Fluorescent probes, methods for evaluating liquid phase polarity and viscosity, and compounds

Abstract

To provide a fluorescent probe capable of evaluating a dynamic state or physical properties of a liquid-liquid phase separation in a cell, a method of evaluating the polarity and viscosity of a liquid phase using the fluorescent probe, and a compound to which the fluorescent probe can be used.SOLUTION: A fluorescent probe for evaluating the polarity and viscosity of a liquid phase, comprising a compound (P) represented by the following general formula (p). 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, and R1 and R2 each independently represents a monovalent organic group. n1 and n2 each independently represent an integer of 1 to 9.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to fluorescent probes, methods and compounds for assessing the polarity and viscosity of liquid phases. [Background technology]

[0002] 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 high concentration of biomolecules locally without the control of protein synthesis. LLPS provides a dynamic biological reaction field within cells.

[0003] Methods proposed to evaluate 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 challenges, such as high background noise, complicated operations, difficulty in sample preparation, and difficulty in data processing. In addition, it is difficult to apply them to the evaluation of the dynamics and physical properties of LLPS in cells.

[0004] Pyrene is a polycyclic aromatic hydrocarbon in which four benzene rings are condensed. Pyrene has dual fluorescent activity that is modulated by the solvent environment. Pyrene can form an excimer by a pyrene monomer in an excited state interacting with another pyrene monomer in the ground state in a concentration-dependent manner (Non-Patent Document 1). Examples of applications of such properties of pyrene to lipid labels (Non-Patent Document 1), telomere repeat RNA detection probes (Non-Patent Document 2), complementary DNA sequence detection probes (Patent Document 1), etc. have been reported. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2005-080637 A [Non-patent literature]

[0006] [Non-Patent Document 1] Pentti Somerharju, Pyrene-labeled lipids as tools in membrane biophysics and cell biology. Chem Phys Lipids. 2002 Jun; 116(1-2):57-74. [Non-Patent Document 2] Yan Xu, Yuta Suzuki, Kenichiro Ito, and Makoto Komiyama. Telomeric repeat-containing RNA structure in living cells. Proc Natl Acad Sci US A. 2010 Aug 17;107(33):14579-84. Summary of the Invention [Problem to be solved by the invention]

[0007] The dynamics and properties of LLPS in cells are important for understanding intracellular vital reactions. In recent years, it has been suggested 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 a technology that can easily detect the dynamics and properties of LLPS.

[0008] Therefore, 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. [Means for solving the problem]

[0009] 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):

[0010] [ka] [In the formula, Py 1 is pyrene (n 1 +1) hydrogen atoms are removed, and Py 2 is pyrene (n 2 +1) hydrogen atoms are removed, L represents a divalent organic group, R 1 and R 2 Each of n independently represents a monovalent organic group. 1 and n 2 each independently represents an integer of 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):

[0011] [ka] [In the formula, L 1 represents a divalent organic group, R 11 and R 12 each independently represents 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):

[0012] [ka] [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 the 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). [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).

[0013] [ka] [In the formula, PEG represents a polyethylene glycol group.] Effect of the Invention

[0014] According to the present invention, there are provided a fluorescent probe capable of evaluating the dynamics or physical properties of LLPS in a cell, a method for evaluating the polarity and viscosity of a liquid phase using the fluorescent probe, and a compound used in the fluorescent probe. [Brief description of the drawings]

[0015] [Figure 1] FIG. 2 is a schematic diagram showing the relationship between the viscosity and polarity of the liquid phase and the monomer luminescence and excimer luminescence of the compound (P). [Diagram 2] The fluorescence spectra of compound (Pyr-A) in various solvents are shown. Excitation wavelength: 358 nm. [Diagram 3]This is a graph plotting the ET(30) of various solvents against the value [I470 (I400 / I470)] which 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). [Figure 4] The fluorescence spectrum of 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) is shown. Excitation wavelength: 358 nm. [Diagram 5] Fluorescence microscopy images of droplets of BRD4-IDR-mEGFP formed in the presence (+) and absence (-) of a compound (Pyr-A) are shown. [Figure 6] Figure 1 shows the fluorescence intensity ratio (fluorescence(e) / fluorescence(m)) between excimer emission (fluorescence(e)) and monomer emission (fluorescence(m)) plotted against droplet size of BRD4-IDR-mEGFP. Pearson r=-0.83, p=0.011. [Figure 7A] Fluorescence microscopy images of cells treated with compound (Pyr-A) are shown, using cells in interphase (upper row) and mitosis (lower row). [Figure 7B] FIG. 1 is a schematic showing centrosome maturation during mitosis. [Figure 8] The results of analyzing the fluorescence intensity ratio (fluorescence (e)) between excimer emission (fluorescence (e)) and monomer emission (fluorescence (m)) in the centrosome are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 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. When describing "optionally having a substituent," it means replacing a hydrogen atom (-H) with a monovalent group or a methylene group (-CH2 -) 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.

[0017] [Fluorescent probe] A first aspect of the present invention is a fluorescent probe (hereinafter also referred to as "fluorescent probe (P)") for evaluating the polarity and viscosity of a liquid phase, which comprises a compound (P) represented by the following general formula (p):

[0018] [ka] [In the formula, Py 1 is pyrene (n 1 +1) hydrogen atoms are removed, and Py 2 is pyrene (n 2 +1) hydrogen atoms are removed, L represents a divalent organic group, R 1 and R 2 Each of n independently represents a monovalent organic group. 1 and n 2 each independently represents an integer of 1 to 9.

[0019] In the formula (p), Py 1 is from pyrene (n 1 +1) hydrogen atoms are removed. 2 is from pyrene (n 2 +1) hydrogen atoms have been removed. n 1 and n 2 Each independently represents an integer of 1 to 9. 1 and n 2 is preferably an integer of 1 to 5, more preferably an integer of 1 to 3, further preferably 1 or 2, and particularly preferably 1. 1 and n 2may be the same or different. From the viewpoint of ease of synthesis, n 1 and n 2 are preferably the same.

[0020] In the formula (p), R 1 and R 2 Each of the groups independently represents 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.

[0021] R 1 and R 2 When the monovalent organic group in 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 straight-chain or branched-chain aliphatic hydrocarbon group, or may be an aliphatic hydrocarbon group containing a ring structure (aliphatic ring).

[0022] A straight-chain or branched aliphatic hydrocarbon group is a group consisting of a methylene group (-CH 2 A part of -) may be substituted with a divalent substituent. Examples of the divalent substituent include -O-, -OC(=O)-, -C(=O)-, -OC(=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 Examples include, but are not limited to, -O- and the like.

[0023] 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.

[0024] R 1 and R 2 When the monovalent organic group in 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 the heteroatom 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 aromatic ring preferably has 5 to 30 carbon atoms, more preferably 5 to 20 carbon atoms, further preferably 6 to 15 carbon atoms, and particularly preferably 6 to 12 carbon atoms. 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 is 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 is removed from an aromatic hydrocarbon ring or aromatic heterocycle is 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.

[0025] In the aliphatic hydrocarbon group and the aromatic hydrocarbon group, a part of 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.

[0026] R 1 and R 2 may be the same or different. From the viewpoint of ease of synthesis, R 1 and R 2 are preferably the same.

[0027] 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. R 1 and R 2 It is preferable that the linear or branched aliphatic hydrocarbon group contains a methylene group (-CH 2It is preferable that a part of -) is substituted with a divalent substituent that imparts hydrophilicity. Examples of the divalent substituent include -O-, -C(=O)-O-, -C(=O)-, -OC(=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 Examples of the divalent substituent include -O-, -C(=O)-O-, -C(=O)-, -OC(=O)-O-, and -C(=O)-NH-, and more preferably -O-, -C(=O)-O-, and -C(=O)-NH-, and further preferably -O-. The linear or branched aliphatic hydrocarbon group is preferably a linear aliphatic hydrocarbon group.

[0028] R 1 and R 2 is preferably a group represented by the following general formula (r).

[0029] [ka] [In the formula, Y 0 represents a hydrocarbon group which may have a substituent, R 0 represents a hydrophilic group. * represents Py 1 or Py 2 represents a bond that bonds to .

[0030] 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. The aliphatic hydrocarbon group and the aromatic hydrocarbon group can be any of the above R 1 and R 2 The aliphatic hydrocarbon group and the aromatic hydrocarbon group in the above formula (I) are respectively the same as those in the above formula (I).

[0031] Y 0 The hydrocarbon group in Y preferably has 1 to 20 carbon atoms, more preferably has 3 to 15 carbon atoms, and still more preferably has 6 to 10 carbon atoms. 0Y 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 still more preferably a group in which an aromatic ring and a linear aliphatic hydrocarbon group are bonded. 0 When Y is an aromatic hydrocarbon group, 0 The number of aromatic rings contained in Y is not particularly limited, but is preferably 1 to 3, more preferably 1 or 2, and even more preferably 1. 0 When Y is a group in which an aromatic ring and a linear aliphatic hydrocarbon group are bonded, 0 The linear aliphatic hydrocarbon group contained in preferably has 1 to 6 carbon atoms, more preferably has 1 to 3 carbon atoms, and further preferably has 1 or 2 carbon atoms.

[0032] In formula (r), R 0 R 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. 0 may be a group containing a cell-penetrating peptide (polyarginine, polylysine, Tat peptide, etc.). When a fluorescent probe (P) is applied to a cell, R 0 is preferably a group having high biocompatibility, more preferably a group having cell membrane permeability. A preferred group having cell membrane permeability is a polyethylene glycol group.

[0033] In the formula (p), L represents a divalent organic group. The divalent organic group preferably has 5 to 50 carbon atoms, more preferably 6 to 40 carbon atoms, and even more preferably 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.

[0034] 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).

[0035] A straight-chain or branched aliphatic hydrocarbon group is a group consisting of a methylene group (-CH 2 A part of the -) may be substituted with a divalent substituent. Examples of the divalent substituent include the same as those mentioned above.

[0036] 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.

[0037] 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 aromatic ring preferably has 5 to 30 carbon atoms, more preferably 5 to 20 carbon atoms, further preferably 6 to 15 carbon atoms, and particularly preferably 6 to 12 carbon atoms. 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; and a group in which an aromatic hydrocarbon ring or an aromatic heterocycle is interposed in the middle of a linear or branched aliphatic hydrocarbon group. Examples of the linear or branched aliphatic hydrocarbon group include the same as those mentioned above.

[0038] 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 as those mentioned above.

[0039] L 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. L preferably contains a linear or branched aliphatic hydrocarbon group which may have a substituent. The linear or branched aliphatic hydrocarbon group is a group in which a methylene group (-CH 2 It is preferable that a part of the -) is substituted with a divalent substituent that imparts hydrophilicity. Examples of the divalent substituent include the same as those described above. As the divalent substituent, -O-, -C(=O)-O-, -C(=O)-, -OC(=O)-O-, and -C(=O)-NH- are preferable, -O-, -C(=O)-O-, and -C(=O)-NH- are more preferable, and -O- is even more preferable.

[0040] The compound (P) is preferably a compound represented by the following general formula (p-1).

[0041] [ka] [In the formula, L 1 represents a divalent organic group, R 11 and R 12 each independently represents a monovalent organic group.

[0042] In the formula, R 11 and R 12 Each of R independently represents a monovalent organic group. 1 and R 2 The monovalent organic groups are the same as those in the above.

[0043] In the formula, L 1 represents a divalent organic group. The divalent organic group is 1 The divalent organic groups are the same as those in the above.

[0044] Specific examples of the compound (P) are shown below, but are not limited thereto.

[0045] [ka] [In the formula, PEG represents a polyethylene glycol group.]

[0046] 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 4,000, more preferably 400 to 2,000, further preferably 600 to 1,500, and particularly preferably 800 to 1,000.

[0047] <Method of 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 scheme, Py 1 , R 1 , n 1 , Py 2 , R 2 , and n 2is the same as in the above formula (p).

[0048] First, compound (a1) is reacted with compound (c1) to obtain compound (b1) (Reaction 1). The reaction is carried out, for example, in a mixed solvent of toluene and ethanol using K 2 CO 3 Tetrakis(triphenylphosphine)palladium (Pd(PPh 3 ) 4 The reaction can be carried out using a catalyst such as .

[0049] [ka]

[0050] Similarly, compound (a2) is reacted with compound (c2) to obtain compound (b2) (Reaction 2). The reaction is carried out, for example, in an organic solvent such as a toluene / ethanol mixed solvent under basic conditions using potassium carbonate or the like with Pd(PPh 3 ) 4 The reaction can be carried out using a palladium catalyst such as palladium.

[0051] [ka]

[0052] 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 [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf) 2 Cl 2 The reaction can be carried out using a palladium catalyst such as .

[0053] [ka]

[0054] 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 using potassium carbonate or the like, using Pd(PPh 3 ) 4 The reaction can be carried out using a palladium catalyst such as palladium.

[0055] [ka]

[0056] In formula (p), (R 1 ) n1 -Py 1 - and (R 2 ) n2 -Py 2 When - and - are the same group, Reaction 1 and Reaction 2 are the same reaction.

[0057] R in formula (p) 1 In the case where a hydrophilic polymer such as a polyethylene glycol group is to be introduced into the compound (c1), the compound (c1') may be used in place of the compound (c1) in Reaction 1. Similarly, 2 When a hydrophilic polymer such as a polyethylene glycol group is to be introduced into the compound (c2), in Reaction 2, compound (c2′) may be used in place of compound (c2).

[0058] [ka] [In the formula, Y 01 and Y 02 each independently represents a hydrocarbon group which may have a substituent, and X represents a functional group.

[0059] Y in the formulas (c1') and (c2') 01 and Y 02 is Y in the formula (r) 0 is the same as: In the formulae (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 hydroxyl group, an amino group, a carboxyl group, an azide group, an alkynyl group, and a thiol group.

[0060] After Reaction 4, a hydrophilic polymer can be introduced into the compound (P) represented by formula (p) by utilizing the functional group represented by X in the formulas (c1') and (c2').

[0061] Compound (P) is a pyrene ring-containing compound having two atomic groups (R 1 ) n1 -Ry 1 - and (R 2 ) n2 -Ry 2 -) linked by a linker (L). In the liquid phase, compound (P) changes between a state in which the two pyrene rings in the molecule associate to form an excimer, and a state in which the two pyrene rings dissociate, depending on the physical properties of the liquid phase. "Excimer emission" is the fluorescence that occurs when the two pyrene rings of compound (P) form an excimer. "Monomer emission" is the fluorescence that occurs when the two pyrene rings of compound (P) do not form an excimer (when the two pyrene rings dissociate).

[0062] As shown in Figure 1, for compound (P) present in the liquid phase, the proportion of molecules 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, for compound (P) present in the liquid phase, the proportion of molecules 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. For compound (P) present in the liquid phase, the proportion of molecules 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 monomer emission increases. On the other hand, for compound (P) present in the liquid phase, the proportion of molecules that form excimers increases as the polarity of the liquid phase increases. Therefore, in a liquid phase with high polarity, the fluorescence intensity of excimer emission increases. Since compound (P) has the above-mentioned properties, a fluorescent probe made of compound (P) can be used to evaluate the viscosity and polarity of a liquid phase.

[0063] The compound (P) is R 1 and R 2 By introducing a hydrophilic group (preferably a group containing a hydrophilic polymer) into R, the cell membrane permeability can be improved. 1 and R 2 A fluorescent probe (P) consisting of a compound (P) having a hydrophilic group introduced therein can be used to evaluate the viscosity and polarity of the liquid phase present within 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). In cells, LLPS is involved in the formation of membrane-free organelles and provides a scaffold for various biological reactions. The dynamics and properties of intracellular LLPS can be easily evaluated by incorporating a fluorescent probe (P) into cells and measuring the excimer and monomer luminescence of the fluorescent probe (P).

[0064] [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.

[0065] (Step (i)) In step (i), fluorescence (m) due to monomer emission of the fluorescent probe (P) is measured in a target sample.

[0066] The target sample is a sample to be evaluated for polarity and viscosity. The target sample is not particularly limited as long as it is a sample containing a liquid phase. Examples of the target sample include cells and polymer compound solutions. The biological species from which the cells are derived is not particularly limited. 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 occurs.

[0067] A fluorescent probe (P) is added to a target sample, and 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 cell by culturing in a medium containing the fluorescent probe (P). The culturing time is not particularly limited, but examples thereof include 15 minutes or more, 30 minutes or more, 45 minutes or more, or 60 minutes or more. The upper limit of the culturing time is not particularly limited, but examples thereof include 180 minutes or less, 150 minutes or less, 120 minutes or less, or 100 minutes or less.

[0068] 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 the compound (P). The wavelength used for measuring the fluorescence (m) is, for example, 380 to 420 nm, preferably 390 to 410 nm. A specific example of the wavelength used for measuring the fluorescence (m) is 400 nm.

[0069] (Step (ii)) In step (ii), fluorescence (e) due to excimer emission of the fluorescent probe (P) is measured in the target sample.

[0070] The target sample is the same as the sample in step (i). After the fluorescent probe (P) is made to exist 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).

[0071] 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 the compound (P). The wavelength used for measuring the fluorescence (e) is, for example, 450 to 490 nm, preferably 460 to 480 nm. A specific example of the wavelength used for measuring the fluorescence (e) is 470 nm.

[0072] For the fluorescent probe (P), the fluorescence intensity of fluorescence (m) increases as the viscosity of the liquid phase increases, and the fluorescence intensity of fluorescence (e) increases as the viscosity of the liquid phase decreases. For the fluorescent probe (P), the fluorescence intensity of fluorescence (e) increases as the polarity of the liquid phase increases, and the fluorescence intensity of fluorescence (m) increases as 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).

[0073] (Optional process) The method of this embodiment 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).

[0074] By calculating the fluorescence intensity ratio between fluorescence (m) and fluorescence (e), the polarity and viscosity of the liquid phase can be grasped more accurately. The fluorescence intensity ratio may be the ratio of fluorescence (e) to fluorescence (m) (fluorescence (e) / fluorescence (m)), or 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, if the value of "fluorescence (m) / fluorescence (e)" is large, the liquid phase can be evaluated as having high viscosity and low polarity. If the value of "fluorescence (m) / fluorescence (e)" is small, the liquid phase can be evaluated as having low viscosity and high polarity.

[0075] By measuring the fluorescence (m) due to monomer emission and the fluorescence (e) due to excimer emission over time in the presence of a fluorescent probe (P) in the liquid phase of a target sample, the changes in the polarity and viscosity properties of the liquid phase can be tracked over time. In addition, by incorporating the fluorescent probe (P) into cells and measuring the fluorescence (m) and fluorescence (e) over time, the dynamics of LLPS in the cells can be tracked over time.

[0076] [Compound] A third aspect of the present invention is a compound represented by the following formula (Pyr-A) (compound (Pyr-A)). [ka] [In the formula, PEG represents a polyethylene glycol group.]

[0077] 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). EXAMPLES

[0078] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0079] [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 through the mixture for 40 minutes. 3 ) 4 (208 mg, 0.18 mmol) was added, and nitrogen gas was bubbled for 10 minutes to deoxygenate the mixture. 2 The mixture was heated to 100° C. under reduced pressure. During heating, deoxygenated ethanol (10 mL) was added to the mixture. After heating for 20 h, the resulting mixture was cooled to room temperature, poured dropwise into water, and dissolved in CH 2 Cl 2 The organic layer was washed with water and extracted with anhydrous Na 2 SO 4 The mixture was dried over 100 ml of water and then evaporated to dryness. The residue was purified by silica gel column chromatography to give compound (1) (1.05 g, 2.7 mmol, 54%) as a pale yellow solid.

[0080] 1 The structure of compound (1) was identified by 1 H NMR. 1 H NMR (400 MHz, CDCl 3 ,TMS standard): δ(ppm)1.77(t,J=5.9Hz,1H,OH),4.86(t,J=5.9Hz,2H,CH 2),7.58(d,J=8.3Hz,2H,ArH),7.63(d,J=8.3Hz,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).

[0081] [ka]

[0082] (Synthesis of compound (2)) N 2 Under the conditions below, 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 hour, and then 4-bromobenzyl bromide (15.36 g, 61.4 mmol) in THF (15 mL) was slowly added at 0° C. Then, it was warmed to room temperature. Anhydrous DMF (50 mL) was added to the mixture, and it was 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 and anhydrous Na 2 SO 4 The mixture was dried over ice and then evaporated to dryness. The residue was purified by silica gel column chromatography (CH 2 Cl 2 The mixture was purified using a solvent mixture (hexane = 5 / 95) to obtain compound (2) as a white solid (6.81 g, 12.9 mmol, 63%).

[0083] 1 The structure of compound (2) was identified by 1 H NMR. 1 HNMR (400MHz, CDCl 3,TMS standard):δ(ppm)0.88(t,J=7.2Hz,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).

[0084] [ka]

[0085] (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 through it for 40 minutes. To the mixture was added [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane adduct (50 mg), and the mixture was deoxygenated by bubbling nitrogen gas through it for 20 minutes. 2 The mixture was heated to 80° C. under reduced pressure. Additional Pd catalyst (50 mg) and bis(pinacolato)diboron (67 mg) were added to the mixture. After heating for 30 min, the resulting mixture was cooled to room temperature, dropped into water, and extracted with a mixture of hexane and ethyl acetate (hexane:ethyl acetate=4:1). The organic layer was washed with brine and diluted with anhydrous Na 2 SO 4 The mixture was dried over ice and then evaporated to dryness. The residue was purified by silica gel column chromatography (ethyl acetate / hexane=5 / 95) to give compound (3) as a clear oil (0.56 g, 0.9 mmol, 45%).

[0086] 1 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,CH3 ), 1.08-1.30(m,12H,CH 2 ), 1.34(s,24H,CH 3 ), 3.26(s,4H,CH 2 ),4.48(s,4H,CH2),7.31(d,J=8.1Hz,4H,ArH),7.77(d,J=8.1Hz,4H,ArH).

[0087] [ka]

[0088] (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 nitrogen gas through the mixture for 30 minutes. 3 ) 4 (45 mg, 0.04 mmol) was added, and nitrogen gas was bubbled for 20 minutes to deoxygenate the mixture. 2 The mixture was refluxed under reduced pressure for 15 h. The mixture was cooled to room temperature and filtered. The residue was dissolved in water, methanol and CH 2 Cl 2 The mixture was washed with water and dried in vacuum to give compound (4) as a pale yellow solid (531 mg, 0.54 mmol, 84%).

[0089] 1 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).

[0090] [ka]

[0091] (Synthesis of compound ((Pyr-A)) NaH (60% dispersion in oil, 20 mg, 0.29 mmol) was added to a mixture of compound (4) (36 mg, 0.06 mmol), dehydrated THF (0.5 mL), and dehydrated DMF (0.6 mL) under N 2 After stirring for 2 hours at 0° C., the mixture was slowly added with poly(ethylene glycol) methyl ether tosylate (Mn=900 gmol -1 , 156 mg, 0.24 mmol) was added, warmed to room temperature and stirred for 35 h. The resulting mixture was diluted with saturated NH 4 Cl aq., and CH 2 Cl 2 The organic layer was washed with water and extracted with anhydrous Na 2 SO 4 The mixture was dried over ice and then evaporated to dryness. The residue was purified by silica gel column chromatography (methanol / CH 2 Cl 2 =2 / 98~5 / 95) and purified by size exclusion chromatography to obtain compound (Pyr-A) as a pale yellow solid (48 mg, 0.019 mmol, 32%).

[0092] 1 The structure of 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).

[0093] [ka]

[0094] [Fluorescence properties of compound (Pyr-A)] The ultraviolet-visible absorption spectrum of compound (Pyr-A) in various solvents was measured using a spectrophotometer (V-750, JASCO Corporation). The fluorescence spectrum of 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 compound (Pyr-A) in the solvent was 1.0 μM. The fluorescence spectrum of compound (Pyr-A) was measured at an excitation wavelength of 358 nm.

[0095] 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).

[0096] Figure 2 shows the fluorescence spectrum 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 thought 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.

[0097] Figure 3 shows the ratio of excimer emission to the total fluorescence intensity of monomer emission (detection wavelength 400 nm) and excimer emission (detection wavelength 470 nm) [I 470 (I 400 / I 470 )] is the solvent polarity parameter E T The results are plotted as a function of (30). [I 470 (I 400 / I 470 The )] value increased with increasing solvent polarity. This result also confirmed that the higher the solvent polarity, the stronger the excimer emission. Excimer emission was more prominent in low-viscosity aprotic polar solvents than in high-viscosity protic polar solvents.

[0098] FIG. 4 shows a mixed solvent of methanol and polyethylene glycol 4000 (PEG4000) (PEG4000 concentration: 0 gL -1 , 25gL -1 , 50gL -1 , 100gL -1 Fluorescence spectra of compound (Pyr-A) in PEG4000. As the concentration of PEG4000 increased, monomer emission was enhanced and excimer emission was suppressed.

[0099] From the above results, it was confirmed that for compound (Pyr-A), excimer emission is dominant in an environment with high polarity and low viscosity, and monomer emission is dominant in an environment with low polarity and high viscosity.

[0100] [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 generated a recombinant fusion protein (BRD4-IDR-mEGFP) containing the IDR of BRD4 (BRD4-IDR) and mEGFP (monomeric enhanced green fluorescent protein), and performed a droplet assay of a compound (Pyr-A).

[0101] 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 to a final concentration of 10 μM in droplet buffer (50 mM Tris-Hcl pH 7.5, 10% glycerol, 1 mM DTT, 10% PEG). The droplet buffer used for dilution was used 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 slip. Droplets were imaged using a Zeiss LSM5 EXCITER microscope equipped with a mercury lamp and a Plan-Apochromat 100× / 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). Image acquisition was performed using Axio Vision software (version 4.8).

[0102] The results of the droplet assay are shown in Figure 5. It was confirmed that the compound (Pyr-A) was incorporated into the droplets of BRD4-IDR-mEGFP.

[0103] Figure 6 shows the relationship between the fluorescence intensity ratio (fluorescence (e)) of excimer emission (fluorescence (e)) and monomer emission (fluorescence (m)) (fluorescence (e) / 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 with maturation of protein droplets.

[0104] [Cell assay] Using a compound (Pyr-A), we observed centrosomes in cells. Centrioles are composed of a pair of centrioles and pericentriolar material (PCM). Centrioles contain centrin-1 and pericentrin. During mitosis, after the nuclear membrane breaks down, specific components of the nuclear pore complex, such as NUP62, localize to the centrosome and are involved in the maturation of the PCM. Centrioles during mitosis are significantly larger than those during interphase. During mitosis, intracellular viscosity is thought to increase and polarity is decreased compared to interphase (see Figure 7B).

[0105] (cell culture) HeLa cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum and penicillin / streptomycin under a humidified atmosphere at 20 °C and 5% CO. 2 , and cultured at 37°C.

[0106] (cDNA Vector and Transformation) The centrin1-EGFP vector was generated by cloning the HeLa cell cDNA into the C1-EGFP vector using the KpnI / BamH1 restriction sites. DNA was introduced into HeLa cells using Lipofectamine 2000 (Life Technologies). After transformation, HeLa cells stably expressing Centrin1-EGFP were selected by G418 treatment.

[0107] (Live cell fluorescence microscopy imaging) Cells were seeded on glass-bottom dishes (Matsunami Glass Industry) and cultured for 24 h to allow attachment. Cells were then synchronized by thymidine block to increase cell density in the mitotic phase and cultured in medium containing 10 μM of compound (Pyr-A). After 1 h 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.

[0108] Fluorescence microscopy images of interphase and mitotic cells are shown in Figure 7A. Both monomer and excimer luminescence were observed in both interphase and mitotic cells. This result confirmed that the compound (Pyr-A) permeated the cells.

[0109] Figure 8 shows the results of analyzing the excimer emission (fluorescence (e)) and monomer emission (fluorescence (m)) in the centrosomes and the fluorescence intensity ratio (fluorescence (e) / fluorescence (m)). In mitotic cells, the "fluorescence (e) / fluorescence (m)" value in the region centered on the centrin1-GFP signal was significantly lower than in interphase cells. These results indicate that during mitosis, centrosomes mature through expansion of the PCM, changing the pericentriolar microenvironment to a more viscous and less polarized state. [Industrial Applicability]

[0110] According to the present invention, there are provided a fluorescent probe capable of evaluating the dynamics or physical properties of LLPS in a cell, 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

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-1): 【Chemistry 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)] 【Chemistry 2】 [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).]

2. L 1 contains 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-; Y 0 includes a linear or branched aliphatic hydrocarbon group in which a part of 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. 3. The fluorescent probe according to claim 2, wherein the compound (P) is a compound represented by the following formula (Pyr-A): 【Chemistry 3】 [In the formula, PEG represents a polyethylene glycol group.]

4. The fluorescent probe according to any one of claims 1 to 3, wherein the liquid phase is a liquid phase present within a cell.

5. 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 assessing the polarity and viscosity of a liquid phase, comprising:

6. 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 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 according to claim 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): 【Chemistry 4】 [In the formula, PEG represents a polyethylene glycol group.]

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