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FLUORESCENT COMPOUND RESPONDING TO MITOCHONDRIAL MEMBRANE POTENTIAL

Foreign code F190009864
File No. (S2018-0102-N0)
Posted date Jul 26, 2019
Country WIPO
International application number 2018JP041417
International publication number WO 2019093400
Date of international filing Nov 8, 2018
Date of international publication May 16, 2019
Priority data
  • P2017-216664 (Nov 9, 2017) JP
Title FLUORESCENT COMPOUND RESPONDING TO MITOCHONDRIAL MEMBRANE POTENTIAL
Abstract The present invention addresses the problem of providing a compound that is a pigment capable of staining cells, is water-soluble, shows a high light emission efficiency, translocates from mitochondria to nucleus depending on mitochondrial membrane potential and exhibits light emission in the red color region. The compound according to the present invention is expressed by formula (1) [in formula (1): X is represented by formula (2) (in formula (2), R1 represents a C1-C10 alkyl group, and Z- represents a counter anion to a pyridinium cation); k and m are an integer of 0-3 and l and n are an integer of 0-2, provided that k, l, m and n do not simultaneously represent 0; X's may be the same or different; R2 represents an electron-donating group or an electron-withdrawing group; a and c are an integer of 0-3 and b and d are an integer of 0-2; R2's may be either the same or different and attached to a carbon atom which is not substituted by X; and the wavy line represents a geometric isomer E or Z].
Outline of related art and contending technology BACKGROUND ART
Mitochondria, while producing energy for the cell involved in the control of apoptosis, cell viability of the cells involved in a small organ. In addition, due to the fault in mitochondrial function, diabetes, stroke, myocardial infarction such as metabolic diseases, neurodegenerative diseases such as Alzheimer's or Parkinson's disease, cancer and the onset of a pointed out. Therefore, these diseases and elucidate the mechanism, also for the development of the method, to observe the change of the mitochondria is important.
Mitochondrial membrane potential is generated along with the energy production, the potential difference between the inside and outside of the mitochondria means, the vitality of the mitochondrial itself (active). That is, when the membrane potential energy are produced, mitochondrial vigor is in a high state. On the other hand, when the membrane potential is lost, not to produce energy, vitality is at a low state. Also, the mitochondrial membrane potential, the state of health of the cells, when the membrane potential of a normal cell, membrane potential and when there is no abnormal cells.
Changes in mitochondrial membrane potential, and the health of the cells due to varying in order to distinguish, on the membrane potential to change the light emission behavior in accordance with the stained mitochondria by the dye, the light emission behavior of a method to observe a change. Mitochondrial membrane potential dye related art in response to, the three types of 2. Is one, the light emission intensity in accordance with the mitochondrial membrane potential change of the dye type, the other one is, to change the emission color of the dye. However, a change in the emission intensity of the dye, and a change in emission intensity caused by mitochondrial membrane potential, a change in emission intensity by photo-bleaching of the dye itself occurs at the same time, change of the fluorescence intensity caused by changes in the membrane potential, due to photo-bleaching of the dye makes it difficult to distinguish, in the membrane potential is not necessarily suitable for the detection of the change.
In addition, the emission color of the other changes in the dye type, due to the change in membrane potential dye in order to observe the color of the emitted light, the fluorescent excitation light source and the detector is also reduced depending on the emission color are to be provided and the system 2, and the excitation light source and the real-time fluorescence detector may need to be adjusted, and the cost of the device, experimental operations becomes extremely complicated. Further, one type of dye can be, from the low solubility of the staining, cells that are harmful to the organic solvent is also a problem that must be used.
The inventors of the present invention, in accordance with the mitochondrial membrane potential, nuclear mitochondrial localization is shifted to the location of the following compounds have the nature of the BP has been found, can be used for the staining of cells and has already been reported (Non-Patent Document 1).
In accordance with the mitochondrial membrane potential, localized in the nucleus compound BP from mitochondria where the properties of the transfer, since it is not a change in fluorescence intensity, its own BP of the photo-bleaching compound without being affected by the changes in the membrane potential to be detected. In addition, since there is no change in the fluorescence color, fluorescence excitation light source and a special device is not required, generally the film by the fluorescent microscope to allow detection of a change of potential. In addition, the water-soluble compound BP is high, the cell without using a harmful organic solvent and is capable of staining of the cells. Therefore, the organic solvent which does not cause cell death, living cells over more than 24 time and can be observed.
However, in the BP compound, the absorption (excitation) by the number of photons absorbed by the compound and the number of photons emitted by fluorescence quantum yield (φ) is a ratio as low as 0.14, may be the luminescence efficiency is not, high sensitivity to detect the fluorescent light emitting efficiency of the compound may have been required.
Therefore, the inventors of the present invention, high quantum yields of light emission efficiency may be further proposed a compound (Patent Document 1 and 2). However, Patent Document 1 and this compound was proposed in 2, light emission of blue or green light and is generated in a region, red region emitting compound was not obtained. A red region of the biological light emission of a high permeability and therefore can be easily identified, has a response to the mitochondrial membrane potential, and the transmission of the biological compounds for emitting light in the red color has been demanded.
Scope of claims (In Japanese)[請求項1]
式(1)で表される化合物。
[化1]

[式(1)中、Xは、
[化2]

(式中、R 1はC1~C10のアルキル基を表し、Z はピリジニウムカチオンに対するカウンターアニオンを表す。)で表され、k及びmは0~3の整数であり、l及びnは0~2の整数であり、k、l、m及びnは同時に0ではなく、Xは同じでも異なっていてもよい。R 2は電子供与性基又は電子求引性基を表し、a及びcは0~3の整数であり、b及びdは0~2の整数であり、R 2は同じでも異なっていてもよく、Xが置換していない炭素原子上に置換する。波線は、幾何異性体E、Zを表す。]

[請求項2]
カウンターアニオンが、ハロゲン化物イオン、ヘキサフルオロリン酸イオン、スルホネート又は過塩素酸イオンであることを特徴とする請求項1記載の化合物。

[請求項3]
請求項1又は2記載の化合物の1又は2以上を含有することを特徴とする蛍光色素組成物。

[請求項4]
請求項1又は2記載の化合物の1又は2以上を用いることを特徴とする、ミトコンドリア膜電位の変化の検出方法。

[請求項5]
請求項1又は2記載の化合物の1又は2以上を用いることを特徴とする、細胞の生死を判別する方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • YAMAGUCHI UNIVERSITY
  • KOCHI UNIVERSITY
  • Inventor
  • KAWAMATA, Jun
  • SUZUKI, Yasutaka
  • ASAMURA, Naoya
  • NIKO, Yosuke
  • SEKI, Hitomi
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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