JP7641003B2 - Fluorescent probes, methods for evaluating liquid phase polarity and viscosity, and compounds - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Publication name: Online Abstracts of the 43rd Annual Meeting of the Molecular Biology Society of Japan, website address: ttps://www.aeplan.co.jp/mbsj2020/, date of publication: November 19, 2020 (2) Meeting name: Online Meeting of the 43rd Annual Meeting of the Molecular Biology Society of Japan, website address: ttps://www.aeplan.co.jp/mbsj2020/, date of publication: December 4, 2020

The present invention relates to a fluorescent probe, a method for evaluating the polarity and viscosity of a liquid phase, and a compound.

Many intracellular compartments are formed by liquid-liquid phase separation (LLPS) of biomolecules. The aggregates produced by LLPS are also called membraneless organelles because they do not have a lipid bilayer. Examples of membraneless organelles include coactivator condensates, centrosomes, stress granules, and nucleoli. In principle, phase-separated aggregates can provide a localized high concentration of biomolecules without the need for control of protein synthesis. LLPS provides a dynamic biological reaction field within cells.

Methods proposed for evaluating the dynamics or physical properties of LLPS include phase separation assays using bright-field imaging or fluorescence microscopy, particle tracking using microrheology, and NMR analysis. However, these methods have various technical issues, such as high background noise, complicated operations, difficulty in sample preparation, and difficulty in data processing. In addition, it is difficult to apply these methods to the evaluation of the dynamics and physical properties of LLPS within cells.

Pyrene is a polycyclic aromatic hydrocarbon with four fused benzene rings. Pyrene has dual fluorescent activity that is modulated by the solvent environment. Pyrene can form excimers by the concentration-dependent interaction of an excited pyrene monomer with another pyrene monomer in the ground state (Non-Patent Document 1). Examples of applications of such properties of pyrene have been reported, such as lipid labels (Non-Patent Document 1), telomere repeat RNA detection probes (Non-Patent Document 2), and complementary DNA sequence detection probes (Patent Document 1).

JP 2005-080637 A

Pentti Somerharju, Pyrene-labeled lipids as tools in membrane biophysics and cell biology. Chem Phys Lipids. 2002 Jun; 116(1-2):57-74. Yan Xu, Yuta Suzuki, Kenichiro Ito, and Makoto Komiyama. Telomeric repeat-containing RNA structure in living cells. Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14579-84.

The dynamics and physical properties of LLPS in cells are important for understanding intracellular vital reactions. In recent years, it has been thought that phase transitions and/or abnormalities in the control of phase transitions are also involved in diseases such as neurodegenerative diseases and cancer. Therefore, there is a demand for the development of technology that can easily detect the dynamics and physical properties of LLPS.

The present invention aims to provide a fluorescent probe capable of evaluating the dynamics or physical properties of LLPS in cells, a method for evaluating the polarity and viscosity of a liquid phase using the fluorescent probe, and a compound used in the fluorescent probe.

The present invention includes the following aspects.
[1] A fluorescent probe for evaluating the polarity and viscosity of a liquid phase, comprising a compound (P) represented by the following general formula (p):

［式中、Ｐｙ^１はピレンから（ｎ^１＋１）個の水素原子を除いた基を表し、Ｐｙ^２はピレンから（ｎ^２＋１）個の水素原子を除いた基を表し、Ｌは２価の有機基を表し、Ｒ^１及びＲ^２は、それぞれ独立に、１価の有機基を表す。ｎ^１及びｎ^２は、それぞれ独立に、１～９の整数を表す。］
［２］前記化合物（Ｐ）が、下記一般式（ｐ－１）で表される化合物である、［１］に記載の蛍光プローブ。

[In the formula, ^Py1 represents a group obtained by removing ( ⁿ¹ +1) hydrogen atoms from pyrene, ^Py2 represents a group obtained by removing ( ⁿ² +1) hydrogen atoms from pyrene, L represents a divalent organic group, ^R1 and ^R2 each independently represent a monovalent organic group, and ⁿ¹ and ⁿ² each independently represent an integer from 1 to 9.]
[2] The fluorescent probe according to [1], wherein the compound (P) is a compound represented by the following general formula (p-1):

［式中、Ｌ^１は２価の有機基を表し、Ｒ^１１及びＲ^１２は、それぞれ独立に、１価の有機基を表す。］
［３］前記化合物（Ｐ）が、下記式（Ｐｙｒ－Ａ）で表される化合物である、［２］に記載の蛍光プローブ。

[In the formula, L ¹ represents a divalent organic group, and R ¹¹ and R ¹² each independently represent a monovalent organic group.]
[3] The fluorescent probe according to [2], wherein the compound (P) is a compound represented by the following formula (Pyr-A):

［式中、ＰＥＧは、ポリエチレングリコール基を表す。］
［４］前記液相が、細胞内に存在する液相である、［１］～［３］いずれか１つに記載の蛍光プローブ。
［５］［１］～［４］いずれか１つに記載の蛍光プローブを用いて、液相の極性及び粘性を評価する方法であって、対象試料中で、前記蛍光プローブのモノマー発光による蛍光（ｍ）を測定する工程（ｉ）と、前記対象試料中で、前記蛍光プローブのエキシマー発光による蛍光（ｅ）を測定する工程（ｉｉ）と、を含む、液相の極性及び粘性を評価する方法。
［６］前記工程（ｉ）で測定された蛍光（ｍ）と、前記工程（ｉｉ）で測定された蛍光（ｅ）との蛍光強度比に基づいて、前記液相の極性及び粘性を評価する工程（ｉｉｉ）をさらに含む、［５］に記載の液相の極性及び粘性を評価する方法。
［７］前記液相が、細胞内に存在する液相である、［５］又は［６］に記載の液相の極性及び粘性を評価する方法。
［８］下記式（Ｐｙｒ－Ａ）で表される化合物。

[In the formula, PEG represents a polyethylene glycol group.]
[4] The fluorescent probe according to any one of [1] to [3], wherein the liquid phase is a liquid phase present within a cell.
[5] A method for evaluating the polarity and viscosity of a liquid phase using the fluorescent probe according to any one of [1] to [4], comprising: a step (i) of measuring, in a target sample, fluorescence (m) due to monomer emission of the fluorescent probe; and a step (ii) of measuring, in the target sample, fluorescence (e) due to excimer emission of the fluorescent probe.
[6] The method for evaluating the polarity and viscosity of a liquid phase described in [5], further comprising a step (iii) of evaluating the polarity and viscosity of the liquid phase based on a fluorescence intensity ratio between the fluorescence (m) measured in the step (i) and the fluorescence (e) measured in the step (ii).
[7] The method for evaluating the polarity and viscosity of a liquid phase described in [5] or [6], wherein the liquid phase is a liquid phase present within a cell.
[8] A compound represented by the following formula (Pyr-A):

［式中、ＰＥＧは、ポリエチレングリコール基を表す。］

[In the formula, PEG represents a polyethylene glycol group.]

The present invention provides a fluorescent probe capable of evaluating the dynamics or physical properties of LLPS in cells, a method for evaluating the polarity and viscosity of a liquid phase using the fluorescent probe, and a compound used in the fluorescent probe.

FIG. 2 is a schematic diagram showing the relationship between the viscosity and polarity of a liquid phase and the monomer luminescence and excimer luminescence of a compound (P). The fluorescence spectra of the compound (Pyr-A) in various solvents are shown. Excitation wavelength: 358 nm. The graph shows the ratio of the fluorescence intensity of excimer emission to the sum of the fluorescence intensity of monomer emission (detection wavelength 400 nm) and the fluorescence intensity of excimer emission (detection wavelength 470 nm) versus E _T (30) for various solvents [I ₄₇₀ (I ₄₀₀ /I ₄₇₀ )]. Fluorescence spectra of compound (Pyr-A) in a mixed solvent of methanol and polyethylene glycol 4000 (PEG4000) (PEG4000 concentrations: 0 gL ⁻¹ , 25 gL ⁻¹ , 50 gL ⁻¹ , 100 gL ⁻¹ ) are shown, excitation wavelength 358 nm. Fluorescence microscopy images taken of droplets of BRD4-IDR-mEGFP formed in the presence (+) and absence (-) of a compound (Pyr-A) are shown. Figure 1 shows a plot of the fluorescence intensity ratio (fluorescence(e)/fluorescence(m)) between excimer emission (fluorescence(e)) and monomer emission (fluorescence(m)) versus droplet size of BRD4-IDR-mEGFP. Pearson r=-0.83, p=0.011. Fluorescence microscopy images of cells treated with compound (Pyr-A) were taken at interphase (upper row) and mitotic phase (lower row). FIG. 1 is a schematic showing centrosome maturation during mitosis. The results of analyzing the fluorescence intensity ratio (fluorescence (e)/fluorescence (m)) between excimer luminescence (fluorescence (e)) and monomer luminescence (fluorescence (m)) in the centrosome are shown.

The term "aromatic hydrocarbon group" refers to a hydrocarbon group having at least one aromatic ring. The aromatic ring is not particularly limited as long as it is a cyclic conjugated system having 4n+2 π electrons, and may be either monocyclic or polycyclic.
The term "aliphatic hydrocarbon group" means a hydrocarbon group that does not contain an aromatic ring.
The phrase "may have a substituent" includes both the case where a hydrogen atom (--H) is replaced with a monovalent group and the case where a methylene group (--CH ₂ -) is replaced with a divalent group.
In the present specification and claims, some structures represented by chemical formulas may have asymmetric carbons, and may have enantiomers or diastereomers. In such cases, a single chemical formula represents all of the isomers. These isomers may be used alone or as a mixture.

[Fluorescent probe]
A first aspect of the present invention is a fluorescent probe for evaluating the polarity and viscosity of a liquid phase (hereinafter also referred to as “fluorescent probe (P)”), which comprises a compound (P) represented by the following general formula (p):

［式中、Ｐｙ^１はピレンから（ｎ^１＋１）個の水素原子を除いた基を表し、Ｐｙ^２はピレンから（ｎ^２＋１）個の水素原子を除いた基を表し、Ｌは２価の有機基を表し、Ｒ^１及びＲ^２は、それぞれ独立に、１価の有機基を表す。ｎ^１及びｎ^２は、それぞれ独立に、１～９の整数を表す。］

[In the formula, ^Py1 represents a group obtained by removing ( ⁿ¹ +1) hydrogen atoms from pyrene, ^Py2 represents a group obtained by removing ( ⁿ² +1) hydrogen atoms from pyrene, L represents a divalent organic group, ^R1 and ^R2 each independently represent a monovalent organic group, and ⁿ¹ and ⁿ² each independently represent an integer from 1 to 9.]

In the formula (p), ^Py1 represents a group in which ( ⁿ¹ +1) hydrogen atoms have been removed from pyrene, and ^Py2 represents a group in which ( ⁿ² +1) hydrogen atoms have been removed from pyrene.
ⁿ¹ and ⁿ² each independently represent an integer of 1 to 9. ⁿ¹ and ⁿ² are preferably integers of 1 to 5, more preferably integers of 1 to 3, further preferably 1 or 2, and particularly preferably 1. ⁿ¹ and ⁿ² may be the same or different. From the viewpoint of ease of synthesis, it is preferable that ⁿ¹ and ⁿ² are the same.

In the formula (p), ^R1 and ^R2 each independently represent a monovalent organic group. The monovalent organic group may be a hydrocarbon group which may have a substituent. The hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group.

When the monovalent organic group in ^R1 and ^R2 is an aliphatic hydrocarbon group which may have a substituent, the aliphatic hydrocarbon group may be saturated or unsaturated. The aliphatic hydrocarbon group may be a linear or branched aliphatic hydrocarbon group, or may be an aliphatic hydrocarbon group containing a ring structure (aliphatic ring).

In the linear or branched aliphatic hydrocarbon group, some of the methylene groups (-CH ₂ -) constituting the hydrocarbon chain may be substituted with a divalent substituent. Examples of the divalent substituent include, but are not limited to, -O-, -O-C(=O)-, -C(=O)-, -O-C(=O)-O-, -C(=O)-NH-, -NH-, -NH-C(=NH)- (H may be substituted with a substituent such as an alkyl group or an acyl group), -S-, -S(=O) ₂ -, -S(=O) ₂ -O-, and the like.

The aliphatic ring contained in the aliphatic hydrocarbon group containing a ring structure may be a monocyclic group or a polycyclic group. The aliphatic ring may be a hydrocarbon ring or a heterocycle containing a heteroatom. Examples of the heteroatom include an oxygen atom, a nitrogen atom, and a sulfur atom. The aliphatic ring preferably has 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, and even more preferably 3 to 6 carbon atoms. The number of carbon atoms in the aliphatic ring does not include the number of carbon atoms of the substituent.
Examples of the aliphatic hydrocarbon group containing a ring structure include a group in which one hydrogen atom is removed from an aliphatic hydrocarbon ring or an aliphatic heterocycle; a group in which a part of the hydrogen atoms of the group in which one hydrogen atom is removed from an aliphatic hydrocarbon ring or an aliphatic heterocycle is substituted with a straight-chain or branched-chain aliphatic hydrocarbon group; a group in which an aliphatic hydrocarbon ring or an aliphatic heterocycle is bonded to the end of a straight-chain or branched-chain aliphatic hydrocarbon group; a group in which an aliphatic hydrocarbon ring or an aliphatic heterocycle is interposed in the middle of a straight-chain or branched-chain aliphatic hydrocarbon group, etc. Examples of the straight-chain or branched-chain aliphatic hydrocarbon group include the same as those mentioned above.

When the monovalent organic group in ^R1 and ^R2 is an aromatic hydrocarbon group which may have a substituent, the aromatic ring contained in the aromatic hydrocarbon group may be an aromatic hydrocarbon ring or an aromatic heterocycle. Examples of heteroatoms contained in the aromatic heterocycle include an oxygen atom, a nitrogen atom, and a sulfur atom. The aromatic ring may be a monocycle or a polycycle.
The number of carbon atoms in the aromatic ring is preferably 5 to 30, more preferably 5 to 20, still more preferably 6 to 15, and particularly preferably 6 to 12. The number of carbon atoms in the aromatic ring does not include the number of carbon atoms of the substituent.
Examples of the aromatic hydrocarbon group include a group in which one hydrogen atom has been removed from an aromatic hydrocarbon ring or aromatic heterocycle; a group in which a part of the hydrogen atoms of the group in which one hydrogen atom has been removed from an aromatic hydrocarbon ring or aromatic heterocycle has been substituted with a linear or branched aliphatic hydrocarbon group; a group in which an aromatic hydrocarbon ring or aromatic heterocycle is bonded to the end of a linear or branched aliphatic hydrocarbon group; a group in which an aromatic hydrocarbon ring or aromatic heterocycle is interposed in the middle of a linear or branched aliphatic hydrocarbon group, etc. Examples of the linear or branched aliphatic hydrocarbon group include the same as those mentioned above.

In the aliphatic hydrocarbon group and the aromatic hydrocarbon group, some of the hydrogen atoms may be substituted with a monovalent substituent. The monovalent substituent is not particularly limited. Examples of the monovalent substituent include, but are not limited to, an amino group, a hydroxy group, a nitro group, a nitrile group, a carboxy group, an oxo group, a thiol group, an alkoxy group, and an acyl group.

R ¹ and R ² may be the same or different. From the viewpoint of ease of synthesis, it is preferable that R ¹ and R ² are the same.

R ¹ and R ² may be a combination of the above-mentioned straight-chain or branched-chain aliphatic hydrocarbon groups, aliphatic hydrocarbon groups having a ring structure, and aromatic hydrocarbon groups.
It is preferable that R ¹ and R ² contain a linear or branched aliphatic hydrocarbon group which may have a substituent. It is preferable that the linear or branched aliphatic hydrocarbon group has a part of the methylene groups (-CH ₂ -) constituting the hydrocarbon chain substituted with a divalent substituent which imparts hydrophilicity. Examples of the divalent substituent include -O-, -C(=O)-O-, -C(=O)-, -O-C(=O)-O-, -C(=O)-NH-, -NH-, -NH-C(=NH)- (H may be substituted with a substituent such as an alkyl group or an acyl group), -S-, -S(=O) ₂ -, or -S(=O) ₂ -O-. The divalent substituent is preferably -O-, -C(=O)-O-, -C(=O)-, -O-C(=O)-O-, or -C(=O)-NH-, more preferably -O-, -C(=O)-O-, or -C(=O)-NH-, and still more preferably -O-. The linear or branched aliphatic hydrocarbon group is preferably a linear aliphatic hydrocarbon group.

R ¹ and R ² are preferably groups represented by the following general formula (r).

［式中、Ｙ^０は置換基を有してもよい炭化水素基を表し、Ｒ^０は親水性基を表す。＊はＰｙ^１又はＰｙ^２に結合する結合手を表す。］

[In the formula, Y ⁰ represents a hydrocarbon group which may have a substituent, R ⁰ represents a hydrophilic group, and * represents a bond bonded to Py ¹ or Py ^2. ]

In formula (r), Y ⁰ represents a hydrocarbon group which may have a substituent. The hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group. Examples of the aliphatic hydrocarbon group and the aromatic hydrocarbon group include the same aliphatic hydrocarbon group and aromatic hydrocarbon group as those in R ¹ and R ² , respectively.

The hydrocarbon group in Y ⁰ preferably has 1 to 20 carbon atoms, more preferably has 3 to 15 carbon atoms, and more preferably has 6 to 10 carbon atoms. Y ⁰ is preferably an aromatic hydrocarbon group, more preferably a group in which an aromatic ring and a linear or branched aliphatic hydrocarbon group are bonded, and more preferably a group in which an aromatic ring and a linear aliphatic hydrocarbon group are bonded. When ^{Y 0} is an aromatic hydrocarbon group, the number of aromatic rings contained in Y ⁰ is not particularly limited, but is preferably 1 to 3, more preferably 1 or 2, and even more preferably 1. When Y ⁰ is a group in which an aromatic ring and a linear aliphatic hydrocarbon group are bonded, the linear aliphatic hydrocarbon group contained in Y ⁰ preferably has 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, and even more preferably 1 or 2 carbon atoms.

In formula (r), R ⁰ represents a hydrophilic group. The hydrophilic group is not particularly limited. Examples of the hydrophilic group include groups containing hydrophilic polymers such as polyethylene glycol, polypropylene glycol, poly(2-oxazoline), polyvinylpyrrolidone, polylactic acid, polycaprolactone, polyamino acid, and polysaccharide. R ⁰ may be a group containing a cell-penetrating peptide (polyarginine, polylysine, Tat peptide, etc.). When the fluorescent probe (P) is applied to a cell, R ⁰ is preferably a group having high biocompatibility, and more preferably a group having cell membrane permeability. As the group having cell membrane permeability, a polyethylene glycol group is preferable.

In the formula (p), L represents a divalent organic group. The divalent organic group preferably has 5 to 50 carbon atoms, more preferably has 6 to 40 carbon atoms, and even more preferably has 8 to 30 carbon atoms. The divalent organic group may be a divalent hydrocarbon group which may have a substituent. The hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group.

When the divalent organic group in L is an aliphatic hydrocarbon group which may have a substituent, the aliphatic hydrocarbon group may be saturated or unsaturated. The aliphatic hydrocarbon group may be a linear or branched aliphatic hydrocarbon group, or may be an aliphatic hydrocarbon group containing a ring structure (aliphatic ring).

In the linear or branched aliphatic hydrocarbon group, some of the methylene groups (-CH ₂ -) constituting the hydrocarbon chain may be substituted with a divalent substituent. Examples of the divalent substituent include the same as those mentioned above.

The aliphatic ring contained in the aliphatic hydrocarbon group containing a ring structure may be a monocyclic group or a polycyclic group. The aliphatic ring may be a hydrocarbon ring or a heterocycle containing a heteroatom. Examples of the heteroatom include an oxygen atom, a nitrogen atom, and a sulfur atom. The aliphatic ring preferably has 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, and even more preferably 3 to 6 carbon atoms. The number of carbon atoms in the aliphatic ring does not include the number of carbon atoms of the substituent.
Examples of the aliphatic hydrocarbon group containing a ring structure include a group in which two hydrogen atoms have been removed from an aliphatic hydrocarbon ring or an aliphatic heterocycle; a group in which a part of the hydrogen atoms of the group in which one hydrogen atom has been removed from an aliphatic hydrocarbon ring or an aliphatic heterocycle has been substituted with a straight-chain or branched-chain aliphatic hydrocarbon group; and a group in which an aliphatic hydrocarbon ring or an aliphatic heterocycle is interposed in the middle of a straight-chain or branched-chain aliphatic hydrocarbon group. Examples of the straight-chain or branched-chain aliphatic hydrocarbon group include the same as those mentioned above.

When the divalent organic group in L is an aromatic hydrocarbon group which may have a substituent, the aromatic ring contained in the aromatic hydrocarbon group may be an aromatic hydrocarbon ring or an aromatic heterocycle. Examples of heteroatoms contained in the aromatic heterocycle include an oxygen atom, a nitrogen atom, and a sulfur atom. The aromatic ring may be a monocycle or a polycycle.
The number of carbon atoms in the aromatic ring is preferably 5 to 30, more preferably 5 to 20, still more preferably 6 to 15, and particularly preferably 6 to 12. The number of carbon atoms in the aromatic ring does not include the number of carbon atoms of the substituent.
Examples of the aromatic hydrocarbon group include a group in which two hydrogen atoms have been removed from an aromatic hydrocarbon ring or an aromatic heterocycle; a group in which a part of the hydrogen atoms of the group in which one hydrogen atom has been removed from an aromatic hydrocarbon ring or an aromatic heterocycle has been substituted with a linear or branched aliphatic hydrocarbon group; a group in which an aromatic hydrocarbon ring or an aromatic heterocycle is present in the middle of a linear or branched aliphatic hydrocarbon group, etc. Examples of the linear or branched aliphatic hydrocarbon group include the same as those mentioned above.

In the aliphatic hydrocarbon group and the aromatic hydrocarbon group, some of the hydrogen atoms may be substituted with a monovalent substituent. Examples of the monovalent substituent include the same ones as described above.

L may be a combination of the above-mentioned straight-chain or branched-chain aliphatic hydrocarbon groups, aliphatic hydrocarbon groups having a cyclic structure, and aromatic hydrocarbon groups.
L preferably contains a linear or branched aliphatic hydrocarbon group which may have a substituent. In the linear or branched aliphatic hydrocarbon group, a part of the methylene groups (-CH ₂ -) constituting the hydrocarbon chain is preferably substituted with a divalent substituent which imparts hydrophilicity. Examples of the divalent substituent include the same as those described above. Examples of the divalent substituent include preferably -O-, -C(=O)-O-, -C(=O)-, -O-C(=O)-O- and -C(=O)-NH-, more preferably -O-, -C(=O)-O- and -C(=O)-NH-, and even more preferably -O-.

Compound (P) is preferably a compound represented by the following general formula (p-1):

［式中、Ｌ^１は２価の有機基を表し、Ｒ^１１及びＲ^１２は、それぞれ独立に、１価の有機基を表す。］

[In the formula, L ¹ represents a divalent organic group, and R ¹¹ and R ¹² each independently represent a monovalent organic group.]

In the formula, ^R11 and ^R12 each independently represent a monovalent organic group. Examples of the monovalent organic group include the same monovalent organic groups as those in ^R1 and ^R2 .

In the formula, ^L1 represents a divalent organic group. Examples of the divalent organic group include the same divalent organic groups as those in ^L1 .

Specific examples of compound (P) are shown below, but are not limited to these.

［式中、ＰＥＧは、ポリエチレングルコール基を表す。］

[In the formula, PEG represents a polyethylene glycol group.]

In the formula (Pyr-A), PEG represents a polyethylene glycol group. The polyethylene glycol group represented by PEG is derived from polyethylene glycol. The number average molecular weight (Mn) of the polyethylene glycol from which the polyethylene glycol group is derived is not particularly limited, but is preferably 200 to 4000, more preferably 400 to 2000, even more preferably 600 to 1500, and particularly preferably 800 to 1000.

<Method for synthesizing compound (P)>
Compound (P) can be synthesized by combining known chemical reactions. An example of a method for synthesizing compound (P) is shown below. In the following reaction formula, Py ¹ , R ¹ , n ¹ , Py ² , R ² , and n ² are the same as those in formula (p).

First, compound (a1) is reacted with compound (c1) to obtain compound (b1) (Reaction 1). The reaction can be carried out, for example, in a mixed solvent of toluene/ethanol under basic conditions such _{as K2CO3} , using a catalyst such as tetrakis(triphenylphosphine)palladium (Pd( _PPh3 ) ₄ ).

Similarly, compound (a2) is reacted with compound (c2) to obtain compound (b2) (Reaction 2). The reaction can be carried out, for example, in an organic solvent such as a toluene/ethanol mixed solvent under basic conditions such as potassium carbonate using a palladium catalyst such as Pd( _PPh3 ) ₄ .

Compound (d) is reacted with compound (e) to obtain compound (f) (Reaction 3). The reaction can be carried out, for example, in an organic solvent such as dioxane under basic conditions such as potassium acetate, using a palladium catalyst such as [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf) _2Cl2 ) _.

Next, compound (f) is reacted with compound (b1) and compound (b2) to obtain compound (P) represented by general formula (p) (Reaction 4). The reaction can be carried out, for example, in an organic solvent such as a toluene/ethanol mixed solvent under basic conditions such as potassium carbonate using a palladium catalyst such as Pd( _PPh3 ) ₄ .

In formula (p), when (R ¹ ) _n1 -Py ¹ - and (R ² ) _n2 -Py ² - are the same group, Reaction 1 and Reaction 2 are the same reaction.

When a hydrophilic polymer such as a polyethylene glycol group is introduced into R ¹ in formula (p), compound (c1') may be used in place of compound (c1) in Reaction 1. Similarly, when a hydrophilic polymer such as a polyethylene glycol group is introduced into R ² in formula (p), compound (c2') may be used in place of compound (c2) in Reaction 2.

［式中、Ｙ^０１及びＹ^０２は、それぞれ独立に、置換基を有してもよい炭化水素基を表し、Ｘは官能基を表す。］

[In the formula, Y ⁰¹ and Y ⁰² each independently represent a hydrocarbon group which may have a substituent, and X represents a functional group.]

Y ⁰¹ and Y ⁰² in the formulae (c1') and (c2') are the same as Y ⁰ in the formula (r).
In the formulas (c1') and (c2'), X is a functional group. The functional group in X can be used to introduce a hydrophilic polymer. Specific examples of X include a hydroxy group, an amino group, a carboxy group, an azide group, an alkynyl group, and a thiol group.

After Reaction 4, a hydrophilic polymer can be introduced into compound (P) represented by formula (p) using the functional group represented by X in formulas (c1') and (c2').

Compound (P) has a structure in which two atomic groups each containing a pyrene ring ((R ¹ ) _n1 -Ry ¹ - and (R ² ) _n2 -Ry ² -) are linked by a linker (L). In a liquid phase, compound (P) changes between a state in which two pyrene rings in the molecule associate to form an excimer and a state in which the two pyrene rings are dissociated, depending on the physical properties of the liquid phase. "Excimer emission" is fluorescence generated when the two pyrene rings of compound (P) form an excimer. "Monomer emission" is fluorescence generated when the two pyrene rings of compound (P) do not form an excimer (when the two pyrene rings are dissociated).

As shown in Figure 1, the proportion of molecules of compound (P) present in the liquid phase that do not form excimers increases as the viscosity of the liquid phase increases. Therefore, in a highly viscous liquid phase, the fluorescence intensity of monomer emission increases. On the other hand, the proportion of molecules of compound (P) present in the liquid phase that form excimers increases as the viscosity of the liquid phase decreases. Therefore, in a low-viscosity liquid phase, the fluorescence intensity of excimer emission increases.
The proportion of molecules of the compound (P) present in the liquid phase that do not form excimers increases as the polarity of the liquid phase decreases. Therefore, in a liquid phase with low polarity, the fluorescence intensity of the monomer emission increases. On the other hand, the proportion of molecules of the compound (P) present in the liquid phase that form excimers increases as the polarity of the liquid phase increases. Therefore, in a liquid phase with high polarity, the fluorescence intensity of the excimer emission increases.
Since the compound (P) has the above-mentioned properties, a fluorescent probe made of the compound (P) can be used to evaluate the viscosity and polarity of a liquid phase.

The cell membrane permeability of the compound (P) can be improved by introducing a hydrophilic group (preferably a group containing a hydrophilic polymer) into R ¹ and R ² in the formula (p). Therefore, a fluorescent probe (P) made of the compound (P) having a hydrophilic group introduced into R ¹ and R ² can be used to evaluate the viscosity and polarity of the liquid phase present in a cell.
LLPS is a phenomenon in which solutes in a solution do not mix uniformly, and the solution separates into two phases according to thermodynamic stability. When solutes are concentrated by LLPS, the viscosity and polarity of the solution change. Such changes in solution properties due to LLPS can also be evaluated by measuring the excimer and monomer luminescence of the fluorescent probe (P).
Intracellular LLPS is involved in the formation of membrane-free organelles and provides a scaffold for various biological reactions. The dynamics and physical properties of intracellular LLPS can be easily evaluated by incorporating the fluorescent probe (P) into cells and measuring the excimer and monomer luminescence of the fluorescent probe (P).

[Method for evaluating polarity and viscosity of liquid phase]
A second aspect of the present invention is a method for evaluating the polarity and viscosity of a liquid phase using the fluorescent probe of the first aspect. The method of this aspect includes a step (i) of measuring fluorescence (m) due to monomer emission of the fluorescent probe in a target sample, and a step (ii) of measuring fluorescence (e) due to excimer emission of the fluorescent probe in the target sample.

(Step (i))
In step (i), fluorescence (m) due to monomer emission of the fluorescent probe (P) is measured in a target sample.

The target sample is a sample to be evaluated for polarity and viscosity. There is no particular limitation on the target sample, so long as it is a sample that contains a liquid phase. Examples of target samples include cells, polymer compound solutions, and the like. There is no particular limitation on the biological species from which the cells are derived. The cells may be any of animal cells, plant cells, fungal cells, protozoan cells, and bacterial cells. Examples of polymer compounds contained in the polymer compound solution include biopolymers such as proteins, peptides, nucleic acids, lipids, and sugars; synthetic polymers, and the like. The target sample is preferably a sample in which LLPS is generated.

The fluorescent probe (P) is added to the target sample, and the fluorescence (m) due to monomer emission of the fluorescent probe (P) is measured. When the target sample is a cell, the fluorescent probe (P) is taken up by the cells by culturing in a medium containing the fluorescent probe (P). The culture time is not particularly limited, but examples include 15 minutes or more, 30 minutes or more, 45 minutes or more, or 60 minutes or more. The upper limit of the culture time is not particularly limited, but examples include 180 minutes or less, 150 minutes or less, 120 minutes or less, or 100 minutes or less.

A target sample containing a fluorescent probe (P) is irradiated with excitation light, and fluorescence (m) due to monomer emission is measured. The wavelength of the excitation light can be set according to the structure of the compound (P). The wavelength of the excitation light is, for example, 300 to 400 nm. The wavelength of the excitation light is preferably 340 to 380 nm. A specific example of the wavelength of the excitation light is 358 nm.
The wavelength used for measuring the fluorescence (m) due to monomer emission can be set according to the structure of compound (P). Examples of the wavelength used for measuring the fluorescence (m) include 380 to 420 nm, and preferably 390 to 410 nm. A specific example of the wavelength used for measuring the fluorescence (m) is 400 nm.

(Step (ii))
In step (ii), fluorescence (e) due to excimer emission of the fluorescent probe (P) is measured in a target sample.

The target sample is the same as the sample in step (i). After the fluorescent probe (P) is present in the liquid phase of the target sample, step (ii) can be carried out before step (i), after step (i), or simultaneously with step (i).

A target sample containing a fluorescent probe (P) is irradiated with excitation light, and fluorescence (e) due to excimer emission is measured. The excitation light may have the same wavelength as in step (i).
The wavelength used for measuring the fluorescence (e) due to excimer emission can be set according to the structure of compound (P). Examples of the wavelength used for measuring the fluorescence (e) include 450 to 490 nm, and preferably 460 to 480 nm. A specific example of the wavelength used for measuring the fluorescence (e) is 470 nm.

The fluorescent probe (P) emits higher intensity of fluorescence (m) when the viscosity of the liquid phase increases, and emits higher intensity of fluorescence (e) when the viscosity of the liquid phase decreases. The fluorescent probe (P) emits higher intensity of fluorescence (e) when the polarity of the liquid phase increases, and emits higher intensity of fluorescence (m) when the polarity of the liquid phase decreases.
Therefore, the viscosity and polarity of the liquid phase can be evaluated based on the fluorescence intensity of the fluorescence due to monomer emission (m) and the fluorescence due to excimer emission (e).

(Optional step)
The method of this aspect may include other steps in addition to the above steps (i) and (ii), such as step (iii) of evaluating the polarity and viscosity of the liquid phase based on the fluorescence intensity ratio between the fluorescence (m) measured in the step (i) and the fluorescence (e) measured in the step (ii).

By calculating the fluorescence intensity ratio between fluorescence (m) and fluorescence (e), the polarity and viscosity of the liquid phase can be more accurately understood. The fluorescence intensity ratio may be the ratio of fluorescence (e) to fluorescence (m) (fluorescence (e)/fluorescence (m)), or may be the ratio of fluorescence (m) to fluorescence (e) (fluorescence (m)/fluorescence (e)). For example, when the value of "fluorescence (e)/fluorescence (m)" is large, the liquid phase can be evaluated as having low viscosity and high polarity. When the value of "fluorescence (e)/fluorescence (m)" is small, the liquid phase can be evaluated as having high viscosity and low polarity.
For example, when the value of "fluorescence(m)/fluorescence(e)" is large, the liquid phase can be evaluated as having high viscosity and low polarity. When the value of "fluorescence(m)/fluorescence(e)" is small, the liquid phase can be evaluated as having low viscosity and high polarity.

By measuring the fluorescence (m) due to monomer emission and the fluorescence (e) due to excimer emission over time with the fluorescent probe (P) present in the liquid phase of the target sample, it is possible to track changes in the polarity and viscosity properties of the liquid phase over time. In addition, by incorporating the fluorescent probe (P) into cells and measuring the fluorescence (m) and fluorescence (e) over time, it is possible to track the dynamics of LLPS within the cells over time.

［式中、ＰＥＧは、ポリエチレングリコール基を表す。］ [Compound]
A third aspect of the present invention is a compound represented by the following formula (Pyr-A) (compound (Pyr-A)).

[In the formula, PEG represents a polyethylene glycol group.]

The compound (Pyr-A) can be synthesized by the method described in the Examples.
The compound (Pyr-A) can be used as a fluorescent probe (P).

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Synthesis Example]
(Synthesis of compound (1))
A mixture of 1,6-dibromopyrene (1.80 g, 5.0 mmol), 4-(hydroxymethyl)phenylboronic acid (0.76 g, 5.0 mmol), potassium carbonate (2 M aqueous solution, 13 mL) and toluene (80 mL) was deoxygenated by bubbling nitrogen gas for 40 min. To the mixture, Pd(Pph ₃ ) ₄ (208 mg, 0.18 mmol) was added, deoxygenated by bubbling nitrogen gas for 10 min and heated to 100° C. under N _2. During heating, deoxygenated ethanol (10 mL) was added to the mixture. After heating for 20 h, the resulting mixture was cooled to room temperature, dropped into water and extracted with a mixture of CH ₂ Cl _2. The organic layer was washed with water, dried over anhydrous Na ₂ SO ₄ and then evaporated to dryness. The residue was purified by silica gel column chromatography to obtain compound (1) as a pale yellow solid (1.05 g, 2.7 mmol, 54%).

The structure of compound (1) was identified by ¹ H NMR.
^1H NMR (400MHz, _CDCl3 , TMS standard): δ (ppm) 1.77 (t, J = 5.9Hz, 1H, OH), 4.86 (t, J = 5.9Hz, 2H, _CH2 ), 7.58 (d, J = 8.3 Hz, 2H, ArH), 7.63 (d, J = 8.3 Hz, 2H, ArH), 7.98 (d, J = 9 .3Hz, 1H, ArH), 8.00 (d, J = 8.3Hz, 1H, ArH), 8.01 (d, J = 7.9Hz, 1H, ArH) , 8.18 (d, J=9.3Hz, 1H, ArH), 8.21 (d, J=9.2Hz, 1H, ArH), 8.25 (d, J=8. 3Hz, 1H, ArH), 8.27 (d, J = 7.9Hz, 1H, ArH), 8.48 (d, J = 9.2Hz, 1H, ArH).

(Synthesis of compound (2))
Under _N2 , 2,2-dibutyl-1,3-propanediol (3.85 g, 20.5 mmol) in THF (70 mL) was slowly added to NaH (60% dispersion in oil, 2.45 g, 61.4 mmol) in THF (15 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h, and then 4-bromobenzyl bromide (15.36 g, 61.4 mmol) in THF (15 mL) was slowly added at 0 °C. It was then warmed to room temperature. To the mixture, anhydrous DMF (50 mL) was added and stirred at room temperature for 1 week. The resulting mixture was added dropwise to ice water and extracted with a mixed solvent of hexane and ethyl acetate (hexane:ethyl acetate = 3:1). The organic layer was washed with water, dried _over anhydrous _Na2SO4 , and then evaporated to dryness. The residue was purified by silica gel column chromatography (CH ₂ Cl ₂ /hexane=5/95) to obtain compound (2) as a white solid (6.81 g, 12.9 mmol, 63%).

The structure of compound (2) was identified by ¹ H NMR.
¹ HNMR (400 MHz, CDCl ₃ , TMS standard): δ (ppm) 0.88 (t, J = 7.2 Hz, 6H, CH ₃ ), 1.08-1.32 (m, 12H, CH ₂ ), 3.25 (s, 4H, CH2), 4.40 (s, 4H, CH ₂ ), 7.15 (d, J=8.3Hz, 4H, ArH), 7.43 (d, J=8.3Hz, 4H, ArH).

(Synthesis of compound (3))
A mixture of compound (2) (1.05 g, 2.0 mmol), bis(pinacolato)diboron (1.12 g, 4.4 mmol), potassium acetate (1.30 g, 13.2 mmol), and dioxane (14 mL) was deoxygenated by bubbling nitrogen gas for 40 minutes. To the mixture, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane adduct (50 mg) was added, deoxygenated by bubbling nitrogen gas for 20 minutes, and heated to 80°C under _N2 . Additional Pd catalyst (50 mg) and bis(pinacolato)diboron (67 mg) were added to the mixture. After heating for 30 minutes, the resulting mixture was cooled to room temperature, dropped into water, and extracted with a mixed solvent of hexane and ethyl acetate (hexane:ethyl acetate = 4: ₁ ). The organic layer was washed with brine, dried over anhydrous _Na2SO4 , and then evaporated to dryness. The residue was purified by silica gel column chromatography (ethyl acetate/hexane=5/95) to obtain compound (3) as a transparent oil (0.56 g, 0.9 mmol, 45%).

The structure of compound (3) was identified by ¹ H NMR.
¹ HNMR (400MHz, CDCl ₃ , TMS standard): δ (ppm) 0.87 (t, J=7.3Hz, 6H, CH ₃ ), 1.08-1.30 (m, 12H, CH ₂ ), 1.34 (s, 24H, CH ₃ ), 3.26 (s, 4H, CH ₂ ), 4.48 (s, 4H, CH 2 ), 7.31 (d, J=8.1Hz, 4H, ArH), 7.77 (d, J=8.1Hz, 4H, ArH).

(Synthesis of compound (4))
A mixture of compound (3) (400 mg, 0.64 mmol), compound (1) (548 mg, 1.41 mmol), potassium carbonate (2 M aqueous solution, 2 mL), ethanol (2 mL), and toluene (4 mL) was deoxygenated by bubbling with nitrogen gas for 30 minutes. To the mixture, Pd( _PPh3 ) ₄ (45 mg, 0.04 mmol) was added, deoxygenated by bubbling with nitrogen gas for 20 minutes, and refluxed under _N2 for 15 hours. The resulting mixture was cooled to room temperature and filtered. The residue was washed with water, methanol, _{and CH2Cl2} , and dried in vacuum to give compound (4) (531 mg, 0.54 mmol, 84%) as a pale yellow solid.

The structure of compound (4) was identified by ¹ H NMR.
¹ HNMR (400MHz, DMSO-d6, TMS standard): δ (ppm) 0.92 (br-t, 6H, CH ₃ ), 1.20-1.38 (m, 12H, CH ₂ ), 3.42 (s, 4H, CH ₂ ), 4.63-4.70 (br-m, 8H, CH ₂ ), 5.36 (br-t, 3H, OH), 7.52-8.73 (m, 32H, ArH).

(Synthesis of compound (Pyr-A))
NaH (60% dispersion in oil, 20 mg, 0.29 mmol) was slowly added to a mixture of compound (4) (36 mg, 0.06 mmol), anhydrous THF (0.5 mL), and anhydrous DMF (0.6 mL) under _N2 at 0°C. After stirring at 0°C for 2 hours, poly(ethylene glycol) methyl ether tosylate (Mn=900 gmol ^-1 , 156 mg, 0.24 mmol) was added to the mixture, which was then warmed to room temperature and stirred for 35 hours. The resulting mixture was added dropwise to a saturated _NH4Cl aq. _and extracted with _CH2Cl2 . The organic layer was washed with water, dried _over anhydrous _Na2SO4 , and then evaporated to dryness. The residue was purified by silica gel column chromatography (methanol/CH ₂ Cl ₂ =2/98 to 5/95) and size exclusion chromatography to obtain compound (Pyr-A) as a pale yellow solid (48 mg, 0.019 mmol, 32%).

The structure of the compound (Pyr-A) was identified by ¹ H NMR.
¹ HNMR (400MHz, CDCl3, TMS standard): δ (ppm) 0.96 (t, J=7.1Hz, 6H, CH ₃ ), 1.24-1.45 (m, 12H, CH ₂ ), 3.36-3.78 (m, CH ₂ , CH ₃ ), 4.69 (s, 4H, CH ₂ ), 4.72 (s, 4H, CH ₂ ), 7.53-7.57 (m, 8H, ArH), 7.59-7.63 (m, 8H, ArH), 7.90-7.99 (m, 8H, ArH), 8.08-8.20 (m, 8H, ArH).

[Fluorescence properties of compound (Pyr-A)]
The ultraviolet-visible absorption spectrum of the compound (Pyr-A) in various solvents was measured using a spectrophotometer (V-750, JASCO Corporation). The fluorescence spectrum of the compound (Pyr-A) in various solvents was measured using a spectrofluorophotometer (FP-8300, JASCO Corporation). The temperature during the measurement was maintained at 25±1° C. The concentration of the compound (Pyr-A) in the solvent was 1.0 μM. The fluorescence spectrum of the compound (Pyr-A) was measured at an excitation wavelength of 358 nm.

Compound (Pyr-A) had a strong absorption band in the range of 320 to 400 nm, similar to that of 1,6-diphenylpyrene derivatives (data not shown).

Figure 2 shows the fluorescence spectra of compound (Pyr-A) in various solvents. Two bands were confirmed in the fluorescence spectrum of compound (Pyr-A) at around 400 nm and 470 nm. These two bands were similar to those of 1,6-diphenylpyrene derivatives and were considered to correspond to the emission of the monomer and excimer, respectively (Imai, K., et al., (2012). Polym. Bull. 68, 158-1601.).
The fluorescence intensity varied depending on the type of solvent: more polar solvents enhanced the excimer emission of compound (Pyr-A) compared to less polar solvents, whereas less polar solvents enhanced the monomer emission compared to more polar solvents.

3 shows the results of plotting the value [I ₄₇₀ (I ₄₀₀ /I ₄₇₀ )], which indicates the ratio of excimer emission to the total value of the fluorescence intensity of monomer emission (detection wavelength 400 nm) and excimer emission (detection wavelength 470 nm), as a function of the solvent polarity parameter E _T (30). The higher the polarity of the solvent, the larger the [I ₄₇₀ (I ₄₀₀ /I ₄₇₀ )] value. This result also confirmed that the higher the polarity of the solvent, the more enhanced the excimer emission. The excimer emission was more prominent in low-viscosity aprotic polar solvents than in high-viscosity protic polar solvents.

4 shows the fluorescence spectrum of the compound (Pyr-A) in a mixed solvent of methanol and polyethylene glycol 4000 (PEG4000) (PEG4000 concentration: 0 gL ⁻¹ , 25 gL ⁻¹ , 50 gL ⁻¹ , 100 gL ⁻¹ ). As the concentration of PEG4000 increased, the monomer emission was enhanced and the excimer emission was suppressed.

These results confirm that for compound (Pyr-A), excimer emission is dominant in environments with high polarity and low viscosity, while monomer emission is dominant in environments with low polarity and high viscosity.

Droplet Assay
BRD4 is a coactivator involved in transcriptional regulation, and its IDR forms droplets in vitro (Wang, ZA, and Cole, PA (2020). Cell Chem. Biol. 27, 953-969.). We therefore constructed a recombinant fusion protein (BRD4-IDR-mEGFP) containing the IDR of BRD4 (BRF4-IDR) and mEGFP (monomeric enhanced green fluorescent protein), and performed a droplet assay of the compound (Pyr-A).

Recombinant mEGFP protein was obtained from ORIGENE (Sku# TP790050). Recombinant mEGFP fusion protein (BRF4aa674-1351; BRD4-IDR-mEGFP) was concentrated using an Amicon Ultra centrifugal filter (50K MWCO, Millipore). The recombinant protein was diluted with droplet buffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT, 10% PEG) to a final concentration of 10 μM. The droplet buffer used for dilution was either with or without 10 μM compound (Pyr-A). Immediately after dilution, the mixture was placed on a glass-bottom dish (Matsunami Glass Industry) and covered with a cover glass. Droplets were imaged using a Zeiss LSM5 EXCITER microscope equipped with a mercury lamp and a Plan-Apochromat 100x/1.4 oil objective. Monomer emission was measured using a customized fluorescence cube I (Ex365/DM390/Em425, Zeiss). Excimer emission was measured using a fluorescence cube II (Ex365/DM400/Em490, Zeiss). Axio Vision software (version 4.8) was used for image acquisition.

Figure 5 shows the results of the droplet assay. It was confirmed that the compound (Pyr-A) was incorporated into the droplets of BRD4-IDR-mEGFP.

Figure 6 shows the relationship between the fluorescence intensity ratio (fluorescence (e)/fluorescence (m)) of excimer emission (fluorescence (e)) and monomer emission (fluorescence (m)) and the droplet size. "Fluorescence (e)/fluorescence (m)" showed a negative correlation with the droplet size. This result suggests that hydrophobicity and viscosity increase as the protein droplets mature.

[Cell assay]
The compound (Pyr-A) was used to observe the centrosomes in cells. The centrosome is composed of a pair of centrioles and pericentriolar material (PCM). Centrin-1 and pericentrin are included in the centrosome. During mitosis, after the nuclear membrane breaks down, specific components of the nuclear pore complex, such as NUP62, localize to the centrosome and participate in the maturation of the PCM. The centrosomes in mitosis are significantly larger than those in interphase. During mitosis, intracellular viscosity is thought to increase and polarity is thought to decrease compared to interphase (see FIG. 7B).

(Cell Culture)
HeLa cells were obtained from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum and penicillin/streptomycin in a humidified atmosphere at 37° C. with 5% CO ₂ .

(cDNA Vector and Transformation)
Centrin1-EGFP vector was prepared by cloning HeLa cell cDNA into C1-EGFP vector using KpnI/BamH1 restriction site. DNA was introduced into HeLa cells using Lipofectamine 2000 (Life Technologies). After transformation, HeLa cells stably expressing Centrin1-EGFP were selected by G418 treatment.

(Live cell fluorescence microscopy imaging)
Cells were seeded on glass-bottom dishes (Matsunami Glass Industry) and cultured for 24 hours to allow attachment. Cells were then synchronized by thymidine block to increase cell density in mitosis and cultured in medium containing 10 μM compound (Pyr-A). After 1 hour of culture, cells were washed twice with PBS and fresh medium was added. Cells were then imaged using a Zeiss LSM5 EXCITER microscope equipped with a mercury lamp and a Plan-Apochromat 100×/1.4 oil objective. Axio Vision software (version 4.8) was used for image acquisition. Approximately 30 image slices (0.2 mm thick) were acquired to identify the region of the centrosome. The position with the maximum centrin1-GFP signal was selected for line profile analysis.

Figure 7A shows fluorescence microscopy images of cells in the interphase and mitosis phase. Both monomer and excimer luminescence were observed in the cells in both the interphase and mitosis phases. This result confirmed that the compound (Pyr-A) penetrates the cells.

Figure 8 shows the results of analyzing the excimer emission (fluorescence (e)) and monomer emission (fluorescence (m)) in the centrosomes, as well as the fluorescence intensity ratio (fluorescence (e)/fluorescence (m)). In cells in mitosis, the "fluorescence (e)/fluorescence (m)" value in the region centered on the centrin1-GFP signal was significantly lower than in cells in interphase. These results indicate that during mitosis, centrosomes mature through expansion of the PCM, changing the microenvironment around the centrosome to a more viscous, less polar state.

The present invention provides a fluorescent probe capable of evaluating the dynamics or physical properties of LLPS in cells, a method for evaluating the polarity and viscosity of a liquid phase using the fluorescent probe, and a compound used in the fluorescent probe.

Claims (8)

A fluorescent probe for evaluating the polarity and viscosity of a liquid phase, comprising a compound (P) represented by the following general formula (p −1 ):

[In the formula, L ¹ represents a divalent hydrocarbon group having 6 to 40 carbon atoms. However, when the divalent hydrocarbon group contains a linear or branched aliphatic hydrocarbon group, a part of the methylene groups constituting the hydrocarbon chain of the aliphatic hydrocarbon group may be substituted with a divalent substituent selected from the group consisting of -O-, -O-C(=O)-, -C(=O)-, -O-C(=O)-O-, -C(=O)-NH-, -NH-, -NH-C(=NH)-, -S-, -S(=O) ₂ -, and -S(=O) ₂ -O-. R ¹¹ and R ¹² each independently represent a group represented by the following general formula (r)]

[In the formula, Y ⁰ represents a divalent hydrocarbon group having 1 to 20 carbon atoms. However, when the divalent hydrocarbon group contains a linear or branched aliphatic hydrocarbon group, a part of the methylene groups constituting the hydrocarbon chain of the aliphatic hydrocarbon group may be substituted with a divalent substituent selected from the group consisting of -O-, -O-C(═O)-, -C(═O)-, -O-C(═O)-O-, -C(═O)-NH-, -NH-, -NH-C(═NH)-, -S-, -S (═O) ₂ -, and -S(═O) ₂ -O-. R 0 ^represents a polyethylene glycol group. * represents a bond bonded to the pyrene ring in general formula (p-1).] The above L ¹ a linear or branched aliphatic hydrocarbon group in which a part of the methylene groups constituting the hydrocarbon chain may be substituted with a divalent substituent selected from the group consisting of -O- and -S-;
The Y ⁰ a linear or branched aliphatic hydrocarbon group in which a part of the methylene groups constituting the hydrocarbon chain may be substituted with a divalent substituent selected from the group consisting of -O- and -S-;
The fluorescent probe according to claim 1 . 3. The fluorescent probe according to claim 2, wherein the compound (P) is a compound represented by the following formula (Pyr-A):

[In the formula, PEG represents a polyethylene glycol group.] The fluorescent probe according to any one of claims 1 to 3, wherein the liquid phase is a liquid phase present within a cell. A method for evaluating polarity and viscosity of a liquid phase using the fluorescent probe according to any one of claims 1 to 4, comprising the steps of:
A step (i) of measuring fluorescence (m) due to monomer emission of the fluorescent probe in a target sample;
(ii) measuring fluorescence (e) due to excimer emission of the fluorescent probe in the target sample;
A method for evaluating the polarity and viscosity of a liquid phase, comprising: The method for evaluating the polarity and viscosity of a liquid phase according to claim 5, further comprising a step (iii) of evaluating the polarity and viscosity of the liquid phase based on the fluorescence intensity ratio between the fluorescence (m) measured in the step (i) and the fluorescence (e) measured in the step (ii). The method for evaluating the polarity and viscosity of a liquid phase according to claim 5 or 6, wherein the liquid phase is a liquid phase present within a cell. A compound represented by the following formula (Pyr-A):

[In the formula, PEG represents a polyethylene glycol group.]

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