethylamine to form a compound represented by the following formula (1).

Figure 112018067993524-pat00003
Figure 112018067993524-pat00004
Figure 112018067993524-pat00003
Figure 112018067993524-pat00004

[화학식 1] [화학식 6]     [Formula 1] [Formula 6]

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 글루타티온 검출용 화합물의 제조방법은 하기 화학식 7로 표시되는 화합물과 염화메틸렌, 2,4-디메틸피롤, 염화포스포릴, 보론트리플루오라이드에터레이트 및 디이소프로필에틸아민을 반응시켜 하기 화학식 2로 표시되는 화합물을 형성하는 것을 특징으로 한다.According to another embodiment of the present invention, a method for preparing a compound for detecting glutathione according to the present invention includes a compound represented by Chemical Formula 7 and methylene chloride, 2,4-dimethylpyrrole, phosphoryl chloride, and boron trifluoride ether. It is characterized by reacting the rate and diisopropylethylamine to form a compound represented by the following formula (2).

Figure 112018067993524-pat00005
Figure 112018067993524-pat00006
Figure 112018067993524-pat00005
Figure 112018067993524-pat00006

[화학식 2] [화학식 7]     [Formula 2] [Formula 7]

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 글루타티온 검출용 화합물의 제조방법에 있어서 상기 화학식 6으로 표시되는 화합물은 디메틸포름아미드에 염화포스포릴를 주입하여 반응시켜 제1반응물을 준비하고, 디클로로에탄에 하기 화학식 5로 표시되는 화합물을 녹여 제2반응물을 준비하고, 제1반응물에 제2반응물을 주입하여 반응시킨 후, 아세트산나트륨을 추가로 넣고 반응시켜 형성되는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for preparing a compound for detecting glutathione according to the present invention, the compound represented by Chemical Formula 6 is reacted by injecting phosphoryl chloride into dimethylformamide to prepare a first reactant, It is characterized by dissolving a compound represented by the following formula (5) in dichloroethane to prepare a second reactant, and then reacting by injecting a second reactant into the first reactant, and then adding sodium acetate to react.

Figure 112018067993524-pat00007
Figure 112018067993524-pat00007

[화학식 5]       [Formula 5]

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 글루타티온 검출용 화합물의 제조방법에 있어서 상기 화학식 7로 표시되는 화합물은 디메틸포름아미드에 염화포스포릴를 주입하여 반응시켜 제1반응물을 준비하고, 디클로로에탄에 하기 화학식 5로 표시되는 화합물을 녹여 제2반응물을 준비하고, 제1반응물에 제2반응물을 주입하여 반응시킨 후, 아세트산나트륨을 추가로 넣고 반응시켜 형성되는 것을 특징으로 한다.  According to another embodiment of the present invention, in the method for preparing a compound for detecting glutathione according to the present invention, the compound represented by Chemical Formula 7 is reacted by injecting phosphoryl chloride into dimethylformamide to prepare a first reactant, It is characterized by dissolving a compound represented by the following formula (5) in dichloroethane to prepare a second reactant, and then reacting by injecting a second reactant into the first reactant, and then adding sodium acetate to react.

Figure 112018067993524-pat00008
Figure 112018067993524-pat00008

[화학식 5]       [Formula 5]

본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 글루타티온 검출용 화합물의 제조방법에 있어서 상기 화학식 5로 표시되는 화합물은 아세토피논과 4-클로로벤젠알데히드를 에탄올에 녹여 교반하고, 수산화리튬일수화물을 첨가하고 반응시키고, 추가로 수산화리튬일수화물과 토실메틸이소시안화물을 넣고 침전이 발생할 때까지 교반하여 형성되는 것을 특징으로 한다. According to another embodiment of the present invention, in the method for preparing the glutathione detecting compound according to the present invention, the compound represented by Chemical Formula 5 is dissolved in acetopinone and 4-chlorobenzenealdehyde in ethanol and stirred, and lithium hydroxide monohydrate It is characterized by being formed by adding and reacting, further adding lithium hydroxide monohydrate and tosylmethylisocyanate, and stirring until precipitation occurs.

본 발명은 앞서 본 실시예에 의해 다음과 같은 효과를 얻을 수 있다.The present invention can obtain the following effects by the present embodiment.

본원발명은 세포 내 고침투성을 가지고 여러 바이오 티올에 대하여 글루타티온에 높은 선택성을 가져 글루타티온을 효과적으로 검출할 수 있는 효과가 있다.The present invention has the effect of being able to effectively detect glutathione by having high permeability in cells and having high selectivity to glutathione for several biothiols.

도 1은 화학식 1로 표시되는 화합물의 글루타티온에 대한 선택성을 확인하기 위한 형광 스펙트럼을 나타내는 도면.
도 2는 화학식 1로 표시되는 화합물의 글루타티온에 대한 선택성을 확인하기 위한 형광세기를 상대비교한 히트맵을 나타내는 도면.
도 3은 화학식 2로 표시되는 화합물의 글루타티온에 대한 선택성을 확인하기 위한 형광 스펙트럼을 나타내는 도면.
도 4는 화학식 2로 표시되는 화합물의 글루타티온에 대한 선택성을 확인하기 위한 형광세기를 상대비교한 히트맵을 나타내는 도면.
도 5는 화학식 2로 표시되는 화합물과 글루타티온이 반응하여 형성된 화합물의 GST와의 반응에 따른 형광 변화의 확인하기 위한 형광 스펙트럼을 나타내는 도면.
도 6은 살아있는 세포에서 화학식 2로 표시되는 화합물의 글루타티온 검출 확인하기 위한 LCMS 분석 결과를 나타내는 도면.
도 7은 화학식 2로 표시되는 화합물을 이용하여 글루타티온의 프랜스로케이션의 시각화를 하기 위한 형광 현미경 사진.
도 8은 화학식 2로 표시되는 화합물의 글루타티온에 대한 colocalization 측정 결과를 나타내는 형광 현미경 사진 및 도표.1 is a view showing a fluorescence spectrum for confirming the selectivity to the glutathione of the compound represented by the formula (1).
FIG. 2 is a diagram showing a heat map of relative comparison of fluorescence intensity for confirming selectivity to glutathione of the compound represented by Chemical Formula 1.
3 is a view showing a fluorescence spectrum for confirming the selectivity to glutathione of the compound represented by the formula (2).
FIG. 4 is a diagram showing a heat map of relative comparison of fluorescence intensity to confirm selectivity for glutathione of the compound represented by Chemical Formula 2.
5 is a view showing a fluorescence spectrum for confirming the change in fluorescence according to the reaction of the compound formed by the reaction of the compound represented by the formula (2) and glutathione GST.
6 is a diagram showing the results of LCMS analysis for confirming the detection of glutathione of a compound represented by Chemical Formula 2 in living cells.
7 is a fluorescence micrograph for visualization of the glutathione translocation using the compound represented by the formula (2).
8 is a fluorescence micrograph and diagram showing the results of colocalization measurement of glutathione of the compound represented by Formula 2.

이하에서는 본 발명에 따른 글루타티온 검출을 위한 다기능성 화합물 및 이의 제조방법을 도면을 참조하여 상세히 설명한다. 특별한 정의가 없는 한 본 명세서의 모든 용어는 본 발명이 속하는 기술분야의 통상의 지식을 가진 기술자가 이해하는 당해 용어의 일반적 의미와 동일하고 만약 본 명세서에 사용된 용어의 의미와 충돌하는 경우에는 본 명세서에 사용된 정의에 따른다. 또한, 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대해 상세한 설명은 생략한다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있는 것을 의미한다.Hereinafter, a multifunctional compound for detecting glutathione according to the present invention and a manufacturing method thereof will be described in detail with reference to the drawings. Unless otherwise specified, all terms in this specification are the same as the general meaning of the term understood by a person skilled in the art to which the present invention belongs, and if there is a conflict with the meaning of the term used in the present specification, It follows the definition used in the specification. In addition, detailed descriptions of well-known functions and configurations that may unnecessarily obscure the subject matter of the present invention are omitted. Throughout the specification, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless otherwise stated.

본 발명의 일 실시예는 글루타티온 검출을 위한 다기능성 화합물에 대한 것으로, 상기 화합물은 화학식 1 또는 화학식 2로 표시된다. 상기 화학식 1 또는 화학식 2로 표시되는 화합물은 세포 내 고침투성을 가지고 여러 바이오 티올에 대하여 글루타티온에 높은 선택성을 가져 글루타티온을 효과적으로 검출할 수 있게 된다.One embodiment of the present invention relates to a multifunctional compound for glutathione detection, the compound is represented by Formula 1 or Formula 2. The compound represented by Chemical Formula 1 or Chemical Formula 2 has high permeability in cells and has high selectivity to glutathione for various biothiols, thereby effectively detecting glutathione.

Figure 112018067993524-pat00009
Figure 112018067993524-pat00010
Figure 112018067993524-pat00009
Figure 112018067993524-pat00010

[화학식 1] [화학식 2]           [Formula 1] [Formula 2]

상기 화학식 1로 표시되는 화합물은 글루타티온과 반응하여 화학식 3으로 표시되는 화합물을 형성하게 되며, 상기 화학식 2로 표시되는 화합물은 글루타티온과 반응하여 화학식 3으로 표시되는 화합물을 형성하게 된다.The compound represented by Formula 1 reacts with glutathione to form a compound represented by Formula 3, and the compound represented by Formula 2 reacts with glutathione to form a compound represented by Formula 3.

Figure 112018067993524-pat00011
Figure 112018067993524-pat00012
Figure 112018067993524-pat00011
Figure 112018067993524-pat00012

[화학식 3] [화학식 4]           [Formula 3] [Formula 4]

본 발명의 다른 실시예는 화학식 1 또는 화학식 2로 표시되는 글루타티온 검출용 화합물의 제조방법에 대한 것으로, 화학식 5로 표시되는 화합물 수득단계, 상기 화학식 6 및 화학식 7로 표시되는 화합물 수득단계, 상기 화학식 1 또는 2로 표시되는 화합물 수득단계를 포함한다.Another embodiment of the present invention relates to a method for preparing a compound for detecting glutathione represented by Chemical Formula 1 or Chemical Formula 2, a compound obtaining step represented by Chemical Formula 5, a compound obtaining step represented by Chemical Formula 6 and Chemical Formula 7, the chemical formula And obtaining a compound represented by 1 or 2.

상기 화학식 5로 표시되는 화합물 수득단계는 하기 반응식 1에 따라 아세토피논(acetophenone)과 4-클로로벤젠알데히드(4-chlorobenzaldehyde)를 에탄올에 녹여 교반하고, 수산화리튬일수화물(lithium hydroxide monohydrate)을 첨가하고 반응시키고, 수산화리튬일수화물과 토실메틸이소시안화물(tosylmethyl isocyanide)을 넣고 침전이 발생할 때까지 교반하고, 침전물을 얼음물과 에탄올 혼합액으로 세척하고 건조하여 화학식 5로 표시되는 화합물을 수득하는 단계이다.In the step of obtaining the compound represented by Chemical Formula 5, acetophenone and 4-chlorobenzaldehyde were dissolved in ethanol and stirred according to Reaction Scheme 1, and lithium hydroxide monohydrate was added. It is a step of reacting, adding lithium hydroxide monohydrate and tosylmethyl isocyanide, stirring until precipitation occurs, washing the precipitate with a mixture of ice water and ethanol and drying to obtain a compound represented by the formula (5).

[반응식 1][Scheme 1]

Figure 112018067993524-pat00013
Figure 112018067993524-pat00013

[화학식 5]                                           [Formula 5]

상기 화학식 6 및 화학식 7로 표시되는 화합물 수득단계는 하기 반응식 2에 따라 디메틸포름아미드(DMF)에 염화포스포릴(POCl3)를 주입하여 반응시켜 제1반응물을 준비하고, 디클로로에탄(dichloroethane)에 화학식 5로 표시되는 화합물을 녹여 제2반응물을 준비한 후, 제1반응물에 제2반응물을 주입하고 환류시킨 후, 아세트산나트륨(sodium acetate)을 추가로 넣고 환류시키고, 포화된 염화나트륨 용액(brine)을 사용해서 합성된 화합물을 유기층으로 추출시키고 칼럼 크로마토그래피를 통해서 화학식 6으로 표시되는 화합물과 화학식 7로 표시되는 화합물을 수득하는 단계이다. 이때, 화학식 6으로 표시되는 화합물이 화학식 7로 표시되는 화합물보다 높을 수율로 수득되게 된다.The step of obtaining the compound represented by Chemical Formulas 6 and 7 is prepared by reacting by injecting phosphoryl chloride (POCl 3 ) into dimethylformamide (DMF) according to the following Reaction Scheme 2 to prepare a first reactant, and dichloroethane. After preparing the second reactant by dissolving the compound represented by the formula (5), the second reactant is injected into the first reactant and refluxed, followed by addition of sodium acetate to reflux, and saturated sodium chloride solution (brine). It is a step of obtaining the compound represented by Formula 6 and the compound represented by Formula 7 by extracting the compound synthesized using the organic layer and performing column chromatography. At this time, the compound represented by Formula 6 is obtained in a higher yield than the compound represented by Formula 7.

[반응식 2][Scheme 2]

Figure 112018067993524-pat00014
Figure 112018067993524-pat00014

[화학식 5] [화학식 6] [화학식 7]   [Formula 5] [Formula 6] [Formula 7]

상기 화학식 1 또는 2로 표시되는 화합물 수득단계는 하기 반응식 3에 따라 화학식 6으로 표시되는 화합물을 무수 염화메틸렌(anhydrous methylene chloride)에 녹인 후, 2,4-디메틸피롤(2,4-dimethyl pyrrole)과 염화포스포릴을 주입하여 반응시키고, 보론트리플루오라이드에터레이트(BF3OEt2)와 디이소프로필에틸아민(DIPEA)을 주입하고 반응시키고, 칼럼 크로마토그래피를 통해 화학식 1로 표시되는 화합물을 수득하는 단계이다. 상기 단계에서 화학식 6으로 표시되는 화합물 대신에 화학식 7로 표시되는 화합물을 사용하는 경우 화학식 2로 표시되는 화합물을 수득할 수 있다.In the step of obtaining the compound represented by Chemical Formula 1 or 2, after dissolving the compound represented by Chemical Formula 6 in anhydrous methylene chloride according to Reaction Scheme 3, 2,4-dimethyl pyrrole And reacting by injecting phosphoryl chloride, injecting and reacting boron trifluoride etherate (BF 3 OEt 2 ) with diisopropylethylamine (DIPEA), and reacting the compound represented by Formula 1 through column chromatography. This is the step of obtaining. When the compound represented by the formula (7) is used instead of the compound represented by the formula (6) in the above step, the compound represented by the formula (2) can be obtained.

[반응식 3][Scheme 3]

Figure 112018067993524-pat00015
Figure 112018067993524-pat00015

[화학식 6] [화학식 1]   [Formula 6] [Formula 1]

본 발명의 또 다른 실시예는 화학식 1 또는 2로 표시되는 화합물을 이용하여 글루타티온을 검출하는 방법을 포함한다. 상기 글루타티온을 검출하는 방법은 상기 화학식 1 또는 2로 표시되는 화합물을 용매에 혼합해 용액을 제조하는 단계, 상기 용액을 세포를 포함하는 시료에 첨가하는 단계, 상기 화학식 1 또는 2로 표시되는 화합물이 첨가된 시료에 일정 파장의 빛을 가하는 단계, 상기 시료에서 방출되는 형광을 측정하는 단계를 포함한다. 상기 화학식 1로 표시되는 화합물은 녹색 형광을 가지고, 상기 화학식 2로 표시되는 화합물은 적색 형광을 가지나, 상기 화학식 1 또는 2로 표시되는 화합물은 여러 바이오 티올 중 선택적으로 글루타티온에 반응하여 형광 세기가 현저하게 감소함으로 글루타티온을 효과적으로 검출할 수 있게 된다. Another embodiment of the present invention includes a method for detecting glutathione using a compound represented by Chemical Formula 1 or 2. The method for detecting the glutathione comprises preparing a solution by mixing a compound represented by Formula 1 or 2 in a solvent, adding the solution to a sample containing cells, and a compound represented by Formula 1 or 2 And adding light of a predetermined wavelength to the added sample, and measuring fluorescence emitted from the sample. The compound represented by Chemical Formula 1 has a green fluorescence, and the compound represented by Chemical Formula 2 has a red fluorescence, but the compound represented by Chemical Formula 1 or 2 reacts to glutathione selectively among several biothiols, so that the fluorescence intensity is remarkable. By decreasing it, glutathione can be effectively detected.

이하, 실시예를 통해서 본 발명을 보다 상세히 설명하기로 한다. 하지만, 이들은 본 발명을 보다 상세하게 설명하기 위한 것일 뿐, 본 발명의 권리범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these are only for explaining the present invention in more detail, and the scope of the present invention is not limited thereto.

<실시예 1> 글루타티온 검출용 화합물의 제조<Example 1> Preparation of glutathione detection compound

1. 화학식 1로 표시되는 화합물의 합성1. Synthesis of Compound Represented by Formula 1

(1) 화학식 5의 화합물 수득(1) Compound 5

아세토피논(1mmol)과 4-클로로벤젠알데히드(1mmol)를 에탄올(3ml)에 녹여 교반하고, 수산화리튬일수화물(0.1mmol)을 첨가하고 상온에서 반응시키고, 수산화리튬일수화물(1.1mmol)과 토실메틸이소시안화물(1.2mmol)을 넣고 침전이 발생할 때까지 교반하고, 침전물을 얼음물과 에탄올 혼합액으로 세척하고 건조하여 화학식 5로 표시되는 화합물을 수득하였다.Acetopinone (1 mmol) and 4-chlorobenzenealdehyde (1 mmol) were dissolved in ethanol (3 ml), stirred, lithium hydroxide monohydrate (0.1 mmol) was added and reacted at room temperature, lithium hydroxide monohydrate (1.1 mmol) and tosyl Methyl isocyanate (1.2 mmol) was added and stirred until precipitation occurred, and the precipitate was washed with a mixture of ice water and ethanol and dried to obtain a compound represented by Chemical Formula 5.

(2) 화학식 6 및 화학식 7로 표시되는 화합물 수득(2) Obtaining Compounds Represented by Formulas 6 and 7

0℃의 디메틸포름아미드(1.2mmol)에 염화포스포릴(1.2mmol)을 천천히 주입하여 상온에서 15분간 반응시켜 제1반응물을 준비하고, 디클로로에탄(15ml)에 화학식 5로 표시되는 화합물(1mmol)을 녹여 제2반응물을 준비한 후, 제1반응물에 제2반응물을 주입하고 30분간 환류시킨 후, 상온으로 온도가 떨어지면 아세트산나트륨(5.5mmol)을 추가로 넣고 30분간 환류시키고, 포화된 염화나트륨 용액(brine)을 사용해서 합성된 화합물을 유기층으로 추출시키고 칼럼 크로마토그래피를 통해서 화학식 6으로 표시되는 화합물과 화학식 7로 표시되는 화합물을 수득하였다.Phosphoryl chloride (1.2 mmol) was slowly injected into 0 ° C dimethylformamide (1.2 mmol) to react for 15 minutes at room temperature to prepare a first reactant, and the compound represented by Formula 5 in dichloroethane (15 ml) (1 mmol) After preparing the second reactant by dissolving, the second reactant was injected into the first reactant and refluxed for 30 minutes. When the temperature dropped to room temperature, sodium acetate (5.5 mmol) was additionally added and refluxed for 30 minutes, and saturated sodium chloride solution ( brine) to extract the synthesized compound into an organic layer and obtain a compound represented by Formula 6 and a compound represented by Formula 7 through column chromatography.

(3) 화학식 1로 표시되는 화합물 수득(3) Obtaining the compound represented by the formula (1)

상기에서 얻은 화학식 6으로 표시되는 화합물(1mmol)을 무수 염화메틸렌(4ml)에 녹이고 영하 5℃로 냉각한 후, 2,4-디메틸피롤(1mmol)과 염화포스포릴(1mmol)을 천천히 주입하고, 보론트리플루오라이드에터레이트(3mmol)와 디이소프로필에틸아민(3mmol)을 주입하고 3시간 동안 반응시키고, 칼럼 크로마토그래피를 통해 녹색이 형광이 발생하는 화학식 1로 표시되는 화합물을 수득하였다. 최종 화합물이 화학식 1로 표기되는 것은 하기의 표 1에 표기된 바와 같은 proton NMR 분광기 및 carbon NMR 분광기 결과를 통해 확인하였다.After dissolving the compound (1 mmol) represented by the formula (6) in anhydrous methylene chloride (4 ml) to 5 ° C below 0, the 2,4-dimethylpyrrole (1 mmol) and phosphoryl chloride (1 mmol) were slowly injected. Boron trifluoride etherate (3mmol) and diisopropylethylamine (3mmol) were injected and reacted for 3 hours to obtain a compound represented by Chemical Formula 1 in which green color fluorescence was generated through column chromatography. The final compound is represented by the formula (1) was confirmed through proton NMR spectroscopy and carbon NMR spectroscopy results as shown in Table 1 below.

1H NMR spectroscopy 1 H NMR spectroscopy (CDCl3 , 400 MHz): 7.94 (s, 1H), 7.81-7.79 (m, 2H), 7.57-7.53 (m, 1H), 7.46-7.35 (m, 6H), 7.15 (s, 1H), 6.33 (s, 1H), 2.69 (s, 3H), 2.30 (s, 3H).(CDCl 3 , 400 MHz): 7.94 (s, 1H), 7.81-7.79 (m, 2H), 7.57-7.53 (m, 1H), 7.46-7.35 (m, 6H), 7.15 (s, 1H), 6.33 (s, 1H), 2.69 (s, 3H), 2.30 (s, 3H). 13C NMR spretroscopy 13 C NMR spretroscopy 190.23, 167.36, 148.09, 141.14, 139.88, 139.19, 138.97, 134.51, 132.28, 131.55, 131.43, 131.04, 129.27, 128.58, 128.34, 126.57, 124.10, 123.30, 15.64, 11.69.(Yield 25%);190.23, 167.36, 148.09, 141.14, 139.88, 139.19, 138.97, 134.51, 132.28, 131.55, 131.43, 131.04, 129.27, 128.58, 128.34, 126.57, 124.10, 123.30, 15.64, 11.69. (Yield 25%) ES-MSES-MS [M+H]+ = 435.1169 (cal.), 435.1208 (exp.).[M + H] + = 435.1169 (cal.), 435.1208 (exp.).

2. 화학식 2로 표시되는 화합물의 합성2. Synthesis of Compound Represented by Formula 2

실시예 1의 (3)에서 화학식 6으로 표시되는 화합물 대신에 화학식 7로 표시되는 화합물을 사용한 것을 제외하고는 다른 조건을 동일하게 하여 적색의 형광이 발생하는 화학식 2로 표시되는 화합물을 수득하였다. 최종 화합물이 화학식 2로 표기되는 것은 하기의 표 2에 표기된 바와 같은 proton NMR 분광기 및 carbon NMR 분광기 결과를 통해 확인하였다.The compound represented by Chemical Formula 2 in which red fluorescence occurs was obtained in the same manner as in other conditions, except that the compound represented by Chemical Formula 7 was used instead of the compound represented by Chemical Formula 6 in (3) of Example 1. The final compound represented by the formula (2) was confirmed through the proton NMR spectroscopy and carbon NMR spectroscopy results as shown in Table 2 below.

1H NMR spectroscopy 1 H NMR spectroscopy (CDCl3 , 400 MHz):7.73-7.71 (m, 3),7.50 (S, 1H), 7.49-7.43 (m, 3H) 7.29- 7.24 (m, 4H), 6.31 (S, 1H), 2.69 (s, 3H), 2.31 (S, 3H).(CDCl 3 , 400 MHz): 7.73-7.71 (m, 3), 7.50 (S, 1H), 7.49-7.43 (m, 3H) 7.29- 7.24 (m, 4H), 6.31 (S, 1H), 2.69 ( s, 3H), 2.31 (S, 3H). 13C NMR spretroscopy 13 C NMR spretroscopy 192.57, 167.88, 148.86, 139.36, 137.40, 134.38, 133.40, 133.15, 132.84, 131.73, 130.90, 129.89, 129.44, 128.57, 128.42, 124.03, 123.15, 15.66, 11.66(Yield 5%)192.57, 167.88, 148.86, 139.36, 137.40, 134.38, 133.40, 133.15, 132.84, 131.73, 130.90, 129.89, 129.44, 128.57, 128.42, 124.03, 123.15, 15.66, 11.66 (Yield 5%) ES-MSES-MS [M+H]+ = 435.1169 (cal.), 435.1208(exp.).[M + H] + = 435.1169 (cal.), 435.1208 (exp.).

<실시예 2> 실시예 1에서 제조된 화합물의 글루타티온에 대한 선택성 확인<Example 2> Confirmation of selectivity for glutathione of the compound prepared in Example 1

1. 화학식 1로 표시되는 화합물의 글루타티온에 대한 선택성 확인1. Confirmation of the selectivity to glutathione of the compound represented by Formula 1

분석 물질인 canonical amino acid(L-Cys, Met, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, Val), homocysteine(Hcy) 및 글루타티온을 각각 PBS(pH=7.4) 버퍼에 녹여서 10mM 농도의 분석 용액을 형성하고, 화학식 1로 표시되는 화합물을 DMSO에 녹여서 100μM 농도의 시료(probe or Ctrl)를 형성하고, DMSO 코솔벤트 버퍼(DMSO:PBS=1:1)를 80μL씩 96 well plate에 옮긴 후, 시료(10μL), 각 분석 용액(10μL)와 시료(10μL)의 혼합물을 각 well에 옮겨담고 30분간 상온에서 처리한 뒤, 형광분광계(λex=460nm)를 이용하여 측정하여, 형광스펙트럼 결과를 도 1에 나타내었고, 시료(Ctrl)의 형광세기를 기준으로 하여 각 혼합물의 형광세기를 상대비교하여 그 결과를 heatmap 형태로 도 2에 나타내었다.Analytes canonical amino acid (L-Cys, Met, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, Val), The homocysteine (Hcy) and glutathione were dissolved in PBS (pH = 7.4), respectively, to form an analytical solution having a concentration of 10 mM, and the compound represented by Formula 1 was dissolved in DMSO to form a sample (probe or Ctrl) with a concentration of 100 μM, DMSO After transferring the cosolvent buffer (DMSO: PBS = 1: 1) to a 96 well plate in 80 μL increments, transfer a mixture of the sample (10 μL), each analytical solution (10 μL) and the sample (10 μL) to each well, and at room temperature for 30 minutes. After treatment, measured using a fluorescence spectrometer (λ ex = 460nm), the fluorescence spectrum results are shown in FIG. 1, and the fluorescence intensity of each mixture is compared relative to the fluorescence intensity of the sample (Ctrl). It is shown in Figure 2 in the form of a heatmap.

2. 화학식 2로 표시되는 화합물의 글루타티온에 대한 선택성 확인2. Confirmation of the selectivity to glutathione of the compound represented by Chemical Formula 2

화학식 1로 표시되는 화합물을 사용한 것 대신에 화학식 2로 표시되는 화합물을 사용한 것을 제외하고는 다른 조건을 실시예 2의 1과 동일하여 실험한 후, 그 결과를 도 3 및 4에 나타내었다.After experimenting in the same manner as in Example 1 in Example 2, except that the compound represented by Formula 2 was used instead of the compound represented by Formula 1, the results are shown in FIGS. 3 and 4.

3. 화학식 2로 표시되는 화합물과 글루타티온이 반응하여 형성된 화합물의 GST와의 반응에 따른 형광 변화의 확인3. Confirmation of the fluorescence change according to the reaction of the compound formed by the reaction of the compound represented by Chemical Formula 2 and glutathione with GST

분석 물질인 글루타티온을 PBS(pH=7.4) 버퍼에 녹여서 10mM 농도의 글루타티온 용액을 형성하고, 화학식 2로 표시되는 화합물을 DMSO에 녹여서 100μM 농도의 시료(probe)를 형성하고, DMSO 코솔벤트 버퍼(DMSO:PBS=1:1)를 80μL씩 96 well plate에 옮긴 후, 글루타티온 용액(10μL)와 시료(10μL)를 각 well에 옮겨담고 반응시켰다(즉, 시료는 GSH(100eq.)와 반응됨). 이후, 각 well에 글루타티온 용액의 일정 배수의 Glutathione S-transferase(1, 5, 10eq.)를 넣고, 상온에서 15분 동안 처리한 후 형광분광계(λex=460nm)를 이용하여 측정하여 그 결과를 도 5에 나타내었다.The analyte glutathione is dissolved in PBS (pH = 7.4) buffer to form a 10 mM glutathione solution, and the compound represented by Formula 2 is dissolved in DMSO to form a 100 μM concentration probe, and DMSO cosolvent buffer (DMSO : PBS = 1: 1) 80 μL was transferred to a 96 well plate, and then glutathione solution (10 μL) and sample (10 μL) were transferred to each well and reacted (ie, the sample was reacted with GSH (100 eq.)). Subsequently, Glutathione S-transferase (1, 5, 10 eq.) Of a certain multiple of glutathione solution was added to each well, treated for 15 minutes at room temperature, and measured using a fluorescence spectrometer (λ ex = 460 nm). It is shown in FIG. 5.

4. 도 1 내지 4를 보면, 시료와 글루타티온을 반응시킨 경우 큰 형광 변화가 일어난데 반해, 시료와 다른 아미노산과 반응시킨 경우 무시할만한 형광 변화가 발생한 것을 확인할 수 있어, 실시예 1에서 제조된 화합물은 바이오 티올 중 글루타티온과 선택적으로 반응함을 알 수 있다. 또한, 도 5를 보면, GST의 추가에 따라 소광된 형광이 점진적으로 회복됨을 확인할 수 있어(즉, 화학식 2로 표시되는 화합물과 글루타티온이 반응하여 형성된 화합물은 GST와 반응함), 실시예 1에서 제조된 화합물은 글루타티온과 선택적으로 반응함을 알 수 있다.4. Referring to Figures 1 to 4, when the sample and glutathione reacted, a large fluorescence change occurred, whereas when reacted with a sample and other amino acids, it was confirmed that a negligible fluorescence change occurred, and the compound prepared in Example 1 It can be seen that silver reacts selectively with glutathione in biothiol. In addition, looking at Figure 5, it can be confirmed that the fluorescence that is gradually extinguished upon the addition of GST (that is, the compound formed by the reaction of the compound represented by Formula 2 and glutathione reacts with GST), in Example 1 It can be seen that the prepared compound reacts selectively with glutathione.

<실시예 3> 살아있는 세포에서 실시예 1에서 제조된 화합물의 글루타티온 검출 확인<Example 3> glutathione detection of the compound prepared in Example 1 in living cells confirmed

1. HeLa 세포에 시료(화학식 2로 표시되는 화합물을 DMSO에 녹여서 10μM 농도를 갖도록 하여 형성)를 처리한 후 2시간 동안 37℃에서 배양하고, 처리된 세포를 용해하여 C18 카트리지로 전처리하고(이때, eluent는 ACN으로 200μL씩 3회 처리하여 씻어냄), 모아진 샘플은 용액을 증발시키고 20μL의 0.1% 포름산을 포함한 물에 다시 녹인후, LCMS를 이용해 그 결과를 도 6에 나타내었다.1. After treating the sample (formed by dissolving the compound represented by Formula 2 in DMSO to have a concentration of 10 μM) in HeLa cells, incubate at 37 ° C. for 2 hours, dissolve the treated cells, and pretreat with a C18 cartridge (at this time) , eluent was washed with 200 μL of ACN three times each), and the collected samples were evaporated and dissolved again in water containing 20 μL of 0.1% formic acid, and the results are shown in FIG. 6 using LCMS.

2. 도 6을 보면, 세포 내에서 화학식 2로 표시되는 화합물이 글루타티온과 반응하여 새로운 화합물을 형성한 것을 확인할 수 있어, 실시예 1에서 제조된 화합물은 살아있는 세포 내에서 글루타티온을 검출할 수 있음을 알 수 있다.2. Referring to FIG. 6, it can be confirmed that the compound represented by Chemical Formula 2 in the cell reacted with glutathione to form a new compound, and the compound prepared in Example 1 was capable of detecting glutathione in living cells. Able to know.

<실시예 4> 실시예 1에서 제조된 화합물을 이용하여 글루타티온의 프랜스로케이션의 시각화<Example 4> Visualization of the glutathione translocation using the compound prepared in Example 1

1. Hela 세포에 화학식 2로 표시되는 화합물(pcBD2-C, 5uM)과 상업적인 세포 소기관 트랙커(1uM)를 동시 처리하고, 현광 현미경을 이용하여 얻은 이미지를 도 7에 표시하였다. 또한, 별도로 Hela 세포에 화학식 2로 표시되는 화합물(pcBD2-C(probe), 5uM)과 미토콘드리아를 트랙킹하는 Mitotracker(1uM)를 동시 처리하고, 현광 현미경을 이용하여 얻은 이미지를 도 8의 a에 표시하고, 도 8의 a의 이미지를 오버래이(overlay)하여 imageJ 프로그램을 이용하여 colocalization 정도를 측정하여 도 8의 b에 나타내었다. 도 7 및 8에서 파란색 형광 이미지를 얻기 위해 사용된 광원은 350/50nm, 필터는 460/50nm이며, 녹색 형광 이미지를 얻기 위해 사용된 광원은 480/40nm, 필터는 527/30nm이고, 빨간색 형광 이미지를 얻기 위해 사용된 사용된 광원 546/10nm, 필터는 584/40nm이다. 또한, 상업적인 세포 소기관 트랙커는 ER-Tracker Blue-White DPX, LyxoTracker Green DND 26, Mitotracker Green FM을 사용하였다.1. Hela cells were treated with a compound represented by Chemical Formula 2 (pcBD2-C, 5 uM) and a commercial organelle tracker (1 uM) at the same time, and the image obtained using a microscope was shown in FIG. 7. In addition, a compound represented by Chemical Formula 2 (pcBD2-C (probe), 5 uM) and Mitotracker (1 uM) for tracking mitochondria were simultaneously treated on Hela cells separately, and the image obtained using a photomicrograph was shown in FIG. 8A. Then, by overlaying the image of FIG. 8A, the degree of colocalization was measured using the imageJ program and shown in FIG. 8B. 7 and 8, the light source used to obtain the blue fluorescent image is 350/50 nm, the filter is 460/50 nm, the light source used to obtain the green fluorescent image is 480/40 nm, the filter is 527/30 nm, and the red fluorescent image The used light source used to obtain 546 / 10nm, the filter is 584 / 40nm. In addition, commercial cell organelle trackers used were ER-Tracker Blue-White DPX, LyxoTracker Green DND 26, and Mitotracker Green FM.

2. 도 7 및 8을 보면, 화학식 2로 표시되는 화합물의 염색 패턴이 미토콘드리아를 염색하는 패턴과 극히 유사함을 확인할 수 있어, 화학식 2로 표시되는 화합물이 글루타티온과 반응하여 형성된 화합물은 미토콘드리아로 트랜스로케이션되어 GST와 반응하였기 때문인 것으로 보인다. 이를 통해, 실시예 1에서 제조된 화합물은 세포기질에서 미토콘드리아로의 GSH의 트랜스로케시션을 시각화할 수 있음을 알 수 있다.2. Referring to FIGS. 7 and 8, it can be confirmed that the dyeing pattern of the compound represented by Chemical Formula 2 is very similar to the pattern of dyeing the mitochondrial, and the compound represented by Chemical Formula 2 reacts with glutathione to transmit to mitochondrial It seems to be because it was located and reacted with GST. Through this, it can be seen that the compound prepared in Example 1 can visualize the translocation of GSH from the cell substrate to the mitochondria.

이상에서, 출원인은 본 발명의 바람직한 실시예들을 설명하였지만, 이와 같은 실시예들은 본 발명의 기술적 사상을 구현하는 일 실시예일 뿐이며 본 발명의 기술적 사상을 구현하는 한 어떠한 변경예 또는 수정예도 본 발명의 범위에 속하는 것으로 해석되어야 한다.In the above, the applicant has described the preferred embodiments of the present invention, but these embodiments are only one embodiment for implementing the technical concept of the present invention and any modification or modification of the present invention as long as the technical concept of the present invention is implemented It should be construed as being within the scope.

Claims (13)

삭제delete 하기 화학식 2로 표시되며, 글루타티온과 반응하여 미토콘드리아로 트랜스로케이션되어 미토콘드리아를 표지할 수 있는 것을 특징으로 하는 화합물.
Figure 112020014818154-pat00017

[화학식 2]A compound represented by Formula 2 below, which is translocated to mitochondria by reacting with glutathione and is capable of labeling mitochondria.
Figure 112020014818154-pat00017

[Formula 2] 삭제delete 삭제delete 삭제delete 제2항에 있어서, 상기 화합물은
적색 형광을 발생시키는 것을 특징으로 하는 화합물.The method of claim 2, wherein the compound
A compound characterized by generating red fluorescence. 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete
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