JP2015218304A - Ratio-type fluorescent probe for intracellular temperature measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorophore-incorporated polyacrylamide fluorescent temperature sensor that is capable of short time quantitative measurement and high time resolution in fluorescence life measurement.SOLUTION: Provided is a temperature-sensitive probe including a copolymer comprising a heat-sensitive unit, cationic unit, and fluorescent unit. The heat-sensitive unit has a repeating structure derived from a monomer represented by formula (a). In the formula (a), R1 is H and C1-3 alkyl; R4 and R5 are independently H and C1-20 alkyl which may have a substituent, or R4 and R5 form a 4 to 8-membered nitrogen-containing heterocyclic ring with the bonded nitrogen atom, and the heterocyclic ring may be substituted by one or more substituent selected from C1-6 alkyl, C1-6 alkoxy, nitro, halogen atom, C1-10 alkylcarbonylamino, and arylcarbonylamino.

Description

  The present invention relates to a novel fluorescent copolymer and a simple method for measuring intracellular temperature using the copolymer.

  Many chemical reactions that occur as cells take up nutrients and extract energy through metabolism are highly temperature dependent. In other words, it can be said that many functions and reactions of cells are controlled by temperature inside and outside the cells. For example, research utilizing the physical property of temperature is often seen in medical research, and abnormal heat generation in cancer cells has been reported (Non-patent Document 1: Scand. J. Haematol. 1986, 36th). Volume, pages 353-357). Heat generation can be used to distinguish cancer cells with high metabolic activity from normal cells that are not. Utilizing this, development of therapeutic methods and drug discovery targeting heat generation is also being studied.

  In addition to medical research, cell metabolic activity control technology by temperature control is important in the food industry using microorganisms such as fermentation. For example, in alcoholic brewing, it has been studied that there is a correlation between the amount of heat generated from yeast and the amount of ethanol produced (Non-patent Document 2: Biotech. Bioeng. 1989, 34, 86-101). .

  On the other hand, in addition to temperature being an important biological indicator, attempts to measure the temperature inside a cell, even though many of the important reactions in the cell take place inside the cell. Are very few. One factor was the lack of established methods for measuring microregional temperatures at the cellular level. However, recent studies have begun to consider the use of molecules whose physical properties change with changes in temperature as temperature sensors (Non-patent Document 3: Photochem. Photobiol. 1995, Vol. 62, pp. 416-425). Non-Patent Document 4: J. Phys.D: Appl.Phys.2004, 37, 2938-2943; Non-patent Document 5: Anal.Chem. 2006, 78, 5094-5101 Non-Patent Document 6: Appl.Phys.Lett.2005, 87, 20112; Non-patent Document 7: Biophys.J.1998, 74, 82-89), especially the smallest functional unit A highly functional molecular temperature sensor with a single molecule has been developed, and its application to cells has begun to be studied.

  As an example of a molecular temperature sensor, a fluorescent temperature sensor based on the principle of incorporating 2,1,3-benzooxadiazolyl group, which is an environmentally responsive fluorophore, into polyacrylamide, which is a thermosensitive polymer, has already been reported. (Non-Patent Document 8: Anal. Chem. 2003, Vol. 75, pages 5926-5935). This fluorescent temperature sensor has the property that the fluorescence intensity increases with increasing temperature in an aqueous solution due to the heat-induced phase transition of polyacrylamide, which is the main chain. Therefore, by measuring the fluorescence intensity, Changes in temperature can be observed.

  In addition, the use of a polyacrylamide microgel incorporating a fluorophore obtained by adding a crosslinking agent during the production of a polymer as a fluorescent temperature sensor has also been reported (Non-Patent Document 9: J. Mater). Chem. 2005, 15, 2796-2800; Non-Patent Document 10: J. Am. Chem. Soc. 2009, 131, 2766-2767). Furthermore, it has been reported that by incorporating an ionic functional group into the polymer, aggregation can be prevented and the measurement temperature range can be widened (Patent Document 1: International Publication No. 2008/029770). .

  In addition, it has already been reported that the temperature range to which the sensor responds in an aqueous solution can be freely changed by changing the structure of the side chain of the main chain acrylamide used as the thermosensitive response site (Non-Patent Document 11: Anal. Chem. 2004, Vol. 76, pp. 1793 to 1798), it was also possible to use a plurality of acrylamide molecules in the thermosensitive response site depending on the temperature range of the measurement object.

  In addition, as a technique for more easily measuring the intracellular temperature, a cationic fluorescent temperature sensor (Patent Document 2: International Publication No. 2013/094748) that can introduce the sensor into the cell simply by contacting with the cell is also available. ,It has been reported.

Scand. J. Haematol. 1986, 36, 353-357 Biotech. Bioeng. 1989, 34, 86-101 Photochem. Photobiol. 1995, 62, 416-425. J. et al. Phys. D: Appl. Phys. 2004, 37, 2938-2943 Anal. Chem. 2006, 78, 5094-5101 Appl. Phys. Lett. 2005, 87, 20112 Biophys. J. et al. 1998, 74, 82-89 Anal. Chem. 2003, 75, 5926-5935 J. et al. Mater. Chem. 2005, Vol. 15, pp. 2796-2800 J. et al. Am. Chem. Soc. 2009, 131, 2766-2767 Anal. Chem. 2004, 76, 1793-1798.

International Publication No. 2008/029770 International Publication No. 2013/094748

  With respect to fluorescent temperature sensors that use linear or cross-linked polyacrylamides with built-in fluorophores, polymers that have already been reported can be introduced simply by mixing with cells. On the other hand, there was a need to perform fluorescence lifetime measurement that is not affected by the polymer concentration in order to perform highly accurate measurement. However, the fluorescence lifetime measurement is not general-purpose because it requires a special device and is not widely used particularly in biological research facilities that handle cells. In addition, since the fluorescence lifetime measurement takes time for quantification, the time resolution is low and it is not suitable for capturing a fast phenomenon in a cell.

  Furthermore, measurement by fluorescence intensity ratio is known as a measurement method that does not depend on the concentration of the sensor other than the fluorescence lifetime, but generally two fluorescent units with different structures must be used, In some cases, such as when there is a large overlap between the two fluorescence wavelengths to be adopted, the sensitivity is low, making it difficult to measure with high sensitivity.

  Further, if pursuing a more general measurement, it is desirable that two fluorescent dyes are simultaneously excited at one excitation wavelength. For example, two wavelengths excitation one wavelength such as Fura-2 used in Ca ion measurement There is also a ratio type measurement method such as a fluorescence detection type, but instruments such as a microscope, a photometer, or a plate reader that simultaneously excite multiple wavelengths are expensive and their use is limited.

  As a result of diligent research to solve the above-mentioned problems, the present inventors can excite at the same wavelength as the 2,1,3-benzooxadiazolyl group conventionally used in acrylamide type fluorescent temperature sensors. In addition, by adding a new fluorescent unit having a fluorescence wavelength different from that of the 2,1,3-benzooxadiazolyl group and incorporating it into the polymer chain, the fluorescence intensity ratio derived from the two fluorescent dyes is taken, It has been found that the intracellular temperature can be measured accurately and easily. Furthermore, by using this copolymer, it is possible to visualize and quantify the temperature distribution in the cell, and it is clear that these measurements are simple methods that can be measured well with a general fluorescence microscope. did. The present invention is based on these findings.

That is, according to the first aspect of the present invention, a temperature-sensitive probe comprising a copolymer comprising a thermosensitive unit, a cationic unit, and a fluorescent unit,
Comprising a first fluorescent unit and a second fluorescent unit having different maximum fluorescence wavelengths from each other;
The thermosensitive unit has the following formula (a):
Wherein R ¹ is selected from a hydrogen atom and C _1-3 alkyl;
R ⁴ and R ⁵ are independently selected from a hydrogen atom and C _1-20 alkyl, wherein the alkyl is substituted with one or more substituents selected from hydroxy, C _1-6 alkoxy, and aryl Or R ⁴ and R ⁵ together with the nitrogen atom to which they are attached form a 4- to 8-membered nitrogen-containing heterocycle, where the heterocycle is C _1-6 alkyl, C _1-6 alkoxy, nitro, halogen atom, optionally substituted by one or more substituents selected from C _1-10 alkylcarbonylamino and arylcarbonylamino]
Is one or two or more repeating structures derived from one or more monomers represented by:
The cationic unit is represented by the following formula (b):
[Wherein R ² is selected from a hydrogen atom and C _1-3 alkyl;
W is an aromatic ring or —X ¹ —C (═O) — (where X ¹ is directly attached to Q ¹ );
X ¹ is O, S, or N—R ¹¹ ;
R ¹¹ is a hydrogen atom, C _1-6 alkyl, or —Q ¹ -Y, wherein the alkyl is hydroxy, halogen atom, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkyl Optionally substituted by one or more substituents selected from sulfinyl, and C _1-6 alkylsulfonyl;
Q ¹ is independently selected from C _1-20 alkylene, C _3-20 alkenylene, or C _3-20 alkynylene, wherein the alkylene is O, S, —O—P () at one or more points. ═O) (— OH) —O— or phenylene may be independently inserted;
Y is independently an ionic functional group that may have one or more positive charges, and is —N ⁺ R ²¹ R ²² R ²³ X _e ⁻ , —C (═NR ⁴¹ ) —NR ⁴² R ⁴³ , and The following formula:
Selected from the group represented by
X _e ⁻ and X _f ⁻ are counter anions;
R ²¹ , R ²² , and R ²³ are independently selected from C _1-10 alkyl and C _7-14 aralkyl, or R ²¹ and R ²² together with the nitrogen atom to which A 7-membered nitrogen-containing heterocycle may be formed;
R ²⁴ is C _1-10 alkyl, or C _7-14 aralkyl;
R ⁴¹ , R ⁴² and R ⁴³ are independently selected from a hydrogen atom and C _1-10 alkyl, or R ⁴¹ and R ⁴² together with the nitrogen and carbon atoms to which each is attached, nitrogen atom may form a 5- to 7-membered heterocyclic ring containing or R ⁴² and R ^43, together with the nitrogen atom to which they are attached may form a 5- to 7-membered nitrogen-containing heterocycle]
A temperature-sensitive probe is provided that is one or more repeating structures derived from one or more monomers represented by:

  Furthermore, according to the second aspect of the present invention, there is provided a method for measuring the temperature in a cell, the method comprising (a) mixing a temperature sensitive probe according to the first aspect of the present invention with a cell in a solvent. A step of introducing the temperature-sensitive probe into the cell, (b) a step of measuring the intensity of fluorescence derived from each of the first fluorescent unit and the second fluorescent unit under irradiation with excitation light, And (c) calculating a ratio of the two measured fluorescence intensities.

  According to the temperature sensitive probe of the present invention, it is possible to measure the temperature in a cell easily and in a short time with high accuracy. Furthermore, according to the temperature sensitive probe of the present invention, the temperature distribution in the cell can be visualized and quantified. In addition, no special equipment is required for these temperature measurements, and even a general fluorescent microscope can be sufficiently measured. The temperature-sensitive probe of the present invention can be suitably used as a fluorescent temperature sensor for measuring the temperature in the cell because it enters the cell without using a special method due to the presence of the cationic group in the copolymer. be able to. Therefore, the present invention can be applied to a wide variety of cells such as microorganisms such as yeast, animal cells, and plant cells. Moreover, according to the temperature sensitive probe of the present invention, it is possible to perform more accurate temperature measurement independent of the copolymer concentration.

Changes in the fluorescence spectrum of Compound 2 in a 150 mM potassium chloride aqueous solution (0.01 w / v%, excitation wavelength 458 nm) are shown at each temperature of 25 ° C. (dotted line), 40 ° C. (solid line), and 55 ° C. (thick line). It is an example. Thermal response test results of fluorescence intensity ratios (580 nm / 515 nm) of compounds 1 to 4 (black circle: compound 1, black rhombus: compound 2, white triangle: compound 3, white circle: compound 4) in a 150 mM potassium chloride aqueous solution ( 0.01 w / v%, excitation wavelength 458 nm). Thermal sensitivity test result of compound 1, 5, 6 (black circle: compound 1, white square: compound 5, black square: compound 6) in a fluorescence intensity ratio (580 nm / 515 nm) in a 150 mM potassium chloride aqueous solution (0.01 w / V%, excitation wavelength 458 nm). Example of thermal response test results (0.01 w / v%, excitation wavelength 458 nm) of fluorescence intensity ratio (580 nm / 515 nm) in 150 mM potassium chloride aqueous solution of compounds 1, 7, 8 (black circle: compound 1) is there. In an example of a thermal sensitivity test (black triangle: pH 6, white circle: pH 7, white square: pH 9) of compound 1 (both 0.01 w / v%, excitation wavelength 458 nm) in 150 mM potassium chloride aqueous solution at pH 6 and 9. is there. Results of thermal response test of Compound 1 (all 0.01 w / v%, excitation wavelength 458 nm, fluorescence intensity ratio (580 nm / 515 nm)) in 125, 150 and 175 mM potassium chloride aqueous solution (black triangle: in 175 mM KCl, White square: 150 mM KCl, black circle: 125 mM KCl). Compounds 1 to 9 were mixed with MOLT-4 (human acute lymphoblastic leukemia T cells) in a 5% glucose solution (25 ° C., 10 minutes) and observed with a microscope (excitation light: 473 nm, fluorescence: 500 nm to It is an example of the photograph which performed 600 nm. It is an example of the result of the thermal sensitivity test of the fluorescence intensity ratio (580 nm / 515 nm) of the compound 1 in MOLT-4 (human acute lymphoblastic leukemia T cell) cells. It is an example of the result of the temperature resolution computed from the thermosensitive response test of the fluorescence intensity ratio (580 nm / 515 nm) of the compound 1 in a MOLT-4 (human acute lymphoblastic leukemia T cell) cell. It is an example of the thermal resolution test result of the fluorescence intensity ratio (560 nm / 580 nm) of the pre-compound 9 in MOLT-4 (human acute lymphoblastic leukemia T cell) cells, and the result of the temperature resolution calculated from the graph. . Compound 1 was introduced into HEK293T (human-derived fetal kidney cells) cells, and observed with a confocal laser microscope (excitation wavelength: 473 nm). A fluorescence image (P1) with a fluorescence wavelength of 490-530 nm and a fluorescence image with a fluorescence wavelength of 570-610 nm ( It is an example of the fluorescence image (P2 / P1) obtained by taking the ratio of P2). FIG. 12 is an example of a cross-sectional view of a part of the cell in FIG. 11 (black line part in the left figure) as a horizontal axis: distance from the left end of the black line, and a vertical axis: black and white value (intensity) of the ratio image. Compound 1 was introduced into HEK293T (human-derived fetal kidney cells) cells and observed with a confocal laser microscope (excitation wavelength: 473 nm). A fluorescence image (P1) with a fluorescence wavelength of 510-520 nm and a fluorescence image with a fluorescence wavelength of 580-590 nm ( This is an example showing the difference in the average fluorescence intensity ratio between the cytoplasm and nucleus (average of 9 cells) of the fluorescence image (P2 / P1) obtained by taking the ratio of P2) according to temperature. C2C12 (derived from mouse myoblast, ECACC) induced to differentiate into myotube cells was mixed with compound 1 in 5% glucose solution containing 0.45 mM CaCl ₂ (final concentration 0.03%) to damage the cells. Compound 1 was introduced without giving Furthermore, it is an example which showed the change of the ratio image (570-670 nm / 485-545 nm) when observing with a confocal laser microscope (excitation wavelength 473 nm) and changing temperature.

Detailed description of the invention

  The copolymer used as a temperature sensitive probe according to the first aspect of the present invention includes a first fluorescent unit and a second fluorescent unit having different maximum fluorescence wavelengths. In the temperature measurement using the temperature-sensitive probe of the present invention, the ratio of the fluorescence intensity derived from the first fluorescence unit and the fluorescence intensity derived from the second fluorescence unit is calculated, and this is actually measured. By matching the temperature, it is possible to measure the temperature with high accuracy in a simple and short time. The copolymer used as the temperature sensitive probe of the present invention contains a cationic unit in addition to the heat sensitive unit and the fluorescent unit. This makes it possible to introduce the copolymer into cells by mixing the copolymer with cells in a solvent.

  It is desirable that the first fluorescent unit and the second fluorescent unit generate fluorescence having different maximum fluorescence wavelengths when irradiated with excitation light having the same wavelength. Also, the difference between the maximum fluorescence wavelength of the first fluorescence unit and the maximum fluorescence wavelength of the second fluorescence unit is sufficiently discriminated by the measuring instrument when simultaneously measuring the fluorescence intensity at the two wavelengths. Although it should just be separated to a certain extent and it does not restrict | limit in particular, Preferably it shall be 50 nm or more.

  According to a preferred embodiment of the present invention, either one of the first fluorescent unit and the second fluorescent unit has an increase in fluorescence intensity with an increase in temperature, and the other has an increase in temperature. The fluorescence intensity does not change or decreases according to the above, and preferably decreases.

According to a particularly preferred embodiment of the invention, the first fluorescent unit has the following formula (c):
[Wherein R ³ is selected from a hydrogen atom and C _1-3 alkyl;
X ² is O, S, or N—R ¹² ;
X ³ is a direct bond, O, S, SO, SO ₂ , N (—R ¹³ ), CON (—R ¹⁶ ), N (—R ¹⁶ ) CO, N (—R ¹⁷ ) CON (—R ¹⁸ ). , SO ₂ N (—R ¹⁹ ) or N (—R ¹⁹ ) SO ₂ ;
Q ² is selected from C _1-20 alkylene, C _3-20 alkenylene, or C _3-20 alkynylene, wherein the alkylene has O, S, or phenylene independently inserted at one or more points. Well;
Ar is selected from a 6 to 18-membered aromatic carbocyclic group or a 5 to 18-membered aromatic heterocyclic group, wherein the aromatic carbocyclic group and the aromatic heterocyclic group include at least one of the included rings. A condensed ring which is an aromatic ring may be included, and —CH ₂ — present as a ring atom in the aromatic carbocyclic group and aromatic heterocyclic group may be substituted with —C (O) —. In addition, the aromatic carbocyclic group and aromatic heterocyclic group include a halogen atom, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylsulfinyl, C _1-6 alkylsulfonyl. , nitro, _{cyano, C 1-6 alkylcarbonyl, C 1-6} alkoxycarbonyl, carboxy, formyl, ^-NR 6 ^{R 7,} and by one or more substituents selected from _-SO 2 ^NR 14 ^{R 15} May be conversion (the _{C 1-6} alkyl, _{where, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, C 1-6} alkylcarbonyl and C _The alkyl contained in _1-6 alkoxycarbonyl is 1 selected from a halogen atom, C _1-6 alkoxy, hydroxy, amino, C _1-6 alkylamino, di (C _1-6 alkyl) amino, aryl, and carboxy. Optionally substituted by the above substituents);
R ⁶ and R ⁷ independently represent a hydrogen atom, C _1-10 alkyl, aryl, C _1-10 alkylcarbonyl, arylcarbonyl, C _1-10 alkylsulfonyl, arylsulfonyl, carbamoyl, N— (C _1-10 Alkyl) carbamoyl, and N, N-di (C _1-10 alkyl) carbamoyl, wherein said C _1-10 alkyl, C _1-10 alkylcarbonyl, C _1-10 alkylsulfonyl, N- (C _{1 -10} alkyl) carbamoyl and alkyl contained in N, N-di (C _1-10 alkyl) carbamoyl are a halogen atom, C _1-6 alkoxy, hydroxy, amino, C _1-6 alkylamino, di (C _{1 -6} alkyl) location amino, aryl, and optionally one or more substituents selected from carboxy May be aryl contained further in the aryl, arylcarbonyl and arylsulfonyl, a substituted, halogen atom, C _1-6 alkyl, C _1-6 alkoxy, and by one or more substituents selected from carboxy Or R ⁶ and R ⁷ together with the nitrogen atom to which they are attached form a 4- to 8-membered nitrogen-containing heterocycle, where the heterocycle is C _1-6 alkyl, Optionally substituted by one or more substituents selected from C _1-6 alkoxy, nitro, halogen atoms, C _1-10 alkylcarbonylamino and arylcarbonylamino;
R ¹² is a hydrogen atom, C _1-6 alkyl, or —Q ² —X ³ —Ar, wherein the alkyl is hydroxy, halogen atom, C _1-6 alkoxy, C _1-6 alkylthio, C ₁ Optionally substituted by one or more substituents selected from _-6 alkylsulfinyl, and C _1-6 alkylsulfonyl;
R ¹³ is hydrogen atom or _{C 1-6} alkyl, wherein said alkyl is hydroxy, halogen _{atom, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6} alkylsulfinyl, and _{C 1-} Optionally substituted by one or more substituents selected from ₆ alkylsulfonyl;
R ¹⁴ and R ¹⁵ are independently selected from a hydrogen atom and C _1-6 alkyl; or R ¹⁴ and R ¹⁵ together with the nitrogen atom to which they are attached, a 4- to 8-membered nitrogen-containing heterocycle Forming;
R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ are independently selected from a hydrogen atom and C _1-6 alkyl, wherein the alkyl is hydroxy, halogen atom, C _1-6 alkoxy, C _1-6 Optionally substituted by one or more substituents selected from alkylthio, C _1-6 alkylsulfinyl, and C _1-6 alkylsulfonyl]
Is a repeating structure derived from a monomer represented by
The second fluorescent unit has the following formula (d):
Wherein R ⁵⁵ is selected from a hydrogen atom and C _1-3 alkyl;
R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ are independently selected from a hydrogen atom and C _1-6 alkyl;
X ⁴ is a direct bond, phenylene, —Q ⁴ —O—C (═O) — (where Q ⁴ is directly bonded to the boron dipyrromethene skeleton), —Q ⁴ —N (—R ⁶¹ ) -C (= O)-(wherein Q ⁴ is directly bonded to the boron dipyrromethene skeleton);
R ⁶¹ is selected from a hydrogen atom and C _1-6 alkyl;
Q ⁴ is selected from C _1-20 alkylene, phenylene, and naphthylene, wherein the phenylene and naphthylene are substituted by one or more substituents selected from a halogen atom, C _1-6 alkoxy, hydroxy, amino, and carboxy It may be done]
It is set as the repeating structure derived from the monomer represented by these.

The thermosensitive unit contained in the copolymer used in the present invention is a repeating structure whose shape changes depending on the temperature, and is derived from one or more monomers represented by the following formula (a). Is one or more repeating structures:
Wherein R ¹ is selected from a hydrogen atom and C _1-3 alkyl;
R ⁴ and R ⁵ are independently selected from a hydrogen atom and C _1-20 alkyl, wherein the alkyl is substituted with one or more substituents selected from hydroxy, C _1-6 alkoxy, and aryl Or R ⁴ and R ⁵ together with the nitrogen atom to which they are attached form a 4- to 8-membered nitrogen-containing heterocycle, where the heterocycle is C _1-6 alkyl, C _1-6 alkoxy, nitro, halogen atom, optionally substituted by one or more substituents selected from C _1-10 alkylcarbonylamino and arylcarbonylamino].

The cationic unit incorporated in the copolymer used in the present invention is a repeating structure derived from a monomer containing an ionic functional group having one or more positive charges, and is represented by the following formula (b) Or one or more repeating structures derived from two or more monomers:

[Wherein R ² is selected from a hydrogen atom and C _1-3 alkyl;
W is an aromatic ring or —X ¹ —C (═O) — (where X ¹ is directly attached to Q ¹ );
X ¹ is O, S, or N—R ¹¹ ;
R ¹¹ is a hydrogen atom, C _1-6 alkyl, or —Q ¹ -Y, wherein the alkyl is hydroxy, halogen atom, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkyl Optionally substituted by one or more substituents selected from sulfinyl, and C _1-6 alkylsulfonyl;
Q ¹ is independently selected from C _1-20 alkylene, C _3-20 alkenylene, or C _3-20 alkynylene, wherein the alkylene is O, S, —O—P () at one or more points. ═O) (— OH) —O— or phenylene may be independently inserted;
Y is independently an ionic functional group that may have one or more positive charges, and is —N ⁺ R ²¹ R ²² R ²³ X _e ⁻ , —C (═NR ⁴¹ ) —NR ⁴² R ⁴³ , and The following formula:
Selected from the group represented by
X _e ⁻ and X _f ⁻ are counter anions;
R ²¹ , R ²² , and R ²³ are independently selected from C _1-10 alkyl and C _7-14 aralkyl, or R ²¹ and R ²² together with the nitrogen atom to which A 7-membered nitrogen-containing heterocycle may be formed;
R ²⁴ is C _1-10 alkyl, or C _7-14 aralkyl;
R ⁴¹ , R ⁴² and R ⁴³ are independently selected from a hydrogen atom and C _1-10 alkyl, or R ⁴¹ and R ⁴² together with the nitrogen and carbon atoms to which each is attached, nitrogen atom may form a 5- to 7-membered heterocyclic ring containing or R ⁴² and R ^43, together with the nitrogen atom to which they are attached may form a 5- to 7-membered nitrogen-containing heterocycle] .

  According to a preferred embodiment of the present invention, the copolymer used in the present invention comprises a monomer represented by the formula (a), a monomer represented by the formula (b), a monomer represented by the formula (c), And a copolymer containing a repeating structure derived from each of the monomers represented by the formula (d).

According to a further preferred embodiment of the present invention, the copolymer used in the present invention has the formula (I), the formula (II), the formula (III) and the formula (XIII):
[Wherein R ¹ , R ⁴ and R ⁵ , and R ² , Y, W and Q ¹ , and R ³ , X ² , X ³ , Q ² and Ar, and R ⁵⁵ , X ⁴ , R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ are as defined above, and a, b, c and d are numbers greater than 0 representing the ratio of each repeating unit]
It is set as the copolymer containing the repeating unit represented by these. In this copolymer, the sum of a and b is 100, b is preferably 2 to 10, c is preferably 0.3 to 2, and d is preferably 0.03 to 1. It is said. Further, this copolymer constitutes one aspect of the present invention as a substance itself.

  According to a preferred embodiment of the present invention, the copolymer includes two or more heat-sensitive units. There are various types of thermosensitive units, and the temperature range showing the highest temperature reactivity varies depending on the type. In this embodiment, it can adjust so that the temperature reactivity of a copolymer may become high in a desired temperature range by combining 2 or more types of thermosensitive units. According to a more preferred embodiment of the present invention, the copolymer includes two or more heat-sensitive units represented by the formula (a). In one embodiment, two types of heat sensitive units are used. For example, it is preferable to use a combination of Nn-propylacrylamide (NNPAM) and N-isopropylacrylamide (NIPAM) for measurement at around 35 ° C., which is a general culture temperature for animal cells. In addition, when it is necessary to measure a temperature range of 25 ° C. or lower for the purpose of monitoring fermentation of microorganisms such as yeast, it is preferable to use a combination of N-tert-butylacrylamide (NTBAM) and NNPAM. .

  A in formula (I) represents the total sum of the thermosensitive units, and when two or more thermosensitive units are used, it means the sum of the ratios of the number of repeating units of all thermosensitive units. .

According to a preferred embodiment of the present invention, in the above-mentioned copolymer, Ar is represented by the following formula:
An aromatic carbocyclic group or an aromatic heterocyclic group selected from the group represented by the formula: These groups are halogen atoms, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 on the ring. _{alkylthio, C 1-6 alkylsulfinyl, C 1-6} alkylsulfonyl, nitro, _{cyano, C 1-6 alkylcarbonyl, C 1-6} alkoxycarbonyl, carboxy, formyl, ^-NR 6 ^{R 7,} and _-SO 2 ^{NR 14} It may be substituted by one or more substituents selected from R ¹⁵ (wherein said C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylsulfinyl, C _{1- 6 alkylsulfonyl,} alkyl, halogen atom contained in _{C 1-6} alkylcarbonyl and _{C 1-6} alkoxycarbonyl, _{C 1 6} alkoxy, hydroxy, _{amino, C 1-6} alkylamino, di _{(C 1-6} alkyl) amino, aryl, and optionally substituted by one or more substituents selected from carboxy);
X ¹⁰ is selected from O, S or Se;
R ⁸ is selected from a hydrogen atom, C _1-10 alkyl, and aryl, wherein the alkyl is
Optionally substituted by one or more substituents selected from a halogen atom, C _1-6 alkoxy, hydroxy, amino, C _1-6 alkylamino, di (C _1-6 alkyl) amino, aryl, and carboxy Furthermore, the aryl may be substituted with one or more substituents selected from a halogen atom, C _1-6 alkyl, C _1-6 alkoxy, and carboxy.

According to a further preferred embodiment of the present invention, Ar is represented by the following formula:
An aromatic carbocyclic group or an aromatic heterocyclic group selected from the group represented by the formula: These groups are halogen atoms, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 on the ring. _{alkylthio, C 1-6 alkylsulfinyl, C 1-6} alkylsulfonyl, _{nitro, C 1-6} alkylcarbonylamino, arylcarbonylamino, cyano, _{formyl, C 1-6 alkylcarbonyl, C 1-6} alkoxycarbonyl, carboxy and It may be substituted with one or more substituents selected from —SO ₂ NR ¹⁴ R ¹⁵ .

In the present specification, “C _1-3 alkyl” means a linear, branched, or cyclic alkyl group having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, cyclo Contains propyl.

As used herein, “C _1-6 alkyl” means a linear, branched, cyclic or partially cyclic alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl. I-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl , 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, and 2-ethylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, and the like, for example, C _1-4 alkyl And C _1-3 alkyl are also included.

In the present specification, “C _1-10 alkyl” means a linear, branched, cyclic or partially cyclic alkyl group having 1 to 10 carbon atoms, for example, C _{1-6 as} defined above. Alkyl and C _1-3 alkyl and the like are included.

In the present specification, the “C _1-20 alkyl” means a linear, branched, cyclic or partially cyclic alkyl group having 1 to 20 carbon atoms. For example, the previously defined C _1-10 Alkyl, C _1-6 alkyl, C _1-3 alkyl and the like are included.

In the present specification, “C _1-6 alkoxy” means an alkyloxy group having an alkyl group having 1 to 6 carbon atoms which has already been defined as an alkyl moiety. For example, methoxy, ethoxy, n-propoxy, i-propoxy N-butoxy, s-butoxy, i-butoxy, t-butoxy, n-pentoxy, 3-methylbutoxy, 2-methylbutoxy, 1-methylbutoxy, 1-ethylpropoxy, n-hexyloxy, 4-methylpen Toxic, 3-methylpentoxy, 2-methylpentoxy, 1-methylpentoxy, 3-ethylbutoxy, cyclopentyloxy, cyclohexyloxy, cyclopropylmethyloxy, etc. are included, for example, C _1-4 alkoxy and C _{1 -3} alkoxy and the like are also included.

  In the present specification, “aryl” means a 6 to 10 membered aromatic carbocyclic group, and includes, for example, phenyl, 1-naphthyl, 2-naphthyl and the like.

In the present specification, “C _7-14 aralkyl” means an arylalkyl group having 7 to 14 carbon atoms including an aryl group, such as benzyl, 1-phenethyl, 2-phenethyl, 1-naphthylmethyl, 2- Naphthylmethyl and the like are included.

  In this specification, examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

In the present specification, “C _1-20 alkylene” means a linear, branched, cyclic or partially cyclic alkylene group having 1 to 20 carbon atoms, such as methylene, ethylene, propylene, butylene. In addition, C _1-10 alkylene and C _1-6 alkylene are included.

In the present specification, “C _3-20 alkenylene” means a linear, branched, cyclic or partially cyclic alkenylene group having 3 to 20 carbon atoms, such as propenylene, butenylene, and the like. _3-10 alkenylene, C _3-6 alkenylene and the like are included.

As used herein, “C _3-20 alkynylene” means a linear, branched, cyclic or partially cyclic alkynylene group having 3 to 20 carbon atoms, such as propynylene, butynylene, and the like. _3-10 alkynylene, C _3-6 alkynylene and the like are included.

As used herein, “C _1-6 alkylthio” means an alkylthio group having an alkyl group having 1 to 6 carbon atoms already defined as the alkyl moiety, and examples thereof include methylthio, ethylthio, n-propylthio, i-propylthio, n-butylthio, s-butylthio, i-butylthio, t-butylthio and the like are included.

In the present specification, “C _1-6 alkylsulfinyl” means an alkylsulfinyl group having an alkyl group having 1 to 6 carbon atoms already defined as an alkyl moiety, such as methylsulfinyl, ethylsulfinyl, n-propylsulfinyl. I-propylsulfinyl, n-butylsulfinyl, s-butylsulfinyl, i-butylsulfinyl, t-butylsulfinyl and the like.

As used herein, “C _1-6 alkylsulfonyl” means an alkylsulfonyl group having an alkyl group having 1 to 6 carbon atoms already defined as the alkyl moiety, and examples thereof include methylsulfonyl, ethylsulfonyl, n-propylsulfonyl. I-propylsulfonyl, n-butylsulfonyl, s-butylsulfonyl, i-butylsulfonyl, t-butylsulfonyl and the like.

  In the present specification, the “6- to 18-membered aromatic carbocyclic group” includes, for example, phenyl, naphthyl, anthracenyl, pyrenyl, indanyl, tetralinyl and the like.

  In the present specification, the “5- to 18-membered aromatic heterocyclic group” is an aromatic heterocyclic ring having one or more heteroatoms selected from oxygen, nitrogen and sulfur. For example, pyrrolyl, pyrazolyl, imidazolyl, pyridyl , Indolyl, quinolyl, quinoxalinyl, quinazolinyl, benzofuranyl, benzothienyl, benzopyranyl, benzochromenyl and the like.

As used herein, “C _2-6 alkenylsulfonyl” means an alkenylsulfonyl group having a C _2-6 alkenyl group already defined as an alkenyl moiety, and includes, for example, vinylsulfonyl, allylsulfonyl and the like.

As used herein, “C _2-6 alkenylcarbonyl” means an alkenylcarbonyl group having a C _2-6 alkenyl group already defined as an alkenyl moiety, and includes, for example, acryloyl, methacryloyl, and the like.

As used herein, “C _2-6 alkynylcarbonyl” means an alkynylcarbonyl group having a C _2-6 alkynyl group which has already been defined as an alkynyl moiety, and includes, for example, ethynylcarbonyl and the like.

As used herein, “C _1-6 alkylcarbonyl” refers to a group —CO (C _1-6 alkyl), where the C _1-6 alkyl is as previously defined.

In the present specification, “C _1-6 alkoxycarbonyl” represents a group —CO (C _1-6 alkoxy), wherein the C _1-6 alkoxy is as defined above.

As used herein, “C _1-6 alkylcarbonylamino” refers to the group —NHCO (C _1-6 alkyl), where C _1-6 alkyl is as previously defined.

As used herein, “C _1-6 arylcarbonylamino” refers to the group —NHCO (aryl), where aryl is as previously defined.

  Examples of the “5- to 7-membered nitrogen-containing heterocycle” in the present specification include saturated heterocycles such as pyrrole ring, pyrrolidine ring, piperidine ring, homopiperidine ring, piperazine ring, homopiperazine ring, morpholine ring, and thiomorpholine ring. included.

  Examples of the “4- to 8-membered nitrogen-containing heterocycle” in the present specification include a pyrrole ring, an azetidine ring, a pyrrolidine ring, a piperidine ring, a homopiperidine ring, a piperazine ring, a homopiperazine ring, a morpholine ring, and a thiomorpholine ring. And 5- to 7-membered nitrogen-containing heterocycles.

  In the present specification, the “5- to 7-membered heterocycle containing two nitrogen atoms” includes, for example, imidazolidine, tetrahydropyrimidine and the like.

In this specification, when alkylene is inserted at one or more sites, the alkylene chain contains an ether bond in the main chain, and the insertion becomes a stable structure. Those skilled in the art should readily understand that the process is performed so as not to form O— and —O—CH ₂ —O— structures. The above also applies to the insertion of S into alkylene.

  In this specification, the copolymer is an aggregate of polymer chains formed by mixing monomers corresponding to each unit and performing a polymerization reaction. The polymer refers to a polymer chain in which monomer units are connected and connected.

  In the present specification, the “counter anion” is not particularly limited as long as it is an anion usually used as a counter anion of an organic compound in the technical field of organic chemistry. For example, a halide anion (chloride ion, bromide ion, fluoride) Ions, iodide ions), organic acid conjugate bases (eg, acetate ions, trifluoroacetate ions), nitrate ions, sulfate ions, carbonate ions, and the like. Preferred counter anions in the present invention include, for example, chloride ions and nitrate ions.

  It should be noted that when the counter anion is divalent or more, it is easily understood by those skilled in the art to form ionic bonds with the corresponding number of ionic functional groups.

In the present invention, R ¹ , R ² , R ³ and R ⁵⁵ are preferably selected from a hydrogen atom and methyl.

^-NR 4 ^{R 5} in the formula (a) and Formula (I) is, is not particularly limited, for example, ^{R 4} is a hydrogen atom, ^{R 5} may be _{C 2-10} alkyl. In addition, when R ⁴ and R ⁵ together with the nitrogen atom to which they are bonded form a 4- to 8-membered nitrogen-containing heterocycle, for example, pyrrolidine ring, piperidine ring, homopiperidine ring, piperazine ring, homopiperazine A ring, a morpholine ring, a thiomorpholine ring, or the like may be formed.

W in the formula (b) and formula (II), the aromatic ring and ^-X 1 -C (= O) - may be any of (here, is ^{X 1} to bind directly to the ^{Q 1)} . The aromatic ring represented by W is preferably a C _6-18 aromatic ring, more preferably a benzene ring. W is preferably —X ¹ —C (═O) — (where X ¹ is directly bonded to Q ¹ ). X ¹ is preferably O, NH or N (C _1-6 alkyl).

-Q ¹ in the formula (b) and Formula (II) - is preferably a _{C 2-10} alkylene.

-X ² -Q ^2- in formula (c) and formula (III) is preferably X ² is O, NH or N (C _1-6 alkyl), and Q ² is C _2-10 alkylene. is there.

-Ar in formula (c) and formula (III) is preferably the following formulas (V) to (XII):
Wherein R ³¹ is selected from a hydrogen atom, a halogen atom, nitro, cyano, and —SO ₂ NR ¹⁴ R ¹⁵ ; R ³² is C _1-6 alkyl; X ¹¹ is N—R ³³ , O Or S; R ³³ is a hydrogen atom or C _1-6 alkyl; X ¹⁰ , R ¹⁴ and R ¹⁵ are as defined above.
Is a group selected from

Preferred X ³ for formula (V) includes, for example, a direct bond, CON (—R ¹⁶ ), N (—R ¹⁶ ) CO, SO ₂ N (—R ¹⁹ ) or N (—R ¹⁹ ) SO _2. It is done.

Preferred X ³ for formula (VI) includes, for example, N—R ¹³ (wherein preferred R ¹³ includes C _1-3 alkyl such as methyl), or S.

Preferred X ³ for formula (VII) includes, for example, a direct bond, CON (—R ¹⁶ ), N (—R ¹⁶ ) CO, SO ₂ N (—R ¹⁹ ) or N (—R ¹⁹ ) SO _2. It is done.

Preferred X ³ for formula (VIII) includes, for example, a direct bond, CON (—R ¹⁶ ), N (—R ¹⁶ ) CO, SO ₂ N (—R ¹⁹ ) or N (—R ¹⁹ ) SO _2. It is done.

Preferred X ³ for formula (IX) includes, for example, a direct bond.

Preferred X ³ for formula (X) includes, for example, a direct bond.

Preferred X ³ for the formula (XI) is, for example, CO, SO ₂ , SO ₂ N (—R ¹⁹ ) or CON (—R ¹⁶ ) (wherein the SO ₂ N (—R ¹⁹ ) and CON (—R) ¹⁶ ) includes a sulfur atom and a carbon atom bonded to Ar, respectively.

Preferred X ³ for formula (XII) is, for example, CO, SO ₂ , SO ₂ N (—R ¹⁹ ) or CON (—R ¹⁶ ) (wherein the SO ₂ N (—R ¹⁹ ) and CON (—R) ¹⁶ ) includes a sulfur atom and a carbon atom bonded to Ar, respectively.

In the present invention, the group —X ³ —Ar functions as an environmentally responsive fluorophore. For example, when a fluorophore of the formula (V) or (VII) is used, the temperature at which the fluorescence intensity decreases with increasing temperature. When the sensor uses a fluorophore of formula (VI) or (VIII) to (XII), a temperature sensor is obtained in which the fluorescence intensity increases as the temperature increases.

R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ in formula (d) and formula (XIII) are preferably independently selected from a hydrogen atom and a methyl group.

Preferred X ⁴ in formula (d) and formula (XIII) is, for example, a direct bond, phenylene, —Q ⁴ —O—C (═O) — (where Q ⁴ is bonded directly to the boron dipyrromethene skeleton. Or —Q ⁴ —NH—C (═O) — (wherein Q ⁴ is directly bonded to the boron dipyrromethene skeleton).

Q ⁴ in formula (d) and formula (XIII) is preferably phenylene.

According to a particularly preferred embodiment of the invention, R ¹ is selected from a hydrogen atom, methyl and ethyl; R ⁴ is selected from n-propyl, isopropyl and t-butyl and R ⁵ is a hydrogen atom; R ² is selected from a hydrogen atom, methyl and ethyl; W is an aromatic ring or —X ¹ —C (═O) — (wherein X ¹ is directly attached to Q ¹ ); X ¹ is O, S, or N—R ¹¹ ; R ¹¹ is a hydrogen atom, C _1-6 alkyl, or —Q ¹ —Y; Q ¹ is C _1-20 alkylene, Here, in the alkylene, O, S, —O—P (═O) (— OH) —O— or phenylene may be independently inserted at one or more points; Y is independently 1 An ionic functional group that can have the above positive charge, and is —N ⁺ R _{^{21 R 22 R 23 X e -}} , -C (= NR 41) -NR 42 R 43, and the following formula:
X _e ⁻ and X _f ⁻ are counter anions; R ²¹ , R ²² , and R ²³ are independently C _1-10 alkyl, or R ²¹ and R ^22. May combine with the nitrogen atom to form a 5-7 membered nitrogen-containing heterocycle; R ²⁴ is C _1-10 alkyl; R ⁴¹ , R ⁴² and R ⁴³ are independently Each selected from a hydrogen atom and C _1-10 alkyl, or R ⁴¹ and R ⁴² together with the nitrogen and carbon atoms to which they are attached, a 5- to 7-membered heterocycle containing two nitrogen atoms, R ⁴² and R ⁴³ together with the nitrogen atom to which they are attached may form a 5-7 membered nitrogen-containing heterocycle; R ³ is a hydrogen atom and C _1-3 Selected from alkyl X ² is O or N—R ¹² ; X ³ is a direct bond, O, N (—R ¹³ ), CON (—R ¹⁶ ), N (—R ¹⁶ ) CO, or N (— R ¹⁷ ) CON (—R ¹⁸ ); Q ² is selected from C _1-20 alkylene, C _3-20 alkenylene, or C _3-20 alkynylene, wherein said alkylene is at one or more points: O, S or phenylene may be inserted independently; Ar is represented by the following formula:
An aromatic carbocyclic group or an aromatic heterocyclic group selected from the group represented by formula (1), and these groups have a halogen atom, C _1-6 alkoxy, nitro, cyano, —NR ⁶ R ⁷ on the ring, And is substituted by one or more substituents selected from —SO ₂ NR ¹⁴ R ¹⁵ and may be further substituted by C _1-6 alkyl; X ¹⁰ is selected from O, S or Se R ⁸ is selected from a hydrogen atom, C _1-10 alkyl, and aryl; R ⁶ and R ⁷ are independently a hydrogen atom, C _1-10 alkyl, aryl, C _1-10 alkylcarbonyl, arylcarbonyl; , C _1-10 alkylsulfonyl, selected arylsulfonyl, and carbamoyl; or R ⁶ and R ⁷ together with the nitrogen atom to which they are attached , To form a 5- to 7-membered nitrogen-containing heterocycle, wherein said heterocycle, C _1-6 alkyl, C _1-6 alkoxy, optionally substituted by one or more substituents selected nitro, and halogen atoms R ¹² is a hydrogen atom, C _1-6 alkyl, or —Q ² —X ³ —Ar, wherein the alkyl is substituted with one or more substituents selected from hydroxy and halogen atoms R ¹³ is a hydrogen atom or C _1-6 alkyl, where the alkyl may be substituted with one or more substituents selected from hydroxy and halogen atoms; R ¹⁴ and ^{R 15} are independently selected from hydrogen, and _{C 1-6} alkyl; or ^{R 14} and ^{R 15} together with the nitrogen atom to which they are attached, 5 Membered nitrogen-containing heterocyclic ring to ^form; R ^{16, R 17} and ^{R 18} are independently selected from hydrogen, and _{C 1-6} alkyl, wherein the alkyl is 1 is selected from hydroxy and halogen atom Optionally substituted by the above substituents; R ⁵¹ , R ⁵² , R ⁵³ , R ⁵⁴ and R ⁵⁵ are independently selected from a hydrogen atom and a methyl group; X ⁴ is a direct bond, phenylene,- Q ⁴ —O—C (═O) — (where Q ⁴ is directly bonded to the boron dipyrromethene skeleton), or —Q ⁴ —NH—C (═O) — (where Boron dipyrro Q ⁴ is directly bonded to the methene skeleton, and Q ⁴ is phenylene.

  In formula (I), formula (II), formula (III) and formula (XIII), a, b, c and d are numbers greater than 0 representing the ratio of each repeating unit in the formula, and are not particularly limited. For example, when a + b = 100, b is, for example, 1 or more, preferably 2 or more, more preferably 2.5 or more, more preferably 3 or more, for example, 15 or less, preferably 10 or less. Further, b when a + b = 100 is, for example, 1 to 15, preferably 2 to 10, and more preferably 3 to 10. c is a ratio to the total amount of a and b (that is, 100), and c is 0.05 to 10, specifically 0.1 to 5, preferably 0.2 to 2, Preferably it is 0.3-1.5. d is a ratio with respect to the total amount (that is, 100) of a and b, and d is 0.01-5, specifically 0.02-2, preferably 0.02-1, and more Preferably it is 0.03-1. Although c / d showing ratio of c and d is not specifically limited, Preferably it is 0.1-30, More preferably, it is 1-20, More preferably, it is 3-10. As described above, a is the sum of the thermosensitive units. For example, when two types of thermosensitive units are used, the ratio of the thermosensitive units is p: ap, using a certain number p. For example, a copolymer is prepared so that b is 2.5 when a + b = 100, and two types of heat-sensitive units (for example, Nn-propylacrylamide and N-isopropylacrylamide) are combined. A is 97.5, and the ratio of Nn-propylacrylamide to N-isopropylacrylamide is represented by p: 97.5-p. Moreover, the weight average molecular weight of the copolymer of this invention is although it does not specifically limit, For example, it is 1000-100000, Preferably it is 5000-50000.

  Depending on the molecular weight of the polymer of the present invention and the amount of monomer used in the fluorescent unit used, two fluorescent units may not necessarily be introduced into one polymer. When this polymer is used as a temperature-sensitive probe, if a combination of general excitation intensity and detection sensitivity is used, it will be used at a somewhat high concentration of 0.001% (w / v) or more in the solution. In addition, since it is used in a cell under the condition that the fluorescence derived from the fluorescent unit is sufficiently observed in the cell, the cell has a relatively high concentration, that is, the same level as 0 in the case of using in a solution. More than 0.001% (w / v) copolymer will be present. In other words, even if two fluorescent units are divided into two polymers, unless there is a special environment where there is only one polymer molecule in the observation field or measurement field, two fluorescent units are included separately. Since the two polymers are placed in almost the same temperature environment, each fluorescent unit will exhibit the same fluorescence intensity as when one fluorescent polymer is included in one polymer. Therefore, the copolymer of the present invention is not limited to the case where two fluorescent units are contained in the same polymer molecule.

  The copolymers of the present invention respond very quickly to ambient temperature changes, with structural changes on the order of a few milliseconds. That is, since the temperature-sensitive fluorescent probe of the present invention changes the fluorescence intensity in response to the temperature change in the cell, when the temperature distribution in the cell is visualized using a microscope or the like, the fluorescence intensity ratio Thus, the intracellular temperature in each minute space in the cell can be quantified.

  The copolymer according to the present invention can be synthesized based on ordinary knowledge in the technical field of polymer synthesis, and can be obtained, for example, as a random copolymer by radical polymerization. The radical initiator that can be used in this case is not particularly limited, but persulfates such as ammonium persulfate, sodium persulfate, and potassium persulfate; 2,2′-azobis (2-methylpropionamidine) Examples thereof include azo compounds such as hydrochloride, 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, and 4,4′-azobis (4-cyanovaleric acid).

  The reaction initiator may be used in an amount of 0.01 mol% or more with respect to the monomer used, and an appropriate amount can be selected as long as the radical synthesis proceeds. For example, a reaction initiator of 0.1 mol% or more, preferably 1 mol% or more can be used.

  The reaction solvent used for the polymerization is not particularly limited, and examples include acetonitrile, dioxane, dimethylformamide, methanol and the like. The radical polymerization is not particularly limited, but can be performed, for example, at a reaction temperature of 0 to 100 ° C., preferably 50 to 70 ° C., and a reaction time of 1 to 48 hours, preferably 2 to 16 hours, for example.

In order to perform temperature measurement without being affected by the pH and salt concentration in the solution containing the copolymer of the present invention, the cationic functional group contained in the copolymer maintains ionicity in a wide range of pH. Is preferable. However, as far as the use for measuring the intracellular temperature is concerned, the intracellular pH is 2 to 9, and it is about 4 to 8 for normal conditions, general animal, plant and microbial cells. Any functional group that is kept cationic at this pH may be used. From this point of view, Y is ^{-N + R 21 R 22 R 23} X e - are preferred.

  The “cell” in the present invention includes both general prokaryotic cells and eukaryotic cells, and is not particularly dependent on the species of the organism. For example, prokaryotic cells are divided into eubacteria and archaea, but eubacteria are broadly divided into gram-positive bacteria such as actinomycetes and gram-negative bacteria such as proteobacteria, depending on the thickness of the peptidoglycan layer. The range in which the copolymer can be applied is not limited. In addition, cells belonging to eukaryotes (protists, fungi, plants, animals) mainly apply to eukaryotic cells. For example, yeast that is generally used in research such as molecular biology and industrially belongs to fungi. Moreover, the temperature sensitive probe of the present invention is suitably used in both floating cells and adherent cells.

  When introducing the copolymer of the present invention into cells, it is desirable to substitute a solution (solvent) having a low ionic strength. Examples of such a solvent include water (preferably pure water), a sorbitol aqueous solution, a glucose solution, and the like. Depending on the type of cell, a solution obtained by adding 0.45 mM calcium chloride to the glucose solution or the like may be used.

  The concentration of the copolymer when the copolymer is introduced into the cells according to the present invention is, for example, a final copolymer concentration of 0.001 to 1% (w / v), preferably 0.01 to 0.5. % (W / v) can be adjusted and mixed with bacterial cells. This applies not only to microbial cells but also to adherent cells.

  The change in the fluorescence intensity due to the thermal sensitivity of the copolymer used in the present invention can be measured by a usual fluorescence intensity measurement method. The excitation wavelength in measurement and the fluorescence wavelength to be measured are not particularly limited. For example, the maximum excitation wavelength when measuring the excitation spectrum of the measurement sample or a wavelength in the vicinity thereof can be used. In addition, the fluorescence wavelength to be measured is not particularly limited, and for example, the maximum fluorescence wavelength when the fluorescence spectrum of the measurement sample is measured at a certain temperature or a wavelength in the vicinity thereof can be used.

  In the present invention, it is desirable to take a method of measuring the fluorescence intensities at two independent fluorescence wavelengths, taking these ratios, and converting the fluorescence intensity ratios into temperatures. This technique eliminates the possibility that the fluorescence intensity emitted from the copolymer is caused by the concentration of the copolymer in the minute space or the intensity of the excited laser, and the ratio of the fluorescence intensity obtained by experiment and the temperature is 1: 1. It is possible to correspond to. This makes it possible to compare not only the temperature in the same cell but also the intracellular temperature of another cell placed under the same condition. For example, it is possible to grasp the physiological state of each yeast cell by measuring the difference in individual cell temperatures in the yeast population.

  The method for calculating the fluorescence intensity ratio is not particularly limited, and the ratio can be calculated from the fluorescence intensities of two regions including different wavelengths. For example, one region is a wavelength region of about 20 nm including the wavelength indicating the maximum intensity of fluorescence generated from the first fluorescent unit, and the integrated value of the fluorescence intensity is S1, and the other region is generated from the second fluorescent unit. In a wavelength region of about 20 nm including the wavelength indicating the maximum intensity of fluorescence, the integrated value of fluorescence intensity may be S2, and S1 / S2 may be the fluorescence intensity ratio. Further, the S1 and S2 regions may have the same width or different widths. For example, if the fluorescence intensity shows a value sufficient to ignore noise, S1 may include a wavelength region with a width of 20 nm, while S2 may have a single wavelength with a width of 1 nm. The wavelength selection criteria are not particularly limited, but in consideration of the fluorescence intensity to be obtained, monomers (for example, represented by the formula (c) or the formula (d)) that give each fluorescent unit contained in the temperature-sensitive probe. It is desirable to select the wavelength from the surrounding wavelengths based on the wavelength indicating the maximum fluorescence intensity when measuring the excitation spectrum of water and a polar solvent close to water at room temperature (about 25 ° C.).

  When obtaining the temperature from the fluorescence intensity ratio obtained in the experiment, it is possible to use a calibration curve created by itself. Specifically, the calibration curve measured under which conditions is not limited, but, for example, a curve plotting the change in fluorescence intensity due to the thermosensitive response of the copolymer in a potassium chloride solution that mimics the inside of a cell. The cell population into which the copolymer has been introduced is subjected to a fluorometer, and the curve plotting the change in fluorescence intensity due to the heat-responsiveness, or the cell population into which the copolymer has been introduced is subjected to a fluorescence microscope to produce heat sensitivity in a plurality of cells. A curve or the like in which an average value of changes in fluorescence intensity due to responsiveness is plotted can be used. More specifically, using a cell population into which the copolymer has been introduced, a thermosensitive response test is performed, and when plotting changes in fluorescence intensity, the cells do not actively undergo metabolic activity, For example, when cells are suspended in water or in a buffer containing a compound that cannot be assimilated, the cells are held at a specific temperature for a certain period of time, and the external temperature and the internal temperature of the cells have reached equilibrium. The method of measuring a fluorescence intensity etc. is mentioned under the situation where it is.

  In order to carry out the method described above, necessary reagents and the like can be collected into a kit. Thus, according to another aspect of the present invention, there is provided a kit for measuring temperature using the method described above, the kit comprising the temperature sensitive probe of the present invention or the copolymer of the present invention. . This reagent kit for temperature measurement can be suitably used for temperature measurement in a minute space, particularly for cell temperature measurement. The reagent kit can be used in research fields such as medicine, biology, and biotechnology, and in medical fields such as diagnosis and treatment.

  The method and temperature measurement kit of the present invention can be applied to various fields of research and development. For example, in the field of biotechnology, in the fermentative production of useful substances using microorganisms, it is expected to improve the efficiency of examination of culture conditions by adding the intracellular temperature, which has been difficult to measure accurately, to analysis parameters. The

  The method and temperature measurement kit of the present invention can be applied to various medical uses. For example, by using the temperature-sensitive probe according to the present invention for a part of a patient's tissue, it is possible to discriminate between cancer cells that are said to have high heat production and normal cells that do not. is there. In addition, it can be used to develop more effective thermotherapy. Alternatively, a material that activates brown adipocytes is screened by introducing a temperature-sensitive probe according to the present invention into brown adipocytes that produce a large amount of heat and measuring the temperature changes caused by adding various materials to the cells. It is also possible to do.

  The method and temperature measurement kit of the present invention can be applied to elucidation of various physiological phenomena. For example, the activity of TRP channels in a different approach by sensing the temperature outside the body and investigating how the TRP channel, which is a receptor that causes a biological response, is related to the intracellular temperature. Can be considered. In addition, by examining the relationship between the intracellular temperature distribution and the biological reaction that occurs inside and outside the cell, it is possible to investigate the effect of the local temperature distribution on the biological reaction. Cells by local heating using an infrared laser, etc. It is also possible to perform control.

  The temperature measurement method and the cell introduction method of the temperature-sensitive probe according to the present invention can be performed both in vitro and in vivo. In one embodiment, these methods are performed in vitro.

  EXAMPLES The present invention will be described in more detail by showing examples below, but the present invention is not limited to these examples.

Reagent and Data Measurement α, α′-Azobisisobutyronitrile (AIBN) was purified by recrystallization using methanol, and N-isopropylacrylamide (NIPAM) was purified by recrystallization using n-hexane. Other reagents were used without further purification.

¹ H-NMR was measured using a BRUKER AVANCE 400 spectrometer (400 MHz), and chemical shifts were expressed in ppm. The number average molecular weight and the weight average molecular weight are obtained from polystyrene standard samples using JASCO GPC system (JASCO PU-2080 pump, JASCO RI-2031 differential refractometer, JASCO CO-2060 column oven, Shodex GPC KD-806M column). Calculated using a calibration curve. For silica gel column chromatography, Kanto Chemical Silica gel 60N (40-50 μm) was used. A JASCO V-550 ultraviolet-visible light spectrophotometer was used for measuring the absorbance.

Example 1-1: 8- (4-acrylamidophenyl) -4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene (BODIPY-AA) Synthesis of

Under argon, 4-acetamidobenzaldehyde (200 mg, 1.23 mmol) and 2,4-dimethylpyrrole (253 μL, 2.45 mmol) were dissolved in dichloromethane (70 mL, dehydrated with molecular sieves 4A), and one drop of trifluoroacetic acid was added. Stir at room temperature for 6 hours. To this reaction solution, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (278.3 mg, 1.23 mmol) was suspended in dichloromethane (5 mL, dehydrated with molecular sieves 4A) and added. The vessel was washed with 3 mL of dichloromethane and added as well. After stirring at room temperature for 30 minutes, the reaction solution was washed with water (100 mL × 2) and saturated brine, dried over Na ₂ SO ₄ , and the solvent was distilled off under reduced pressure. The obtained crude product was subjected to alumina chromatography (first dichloromethane → dichloromethane: methanol = 100: 1 → 50: 1, second dichloromethane → dichloromethane: methanol = 100: 1, third dichloromethane: methanol = 200: 1). Purification gave the desired product (Compound 1a) as light brown crystals (31 mg, 7.5%).

1H NMR (400 MHz, CDCl ₃ ) of Compound 1a is as follows.
δ 7.61 (d, 2H, J = 8.4 Hz), 7.29 (br, 1H), 7.24 (d, 2H, J = 8.4 Hz), 5.89 (s, 2H), 2.34 (s, 6H), 2.21 (s, 3H), 1.34 (s, 6H)

The obtained compound 1a (66 mg, 0.20 mmol) was dissolved in 10 mL of methanol, 10 mL of 1N hydrochloric acid was added, and the mixture was heated to reflux for 4 hours. After cooling to room temperature, it was neutralized with 2N NaOH solution. After extraction with dichloromethane (100 mL × 3), the organic layer was washed with water and saturated brine, dried over Na ₂ SO ₄ , and the solvent was distilled off under reduced pressure. The obtained crude product was purified by alumina chromatography (dichloromethane: methanol = 20: 1) to obtain the desired product (compound 1b) as light brown crystals (46 mg, 79%).

1H NMR (400 MHz, CDCl ₃ ) of Compound 1b is as follows.
δ 7.05-7.03 (m, 2H), 6.75-6.73 (m, 2H), 5.89 (s, 2H), 3.76 (br, 2H), 2.34 (s, 6H), 1.42 (s, 6H)

Under argon, compound 1b (20 mg, 0.069 mmol) was dissolved in 25 mL of dichloromethane, and triethylamine (200 μL) and boron trifluoride-ethyl ether complex (200 μL) were sequentially added thereto, followed by stirring at room temperature for 2 hours. The reaction solution was poured into 80 mL of water to stop the reaction, and 2N NaOH solution was added to make it alkaline. This was extracted with dichloromethane, the organic layer was washed with saturated brine, dried over Na ₂ SO ₄ , and the solvent was distilled off under reduced pressure. The obtained crude product was purified by alumina chromatography (dichloromethane: hexane = 1: 1) to obtain the desired product (compound 1c) as red crystals (22 mg, 96%).

1H NMR (400 MHz, CDCl ₃ ) of Compound 1c is as follows.
δ 7.02-7.00 (m, 2H), 6.79-6.77 (m, 2H), 5.97 (2H), 3.83 (br, 2H), 2.55 (s, 6H) , 1.50 (s, 6H).

The 13C NMR (100 MHz, CDCl ₃ ) of compound 1c is as follows.
δ155.0, 147.0, 143.2, 142.6, 132.0, 129.0, 124.7, 120.9, 115.4, 14.6, 14.5.

Compound 1c (10 mg, 0.029 mmol) was dissolved in 2 mL of acetonitrile, triethylamine (4.1 μL, 0.029 mmol) was added, and then acrylic acid chloride (3.1 μL, 0.038 mmol) was added at 0 ° C. After stirring at room temperature for 2 hours, K ₂ CO ₃ was added and the salt was filtered. The crude product obtained by distilling off the filtrate under reduced pressure was purified by silica gel chromatography (dichloromethane: hexane = 3: 1 → dichloromethane 100%) to give the title compound (BODIPY-AA) as orange crystals. (11 mg, 97%).

The results of 1H NMR (400 MHz, methanol-d4) of BODIPY-AA are as follows.
δ 7.88-7.86 (m, 2H), 7.31-7.29 (m, 2H), 6.51-6.37 (m, 2H), 6.07 (s, 2H), 5. 81 (dd, 1H, J = 2.4, 9.6 Hz), 2.49 (s, 6H), 1.48 (s, 6H).

The results of 13C NMR (100 MHz, CDCl ₃ ) of BODIPY-AA are as follows. δ 163.5, 155.5, 143.1, 141.1, 138.6, 131.6, 130.9, 128.8, 128.5, 121.2, 120.1, 14.6.

Example 1-2: 4,4-Difluoro-8- (4-isobutyramidephenyl) -1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene (BODIPY- Synthesis of IA)

Compound 1c (10 mg, 0.029 mmol) synthesized in Example 1-1 was dissolved in 1 mL of acetonitrile, and triethylamine (4.9 μL, 0.035 mmol) and isobutyric anhydride (7.4 μL, 0.044 mmol) were added at 0 ° C. ) And stirred at room temperature for 3 hours. Further, triethylamine (4.9 μL, 0.035 mmol) and isobutyric anhydride (7.4 μL, 0.044 mmol) were added, stirred for 30 minutes, then stirred at 40 ° C. for 2 hours, then at 50 ° C. for 3 hours, and then at room temperature overnight. Stir. After adding Na ₂ SO ₄ to the reaction solution and filtering, the filtrate was distilled off under reduced pressure, and the resulting crude product was purified by silica gel chromatography (dichloromethane) to obtain the desired product as red crystals. (12 mg, 95%).

The result of 1H NMR (400 MHz, CDCl ₃ ) of BODIPY-IA is as follows.
δ 7.69 (d, 2H, J = 8.2 Hz), 7.31 (br, 1H), 7.22 (d, 2H, 8.2 Hz), 5.98 (s, 2H), 2. 55 (s, 6H), 1.43 (s, 6H), 1.29 (d, 6H, J = 6.8 Hz), 1.25 (br, 1H).

The results of 13C NMR (100 MHz, CDCl ₃ ) of BODIPY-IA are as follows.
δ 175.3, 155.5, 143.1, 141.3, 138.9, 131.6, 130.5, 128.8, 121.2, 119.9, 36.9, 19.6, 14. 64, 14.56.

Example 1-3: Calculation of quantum yield depending on the solvent of BODIPY-IA BODIPY-IA synthesized in Example 1-2 was acetonitrile, methanol, hexane, ethyl acetate and water 75 v / v% dioxane 25 v / v%, respectively. The absolute fluorescence quantum yield was calculated using a fluorimeter FP8500 (JASCO) which was dissolved in 10 μM and provided with an integrating sphere unit ILF835. The excitation wavelength was selected to be 470 nm. Calculate the incident light area S ₀ , the sample scattered light area S ₁ , and the peak area S ₂ of the fluorescence wavelength in the absence of the sample,
Quantum yield _{= S 2 / (S 0 -S} 1) × 100
Table 1 shows the result of calculation using the equation.

  As can be seen from the results in Table 1, the BODIPY-AA fluorescent unit newly used for the temperature probe has a tendency that the quantum yield is low in a hydrophobic environment such as acetonitrile and the quantum yield is high in water. When this unit is incorporated into an acrylamide polymer, at low temperatures (in water) (the acrylamide chain is in a string state), the BODIPY-AA is placed in a hydrophilic environment, the fluorescence quantum yield is high, and relatively strong fluorescence is emitted. When the temperature is high (the acrylamide chain is aggregated), the periphery of BODIPY-AA is placed in a hydrophobic environment, the fluorescence quantum yield is reduced, and relatively weak fluorescence is emitted. In a fluorescence temperature sensor using a fluorescent dye having a benzofurazan skeleton, it is known that the fluorescence intensity increases as the temperature rises. That is, it was predicted that by using both BODIPY and the benzofurazan skeleton fluorescent dye, a change in the fluorescence intensity ratio accompanying a change in temperature can be detected with high sensitivity.

Example 2: Synthesis of Nn-propylacrylamide Nn-propylamine and triethylamine (1.2 eq) were added to benzene, and the mixture was stirred well in an ice bath using a stirrer. .2 eq) was added in 3 portions every 10 minutes and allowed to warm to room temperature. The reaction solution was filtered and separated with 0.1N hydrochloric acid, and the upper benzene fraction was extracted. After adding anhydrous sodium sulfate, it was dried under reduced pressure, and silica gel column chromatography (dichloromethane-methanol = developing solvent = 50: 1 was used) for the first purification. The fractions containing the target compound were collected again, dried under reduced pressure, and purified a second time by silica gel column chromatography (using ethyl acetate-hexane = 1: 2 as a developing solvent), and the title compound was colorless and transparent Obtained as a liquid.

Example 3: N- (2-{[7- (N, N-dimethylaminosulfonyl) -2,1,3-benzothiadiazol-4-yl]-(methyl) amino} ethyl) -N-methylacrylamide ( DBThD-AA) was synthesized according to the method described in Document A (Chemistry A European Journal 2012, Vol. 18, pages 9552-9563). 3-Chloro-1,2-phenylenediamine was dissolved in toluene and N-thionylaniline was added. After refluxing at 130 ° C. for 3 hours, the mixture was dried under reduced pressure and purified by silica gel chromatography to obtain 4-chloro-2,1,3-benzothiadiazole. This was dissolved in concentrated sulfuric acid at 0 ° C. and stirred at 0 ° C. for 1.5 hours, then heated to 150 ° C. and stirred for 2.5 hours. After completion of the reaction, the reaction solution was poured into ice water and extracted with dichloromethane. The organic layer was dried over Na ₂ SO ₄ , dried under reduced pressure, and purified by silica gel chromatography to obtain 7-chloro-2,1,3-benzothiadiazole 4-sulfonyl chloride.

  Further, the obtained substance was dissolved in dichloromethane, and an aqueous dimethylamine solution dissolved in acetonitrile was added. After reacting at room temperature for 30 minutes, it was dried under reduced pressure and purified by silica gel chromatography to obtain 7-chloro-N, N-dimethyl-2,1,3-benzothiadiazole 4-sulfonamide. This material was dissolved in acetonitrile and this solution was added to N, N'-dimethylethylenediamine. After reacting at 80 ° C. for 1 hour, the mixture was dried under reduced pressure and purified by silica gel chromatography to obtain N, N-dimethyl-7- [methyl {2- (methylamino) ethyl} amino] -2,1, 3-Benzothiadiazole-4-sulfonamide was obtained.

The obtained N, N-dimethyl-7- [methyl {2- (methylamino) ethyl} amino] -2,1,3-benzothiadiazole-4-sulfonamide was dissolved in acetonitrile, triethylamine (1 eq), acrylic After acid chloride (1.3 eq) was added at 0 ° C., the mixture was stirred at 0 ° C. for 1 hour. The reaction solution was added with Na ₂ CO ₃ (1 g), filtered, dried under reduced pressure, and purified by silica gel column chromatography (using dichloromethane / methanol as a developing solvent) to give the title compound DBThD-AA as orange crystals. Got as.

Example 4: Nn-propylacrylamide / (3-acrylamidopropyl) trimethylammonium / N- {2-[(7-N, N-dimethylaminosulfonyl) -2,1,3-benzooxadiazole-4 Preparation of -yl] (methyl) amino} ethyl-N-methylacrylamide / BODIPY-AA copolymer NNPAM (543 mg, 4.8 mmol), (3-acrylamidopropyl) trimethylammonium chloride (55.1 mg, 0.2 mmol, (Hereinafter also referred to as “APTMA chloride”), DBThD-AA (9.59 mg, 25 μmol), BODIPY-AA (1.97 mg, 5 μmol), AIBN (8.21 mg, 50 μmol) are dissolved in DMF (10 ml) for 30 minutes. Remove dissolved oxygen by passing argon gas through It was. Then, it was made to react at 60 degreeC for 4 hours, and it cooled to room temperature. This solution was poured into diethyl ether (300 ml) with stirring. The obtained crystals were collected by filtration, dried under reduced pressure, further dissolved in MeOH (1 mL), reprecipitated, then dissolved in pure water, and a Visking tube (cellulose tube for dialysis) having a diameter of 28.6 mm was attached. Used, the dialysis external solution was 1000 ml, and was sufficiently dialyzed for purification. The purified product was lyophilized to give the title copolymer as a pale yellow powder (254.4 mg, 42%).

The number average molecular weight is 4537, the weight average molecular weight is 9264, and the composition ratio of each unit in the copolymer is NNPAM: APTMA: DBThD-AA: BODIPY-AA = 94.5: 5.5: 0.579: 0.071. In addition, the ratio of the NNPAM unit and the APTMA unit in the copolymer is based on the integrated value in ¹ H-NMR measurement, and the ratio of the DBThD-AA unit is the absorbance in methanol of 4-N, N- (dimethylamino) -7-N. , N-dimethylaminosulfonyl-2,1,3-benzothiadiazole was calculated by comparison. The ratio of BODIPY-AA unit was calculated by comparing the absorbance in methanol with BODIPY-IA.

Example 5 Production of Copolymer with Changed Temperature Sensitive Unit NIPAM and N-tert-butyl causing structural changes at higher and lower temperatures than the temperature sensitive unit NNPAM used in Example 4 Copolymers were synthesized using acrylamide (NTBAM). A list of synthesized compounds is shown in Table 2. The synthesis was performed in the same manner as in Example 4 to obtain a pale yellow powder.

As shown in Table 2, all the desired copolymers could be synthesized. The composition ratio of the copolymer was calculated as shown in Example 4, and the molecular weight of the copolymer was also calculated in the same manner as in Example 4. The ratio of the thermosensitive unit and the APTMA unit in the copolymer is based on the integral value in ¹ H-NMR measurement, and the ratio of the DBD-AA unit and the BODIPY-AA unit is the absorbance in methanol of 4-N, N- It was calculated from the molar extinction coefficient of 447 nm and 498 nm of (dimethylamino) -7-N, N-dimethylaminosulfonyl-2,1,3-benzothiadiazole and BODIPY-IA, respectively.

Example 6 Production of Copolymer with Changed Cationic Unit A copolymer was produced that used APTMA, which is a cationic unit used in Example 4, in a higher ratio. A copolymer using VBTMA instead of APTMA as a cationic unit which is a quaternary ammonium salt was synthesized. A list of synthesized compounds is shown in Table 3. The synthesis was performed in the same manner as in Example 4 to obtain a pale yellow powder.

As shown in Table 3, all the desired copolymers could be synthesized. The composition ratio of the copolymer was calculated as shown in Example 4, and the molecular weight of the copolymer was also calculated in the same manner as in Example 4. In addition, the ratio of the NNPAM unit and the cationic unit in the copolymer is the integrated value in ¹ H-NMR measurement, and the ratio of the DBD-AA unit and the BODIPY-AA unit is the absorbance in methanol of 4-N, N- ( It was calculated from the molar extinction coefficients of 447 nm and 498 nm of dimethylamino) -7-N, N-dimethylaminosulfonyl-2,1,3-benzothiadiazole and BODIPY-IA, respectively.

Example 7: Production of Copolymer with Changed Fluorescent Unit Ratio A copolymer with the two fluorescent unit ratios of the copolymer synthesized in Example 4 changed was synthesized. Table 4 shows a list of synthesized compounds. The synthesis was performed in the same manner as in Example 4 to obtain a pale yellow powder.

As shown in Table 4, all the desired copolymers could be synthesized. The composition ratio of the copolymer was calculated as shown in Example 4, and the molecular weight of the copolymer was also calculated in the same manner as in Example 4. The ratio of the NNPAM unit and the APTMA unit in the copolymer is based on the integral value in ¹ H-NMR measurement. The ratio of the DBD-AA unit and the BODIPY-AA unit is the absorbance in methanol of 4-N, N- (dimethyl Amino) -7-N, N-dimethylaminosulfonyl-2,1,3-benzothiadiazole and BODIPY-IA were calculated from the molar extinction coefficients of 447 nm and 498 nm, respectively.

Example 8: Thermal sensitivity test The thermal sensitivity test of compounds 1 to 8 in 150 mM KCl was performed according to the following procedure. Using a JASCO FP-6500 spectrofluorometer, KCl purchased from Wako Pure Chemicals was dissolved to a concentration of 150 mM using ultrapure water obtained from Millipore Milli-Q reagent system as a solvent. A thing was used. The initial concentration of the compound in this experiment was 0.01 w / v%, and the excitation wavelength was 458 nm. A JASCO ETC-273T water-cooled Peltier thermostatic cell holder was used for temperature control of the solution, and the temperature was measured with an attached thermocouple. The solution temperature was increased in increments of 1 ° C. or 2 ° C., and a fluorescence spectrum of 450 to 850 nm at each temperature was measured.

  An example of a spectrum change when the temperature of the compound 2 is changed is shown in FIG. From this result, it was confirmed that the fluorescence intensity at 500 to 520 nm, which is a fluorescence spectrum derived from BODIPY, decreases with increasing temperature, and the fluorescence intensity at 540 to 600 nm, which is a fluorescence spectrum derived from DBThD, increases. By taking the ratio of these two wavelength regions, the structural change of the copolymer accompanying the temperature change can be captured. That is, it was confirmed that the temperature around the copolymer can be expressed by the fluorescence intensity ratio. From the result of this spectrum change, it was found that the change in the fluorescence intensity ratio with respect to the temperature change is increased by taking the fluorescence intensity ratio (FI580 / FI510) of 580 nm and 515 nm.

  2 to 4 show the fluorescence intensity ratio (FI580 / FI510) of 580 nm and 515 nm at each temperature of each compound. As shown in FIG. 2, the temperature sensitive unit responds in the order of NTBAM, NNPAM, and NIPAM, but the temperature range of response is freely controlled according to the nature of the temperature sensitive response site to be used. It was found that the temperature could be quantified by the ratio. In addition, as shown in FIG. 3, an increase in the fluorescence intensity ratio with respect to temperature was also observed in the copolymer (compound 6) using VBTMA as the cationic unit. Further, even when the composition ratio of APTMA was changed (Compound 5), an increase in the fluorescence intensity ratio with respect to temperature was observed. Further, FIG. 4 shows that the temperature can be quantified in the same manner with respect to a copolymer in which the ratio of two fluorescent units is changed.

Example 9: Evaluation of dependence of potassium chloride concentration on thermal response test The dependence of KCl on thermal sensitivity of compound 1 was examined and investigated by the following procedure. The experimental procedure was the same as in Example 8. The solvent was prepared so that the initial concentration of Compound 1 in this experiment was 0.005 w / v% and the KCl concentrations were 125 mM, 150 mM, and 175 mM.

  The test results are shown in FIG. In general, the intracellular potassium concentration is estimated to be about 140 mM, and it is known that the change in intracellular potassium concentration is small unless there is a special environmental change. Although a slight change in temperature responsiveness to changes in the concentration of potassium chloride solution within the range of 125 to 175 mM is observed, the change in intracellular potassium concentration is at a level lower than this. It was suggested that there was almost no influence on the measurement.

Example 10: Evaluation of pH responsiveness The pH dependence of the thermal responsiveness of Compound 1 was tested and investigated by the following procedure. Using 150 mM KCl as a solvent, the pH was adjusted to 6 with hydrochloric acid, and the pH was adjusted to 9 with potassium hydroxide. The experimental method is the same as in Example 8. The initial concentration of Compound 1 in this experiment was 0.005 w / v%.

  The test results are shown in FIG. In the general cell temperature range (25 ° C. to 45 ° C.), almost no change in fluorescence intensity due to pH change was observed. That is, it became clear that the temperature responsiveness was not substantially affected by the pH change, and the fluorescence intensity reflected the temperature with higher sensitivity.

Example 11: Introduction of temperature probe into cultured cells (floating cells) MOLT-4 (human acute lymphoblastic leukemia (T-cell)) was cultured in RPMI 1640 medium (10% FBS) in a 100 mm dish (seeding). 1 × 10 ⁴ cells / ml). Two days later, 3 ml of the culture broth was centrifuged (300 g, 2 minutes), the medium was removed, washed with 5% glucose, and then suspended again with 1 ml of 5% glucose to give each compound 1-9 at a final concentration of 0.05%. Was added as follows. After 10 minutes at 25 ° C., the mixture was centrifuged (300 g, 2 minutes), the supernatant was removed, washed with PBS, suspended in PBS, and the presence of the probe was confirmed by microscopic observation. Observe with confocal laser microscope (FV1000, Olympus), 40x objective lens (UplanSApo, Olympus), apply 473 nm laser (Multi Ar laser) to cells and observe fluorescence images for fluorescence wavelengths from 500 nm to 600 nm did.

  The results are shown in FIG. With each compound, the introduction of the probe could be confirmed even in MOLT4 which is a floating cell. For compound 1, the proportion of cells in which the fluorescence derived from the probe could be confirmed in the cytoplasm in about 50 to 100 cells (excluding the autofluorescence of the cells) was calculated, and 99.2 ± 1.1 (%) Met.

Example 12: Fluorescence intensity response of Compound 1 to cultured cells (floating cells) As in Example 11, Compound 1 was introduced into MOLT-4 and placed in a cuvette suspended in PBS. Furthermore, a spherical stirring bar having a diameter of 2 mm was placed in the cuvette. The cuvette was set on a JASCO FP-6500 spectrofluorometer and rotated at a speed of about 800 rpm to measure the fluorescence spectrum while preventing the cells from sinking. The excitation wavelength was 458 nm. A JASCO ETC-273T water-cooled Peltier thermostatic cell holder was used for temperature control of the solution, and the temperature was measured with an attached thermocouple. The solution temperature was increased in increments of 2 ° C., and the temperature was increased for 2 minutes after the temperature was increased so that the temperature inside the cell and the temperature outside the cell were kept constant.

  The results are shown in FIG. Compound 1 was able to calculate the intracellular temperature. It was confirmed that the intracellular temperature could be measured in a wide temperature range of 25 to 40 ° C., which is a general mammalian cell growth temperature.

Example 13: Evaluation of temperature resolution As in the result of Example 8, it is assumed that temperature (T) is plotted on the horizontal axis and fluorescence intensity ratio (R) is plotted on the vertical axis. When ∂ is a minute amount and δ is an error, the following relationship is established.

In other words, the temperature resolution δT indicating how many degrees of temperature difference can be detected is
It is indicated. Here, since ∂ represents a minute amount,
Indicates the slope of the tangent of the curve of the graph with temperature (T) on the horizontal axis and fluorescence intensity ratio (R) on the vertical axis. Since δ represents an error, δR is an error in the fluorescence intensity ratio. Here, the standard deviation was used as the error value.

  That is, (temperature resolution) = (reciprocal of the slope of the tangent of the curve of the graph with temperature (T) on the horizontal axis and fluorescence intensity ratio (R) on the vertical axis) × (error of fluorescence intensity ratio). Can do.

  As a result of evaluating the temperature resolution of FIG. 8 which is the result of Example 12, the result is as shown in FIG. For example, Compound 1 has a temperature resolution of about 0.2 ° C. at a general mammalian cell physiological temperature of 30 to 44 ° C., and it was found that a very wide temperature range can be quantified with high sensitivity. 9 shows the result of analyzing the temperature resolution of FIG. 4 which is the result of the temperature responsiveness test in a 150 mM KCl solution of three compounds (compound 1, compound 7 and compound 8) having two different fluorescent unit ratios. It was shown to. Each probe was shown to have a high temperature resolution of about 0.1 ° C. in a wide temperature range from 25 ° C. to 45 ° C.

Comparative Example 1: Compound 9 (hereinafter referred to as “pre-compound 9”) described in a known publication (International Publication No. 2013/094748) of a temperature resolution in a cell of a fluorescent temperature probe consisting of only a fluorescent unit: It has been found that the temperature range in which the response in the cell is sensitive is in the range of ± 5 ° C. centering around 35 ° C., and the intracellular temperature can be measured with high sensitivity. The cultured MOLT-4 was washed with 5% glucose and then suspended again with 1 ml of 5% glucose, and the pre-compound 9 was added to a final concentration of 0.05%. After 10 minutes at 25 ° C, the mixture is centrifuged (300 g, 2 minutes), the supernatant is removed, washed with PBS, suspended in PBS, and each temperature (25-40 ° C, 1 ° C as in Example 12). The fluorescence spectrum at the step) was measured.

  FIG. 10 shows a graph of changes in the fluorescence intensity ratio and the temperature resolution when the ratio of the fluorescence intensity of 565 nm (maximum fluorescence wavelength at high temperature) and 580 nm (maximum fluorescence wavelength at low temperature) is taken. Even in the pre-compound 9, which was thought to have high sensitivity, the maximum sensitivity in the range from 25 to 40 ° C is 0.05 ° C at 40 ° C, but a temperature difference smaller than 0.1 ° C can be detected. This is the only temperature. In the temperature range where the sensitivity can be measured at around 35 ° C., a temperature resolution greater than 0.2 ° C. was exhibited, and the average temperature resolution from 30 ° C. to 40 ° C. was 0.45 ° C. On the other hand, compound 1 in FIG. 9 has an average temperature resolution of 0.07 ° C. in the same temperature region, and thus it has been clarified that the novel probe synthesized this time can measure temperature with extremely high sensitivity.

Example 14: Application of this probe to adherent cells Human-derived fetal kidney cells HEK293T were cultured in a DMEM medium (5% FBS, 1% penicillin-streptomycin) in a 35 mm glass bottom dish (seeding 1 × 10 ³ cells / cm). ² ). One day later, the medium was replaced with 5% glucose, Compound 1 was added to a final concentration of 0.05%, and the mixture was allowed to stand at 25 ° C. for 10 minutes. Thereafter, the probe was removed, washed with PBS, replaced with phenolic-free DMEM medium, and observed under a microscope at 37 ° C. Microscopic observation was performed using a confocal laser microscope (FV1000, Olympus) and a 60 × oil immersion objective lens (UplanSApo NA 1.40, Olympus). The cells were irradiated with a 473 nm laser (Multi Ar laser) to obtain a 490-530 nm fluorescent image (P1) and a 560-610 nm fluorescent image (P2). P1 and P2 were subjected to image processing to subtract the fluorescence intensity of a certain area without cells as a background.

  The results are shown in FIG. As is clear from the results of P1 and P2, the probe could be easily introduced into adherent cells such as HEK293T. Further, a graph obtained by dividing the fluorescence intensity value of each pixel of P2 by the fluorescence intensity value of P1 is shown as P2 / P1. As a result, it became clear that the temperature distribution in the cell can be visualized and quantified. Furthermore, the ratio intensity (black and white intensity value) of the sectional view of a part of the cells is shown in FIG. As a result, it was clear that there was a clear difference in the ratio value even in the cells. From these results, it became clear that the temperature distribution in the cell can be visualized even with a microscope capable of taking a fluorescence intensity ratio, and this is a very versatile technology that could not be realized with conventional fluorescent temperature sensors. Indicated.

Example 15: Evaluation of temperature according to organelle by microscope As in Example 14, Compound 1 was introduced into HEK293T cells, replaced with DMEM medium, and maintained at 27 ° C, 32 ° C, and 37 ° C. Microscopic observation was performed at temperature. Microscopic observation was performed using a confocal laser microscope (FV1000, Olympus) and a 60 × oil immersion objective lens (UplanSApo NA 1.40, Olympus). Excitation was performed with a 473 nm laser (Multi Ar laser), and a fluorescence image (P1) of 510 to 520 nm and a fluorescence image (P2) of 580 to 590 nm were obtained via an 80/20 reflector.

  Based on the obtained differential interference image of the cells, the whole cell and nucleus of the fluorescence image were surrounded by the region of interest (ROI), and the average fluorescence intensity signal in each ROI was calculated by FV10-ASW analysis software (Olympus). The cytoplasmic mean fluorescence intensity signal was calculated by subtracting the nuclear signal from the whole cell signal. Further, by dividing the P2 signal by the P1 signal, the respective average ratio signals of the cytoplasm and nucleus were calculated. The average value of 9 cells is shown in FIG. As a result, it was shown that the intracellular temperature differs between cytoplasm and nucleus. Together with the results of Example 14, it was found that the temperature distribution in the cells can be quantified with high sensitivity.

Example 16: Toxicity evaluation of probe As in Example 11, Compound 1 was introduced into MOLT-4 cells and suspended in RPMI medium. Further, as in Example 14, Compound 1 was introduced into HEK293T cells and replaced with DMEM medium. Thereafter, propidium iodide (PI), which is a non-membrane-permeable fluorescent reagent, was added to the medium to a final concentration of 0.67 μg / ml and treated at 37 ° C. for 30 minutes. Observation was performed with a microscope. Compound 1 was excited by a 473 nm laser, PI was excited by a 559 nm laser, and observation was performed at fluorescence wavelengths of 490 to 550 nm and 655 to 755 nm, respectively. In addition, the photomultiplier tube sensitivity and the laser intensity of the camera at the time of observation were adjusted by using cells that had been heat-treated at 95 ° C. for 1 minute as controls for dead cells.

  About 100 cells in which the fluorescence of Compound 1 was observed under the microscope were selected, and the number of cells in which the fluorescence of PI was observed was counted as dead cells. As a result, in MOLT-4 cells, the survival rate was 97.9 ± 1.7 (%), and the survival rate of HEK293T cells was 100.0 ± 0.0 (%). It was found that the toxicity of the probe was very low.

Example 17: Probe introduction test into myotube cells and evaluation of temperature responsiveness C2C12 (derived from mouse myoblast, ECACC) was cultured in a growth medium (DMEM, 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin) Thereafter, the cells were seeded in a plastic bottom dish (IBIDI) to ³ × 10 ³ / cm ² and replaced with differentiation medium (DMEM, 2% horse serum, 100 U / ml penicillin, 100 μg / ml streptomycin) after 3 days. Cultured for days.

The cultured cells were washed with 5% glucose containing 0.45 mM CaCl _2, Compound 1 suspension was dissolved in 5% glucose containing 0.45 mM CaCl ₂ (final concentration 0.03%), the dish center 350 μl was added to the hole part (observation part) and treated at room temperature for 10 min. After washing twice with PBS containing 0.45 mM CaCl ₂ , the medium was replaced with a microscope observation medium. The observation was performed using a confocal laser microscope (FV1000, Olympus) and a 20 × objective lens (UPLSAPO, NA 0.75, Olympus), and the cells were irradiated with a 473 nm laser (Multi Ar laser), and 485- Fluorescence images for two fluorescence wavelengths of 545 nm (P1) and 570 to 670 nm (P2) were observed. A ratio image of P2 / P1 was also created in the same manner as in Example 14.

  The results are shown in FIG. It was found that muscle cells were damaged when Ca was removed from the medium, and that the probe could be introduced without damaging the cells by adding a small amount of Ca to the glucose solution at the time of introduction. Furthermore, it was also confirmed that the ratio value was changed by changing the external temperature. It was shown that the probe can be applied to mature cells that are differentiated in this way, and the temperature inside the cell can be measured.

Claims (10)

A temperature sensitive probe comprising a copolymer comprising a thermosensitive unit, a cationic unit and a fluorescent unit,
Comprising a first fluorescent unit and a second fluorescent unit having different maximum fluorescence wavelengths from each other;
The thermosensitive unit has the following formula (a):
Wherein R ¹ is selected from a hydrogen atom and C _1-3 alkyl;
R ⁴ and R ⁵ are independently selected from a hydrogen atom and C _1-20 alkyl, wherein the alkyl is substituted with one or more substituents selected from hydroxy, C _1-6 alkoxy, and aryl Or R ⁴ and R ⁵ together with the nitrogen atom to which they are attached form a 4- to 8-membered nitrogen-containing heterocycle, where the heterocycle is C _1-6 alkyl, C _1-6 alkoxy, nitro, halogen atom, optionally substituted by one or more substituents selected from C _1-10 alkylcarbonylamino and arylcarbonylamino]
Is one or two or more repeating structures derived from one or more monomers represented by:
The cationic unit is represented by the following formula (b):
[Wherein R ² is selected from a hydrogen atom and C _1-3 alkyl;
W is an aromatic ring or —X ¹ —C (═O) — (where X ¹ is directly attached to Q ¹ );
X ¹ is O, S, or N—R ¹¹ ;
R ¹¹ is a hydrogen atom, C _1-6 alkyl, or —Q ¹ -Y, wherein the alkyl is hydroxy, halogen atom, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkyl Optionally substituted by one or more substituents selected from sulfinyl, and C _1-6 alkylsulfonyl;
Q ¹ is independently selected from C _1-20 alkylene, C _3-20 alkenylene, or C _3-20 alkynylene, wherein the alkylene is O, S, —O—P () at one or more points. ═O) (— OH) —O— or phenylene may be independently inserted;
Y is independently an ionic functional group that may have one or more positive charges, and is —N ⁺ R ²¹ R ²² R ²³ X _e ⁻ , —C (═NR ⁴¹ ) —NR ⁴² R ⁴³ , and The following formula:
Selected from the group represented by
X _e ⁻ and X _f ⁻ are counter anions;
R ²¹ , R ²² , and R ²³ are independently selected from C _1-10 alkyl and C _7-14 aralkyl, or R ²¹ and R ²² together with the nitrogen atom to which A 7-membered nitrogen-containing heterocycle may be formed;
R ²⁴ is C _1-10 alkyl, or C _7-14 aralkyl;
R ⁴¹ , R ⁴² and R ⁴³ are independently selected from a hydrogen atom and C _1-10 alkyl, or R ⁴¹ and R ⁴² together with the nitrogen and carbon atoms to which each is attached, nitrogen atom may form a 5- to 7-membered heterocyclic ring containing or R ⁴² and R ^43, together with the nitrogen atom to which they are attached may form a 5- to 7-membered nitrogen-containing heterocycle]
A temperature-sensitive probe, which is one or more repeating structures derived from one or more monomers represented by the formula:   The temperature-sensitive probe according to claim 1, wherein the first fluorescent unit and the second fluorescent unit generate fluorescence having different maximum fluorescence wavelengths by irradiation with excitation light having the same wavelength.   The temperature-sensitive probe according to claim 1, wherein the difference between the maximum fluorescence wavelength of the first fluorescence unit and the maximum fluorescence wavelength of the second fluorescence unit is 50 nm or more.   Either one of the first fluorescent unit and the second fluorescent unit increases in fluorescence intensity as the temperature increases, and the other increases or decreases in fluorescence intensity as the temperature increases. The temperature sensitive probe of claim 1, wherein the temperature sensitive probe is one that is and preferably descends. The first fluorescent unit has the following formula (c):
[Wherein R ³ is selected from a hydrogen atom and C _1-3 alkyl;
X ² is O, S, or N—R ¹² ;
X ³ is a direct bond, O, S, SO, SO ₂ , N (—R ¹³ ), CON (—R ¹⁶ ), N (—R ¹⁶ ) CO, N (—R ¹⁷ ) CON (—R ¹⁸ ). , SO ₂ N (—R ¹⁹ ) or N (—R ¹⁹ ) SO ₂ ;
Q ² is selected from C _1-20 alkylene, C _3-20 alkenylene, or C _3-20 alkynylene, wherein the alkylene has O, S, or phenylene independently inserted at one or more points. Well;
Ar is selected from a 6 to 18-membered aromatic carbocyclic group or a 5 to 18-membered aromatic heterocyclic group, wherein the aromatic carbocyclic group and the aromatic heterocyclic group include at least one of the included rings. A condensed ring which is an aromatic ring may be included, and —CH ₂ — present as a ring atom in the aromatic carbocyclic group and aromatic heterocyclic group may be substituted with —C (O) —. In addition, the aromatic carbocyclic group and aromatic heterocyclic group include a halogen atom, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylsulfinyl, C _1-6 alkylsulfonyl. , nitro, _{cyano, C 1-6 alkylcarbonyl, C 1-6} alkoxycarbonyl, carboxy, formyl, ^-NR 6 ^{R 7,} and by one or more substituents selected from _-SO 2 ^NR 14 ^{R 15} May be conversion (the _{C 1-6} alkyl, _{where, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, C 1-6} alkylcarbonyl and C _The alkyl contained in _1-6 alkoxycarbonyl is 1 selected from a halogen atom, C _1-6 alkoxy, hydroxy, amino, C _1-6 alkylamino, di (C _1-6 alkyl) amino, aryl, and carboxy. Optionally substituted by the above substituents);
R ⁶ and R ⁷ independently represent a hydrogen atom, C _1-10 alkyl, aryl, C _1-10 alkylcarbonyl, arylcarbonyl, C _1-10 alkylsulfonyl, arylsulfonyl, carbamoyl, N— (C _1-10 Alkyl) carbamoyl, and N, N-di (C _1-10 alkyl) carbamoyl, wherein said C _1-10 alkyl, C _1-10 alkylcarbonyl, C _1-10 alkylsulfonyl, N- (C _{1 -10} alkyl) carbamoyl and alkyl contained in N, N-di (C _1-10 alkyl) carbamoyl are a halogen atom, C _1-6 alkoxy, hydroxy, amino, C _1-6 alkylamino, di (C _{1 -6} alkyl) location amino, aryl, and optionally one or more substituents selected from carboxy May be aryl contained further in the aryl, arylcarbonyl and arylsulfonyl, a substituted, halogen atom, C _1-6 alkyl, C _1-6 alkoxy, and by one or more substituents selected from carboxy Or R ⁶ and R ⁷ together with the nitrogen atom to which they are attached form a 4- to 8-membered nitrogen-containing heterocycle, where the heterocycle is C _1-6 alkyl, Optionally substituted by one or more substituents selected from C _1-6 alkoxy, nitro, halogen atoms, C _1-10 alkylcarbonylamino and arylcarbonylamino;
R ¹² is a hydrogen atom, C _1-6 alkyl, or —Q ² —X ³ —Ar, wherein the alkyl is hydroxy, halogen atom, C _1-6 alkoxy, C _1-6 alkylthio, C ₁ Optionally substituted by one or more substituents selected from _-6 alkylsulfinyl, and C _1-6 alkylsulfonyl;
R ¹³ is hydrogen atom or _{C 1-6} alkyl, wherein said alkyl is hydroxy, halogen _{atom, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6} alkylsulfinyl, and _{C 1-} Optionally substituted by one or more substituents selected from ₆ alkylsulfonyl;
R ¹⁴ and R ¹⁵ are independently selected from a hydrogen atom and C _1-6 alkyl; or R ¹⁴ and R ¹⁵ together with the nitrogen atom to which they are attached, a 4- to 8-membered nitrogen-containing heterocycle Forming;
R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ are independently selected from a hydrogen atom and C _1-6 alkyl, wherein the alkyl is hydroxy, halogen atom, C _1-6 alkoxy, C _1-6 Optionally substituted by one or more substituents selected from alkylthio, C _1-6 alkylsulfinyl, and C _1-6 alkylsulfonyl]
Is a repeating structure derived from a monomer represented by
The second fluorescent unit is represented by the following formula (d):
Wherein R ⁵⁵ is selected from a hydrogen atom and C _1-3 alkyl;
R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ are independently selected from a hydrogen atom and C _1-6 alkyl;
X ⁴ is a direct bond, phenylene, —Q ⁴ —O—C (═O) — (where Q ⁴ is directly bonded to the boron dipyrromethene skeleton), or —Q ⁴ —N (—R ⁶¹ ) —C (═O) — (wherein Q ⁴ is directly bonded to the boron dipyrromethene skeleton);
R ⁶¹ is selected from a hydrogen atom and C _1-6 alkyl;
Q ⁴ is selected from C _1-20 alkylene, phenylene, and naphthylene, wherein the phenylene and naphthylene are substituted by one or more substituents selected from a halogen atom, C _1-6 alkoxy, hydroxy, amino, and carboxy. It may be]
The temperature-sensitive probe according to claim 1, which has a repeating structure derived from a monomer represented by the formula: The copolymer is of formula (I), formula (II), formula (III) and formula (XIII):
[Wherein R ¹ , R ⁴ and R ⁵ , and R ² , Y, W and Q ¹ are as defined in claim 1, and R ³ , X ² , X ³ , Q ² and Ar, and R ⁵⁵ , X ⁴ , R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ are as defined in claim 5, and a, b, c and d are numbers greater than 0 representing the ratio of each repeating unit. is there]
The temperature-sensitive probe according to any one of claims 1 to 5, which comprises a repeating unit represented by: wherein the sum of a and b is 100, and b is 2 to 10.   The temperature-sensitive probe according to any one of claims 1 to 6, wherein the copolymer includes two or more types of heat-sensitive units represented by the formula (a). Formula (I), Formula (II), Formula (III) and Formula (XIII):
[Wherein R ¹ , R ⁴ and R ⁵ , and R ² , Y, W and Q ¹ are as defined in claim 1, and R ³ , X ² , X ³ , Q ² and Ar, and R ⁵⁵ , X ⁴ , R ⁵¹ , R ⁵² , R ⁵³ and R ⁵⁴ are as defined in claim 5, and a, b, c and d are numbers greater than 0 representing the ratio of each repeating unit. is there]
A copolymer comprising a repeating unit represented by: wherein the sum of a and b is 100 and b is 2 to 10. A method for measuring the temperature in a cell,
(A) a step of introducing the temperature-sensitive probe into a cell by mixing the temperature-sensitive probe according to any one of claims 1 to 7 with the cell in a solvent;
(B) a step of measuring the intensity of fluorescence derived from each of the first fluorescent unit and the second fluorescent unit under irradiation with excitation light; and (c) calculating a ratio of the two measured fluorescence intensities. A method comprising the steps.   A kit for measuring a temperature in a cell, comprising the temperature-sensitive probe according to any one of claims 1 to 7 or the copolymer according to claim 8.