JP5441098B2 - Fluorescent molecular thermometer - Google Patents

Description

  The present invention relates to a novel fluorescent polymer and a temperature measurement method using the polymer.

  The use of molecules whose physical properties change with changes in temperature as a temperature sensor has been studied (Non-Patent Documents 1 to 5). As an example, a fluorescent temperature sensor based on the principle of incorporating a 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 6). 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 7). 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. Such a temperature sensor has attracted attention in recent years because it has a minimum functional unit of one molecule and enables temperature measurement in a minute space such as inside a microreactor.

The use of polyacrylamide as a logic gate that responds to the temperature and pH of the surrounding environment has also been studied (Non-Patent Document 9).
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 Anal. Chem. 2004, 76, 1793-1798. J. et al. Am. Chem. Soc. 2004, 126, 3032-3033

  Regarding fluorescent temperature sensors using linear or cross-linked polyacrylamide with a built-in fluorophore, already reported polymers have the phenomenon that the fluorescence intensity at the same temperature is different when the temperature rises and falls. It has been confirmed that it has a problem that the temperature cannot be uniquely determined with respect to the fluorescence intensity. In addition, these polymers have a problem that the temperature range that can be measured is narrow because the thermal phase transition of polyacrylamide is sensitive.

  Furthermore, since these polymers aggregate in a solution having a salt concentration of a certain level or more, they are not suitable for temperature measurement in a reaction system containing cells or in cells. For example, Non-Patent Document 7 The disclosed microgel has a problem of salting out with increasing temperature in a phosphate buffer (PBS).

  The inventor has conducted extensive research to solve the above problems, and has completed the present invention which can be used as a more practical fluorescent temperature sensor by introducing an ionic unit into the polymer. I let you.

  That is, according to one aspect of the present invention, the 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 optionally substituted by one or more substituents selected from alkoxy, nitro, halogen atom, C _1-10 alkylcarbonylamino and arylcarbonylamino]
And a monomer represented by formula (b):

[Wherein R ² is selected from a hydrogen atom and C _1-3 alkyl;
X ¹ is O, S, or N—R ¹¹ ;
R ¹¹ is a hydrogen atom, C _1-6 alkyl, or —Q ¹ -Y, where 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 independently inserted with O, S or phenylene at one or more points. May be;
Y is independently an ionic functional group that can have one or more positive or negative charges, and is —SO ₃ X _a , —PO (OX _b ) (OX _c ), —COOX _d , —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
Wherein X _a , X _b , X _c , and X _d are independently selected from a hydrogen atom or a counter cation, and 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 they are 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]
And / or a crosslinker monomer containing two or more double bonds in the molecule: and 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) —. Furthermore, 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. Substituted by one or more substituents selected from nitro, cyano, C _1-6 alkylcarbonyl, C _1-6 alkoxycarbonyl, carboxy, formyl, —NR ⁶ R ⁷ , and —SO ₂ NR ¹⁴ R ¹⁵ at best (the C _1-6 a where Kill, C _1-6 alkoxy, C _1-6 alkylthio, C _1-6 alkylsulfinyl, C _1-6 alkylsulfonyl, alkyl contained in C _1-6 alkylcarbonyl and C _1-6 alkoxycarbonyl, halogen atom, Optionally substituted by one or more substituents selected from C _1-6 alkoxy, hydroxy, amino, C _1-6 alkylamino, di (C _1-6 alkyl) amino, aryl, and carboxy);
R ⁶ and R ⁷ are independently 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) amino, aryl, and one or more may be substituted by a substituent selected from carboxy, further wherein the aryl, arylcarbonyl, and a Aryl contained in Rusuruhoniru a halogen atom, C _1-6 alkyl, C _1-6 alkoxy, and one or more may be substituted by a substituent selected from carboxy; or R ⁶ and R ⁷ are those Together with the nitrogen atom to be bonded, a 4- to 8-membered nitrogen-containing heterocycle is formed, where the heterocycle is a C _1-6 alkyl, C _1-6 alkoxy, nitro, halogen atom, C _1-10 Optionally substituted by one or more substituents selected from 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 with one or more substituents selected from _-6 alkylsulfinyl, and C _1-6 alkylsulfonyl;
R ¹³ is a hydrogen atom or C _1-6 alkyl, where the 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]
A copolymer containing a repeating structure derived from each of the monomers represented by the formula (provided that the copolymer does not contain a repeating structure derived from the monomer represented by the formula (b), , Prepared by a copolymerization reaction using an initiator in an amount of 7 mol% or more based on the monomer used, and the initiator is represented by the formula (d):
Y ^1- (C _1-10 alkylene) -N═N— (C _1-10 alkylene) -Y ¹
{ _Wherein C _1-10 alkylene may be independently substituted with one or more substituents selected from halogen atoms and cyano; Y ¹ is independently one or more positive or negative charges. Selected from —COOX _d , and —C (═NR ⁴¹ ) —NR ⁴² R ⁴³ ; X _d , R ⁴¹ , R ⁴² , and R ⁴³ are as defined above. Selected from azo compounds represented by the following formula: or persulfate.

  According to another aspect of the invention, formulas (I), (II) and (III):

[Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , Y, X ¹ , X ² , X ³ , Q ¹ , Q ² and Ar are as defined above]
The copolymer containing the repeating unit represented by this is provided.

  According to yet another aspect of the present invention, a copolymer as defined herein, wherein Ar is represented by the formula:

An aromatic carbocyclic group or an aromatic heterocyclic group selected from the group represented by the formula: These groups have a halogen atom, 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 with 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 contained in C _1-6 alkylcarbonyl and C _1-6 alkoxycarbonyl, halogen atom, C _1-6 alkoxy, hydroxy, amino, C _1-6 alkylamino, di (C _1-6 Al ) 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 a halogen atom, C _1-6 alkoxy, hydroxy, amino, C _1-6 alkylamino, di (C _1-6 alkyl) ) Optionally substituted by one or more substituents selected from amino, aryl, and carboxy, wherein said aryl is selected from a halogen atom, C _1-6 alkyl, C _1-6 alkoxy, and carboxy There is provided the copolymer, which may be substituted with one or more substituents.

  In one embodiment of the above aspect of the 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 have a halogen atom, 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 .

  According to another aspect of the present invention, a temperature measurement method using the above copolymer is provided. According to still another aspect of the present invention, there is provided a temperature measurement method including the step of dissolving the above copolymer in a liquid; and the step of measuring fluorescence intensity under excitation light irradiation. A temperature measurement method comprising: dissolving the above copolymer in a liquid; and measuring an average fluorescence lifetime under irradiation with excitation light. The temperature measurement method can be used, for example, for intracellular temperature measurement.

  The copolymer of the present invention can be used as a fluorescent temperature sensor that maintains high temperature resolution over a wide temperature range. Further, since salting-out does not occur even in a solution having a high salt concentration, it can be used for temperature measurement in a reaction system containing cells or in cells.

It is an example of the result of the heat-sensitive response test of the fluorescence intensity of compounds 1 to 4 (0.01 w / v% in pure water) (excitation wavelength: 456 nm; It is a schematic diagram of the relationship between the temperature of a fluorescent temperature sensor and fluorescence intensity. It is an example of the temperature resolution evaluation result (+/- standard deviation, n = 3) of the compounds 1-3. 2 is an example of a temperature resolution evaluation result of Compound 4. It is an example of the thermal sensitivity test result (0.01 w / v%, excitation wavelength 456 nm) of the fluorescence intensity of the compounds 5-7 (● compound 5, ▲ compound 6, ♦ compound 7) as comparative examples. It is an example of the result of the temperature resolution evaluation of the compounds 5-7. Nanogel 1 (0.01 w / v% in pure water, fluorescence wavelength 550 nm; when temperature rises: ●, when temperature falls: ○) and nanogel 3 (0.01 w / v% in pure water, fluorescence wavelength 554 nm; temperature) It is an example of the result of a heat-sensitive response test of fluorescence intensity when rising: ▲, when temperature falls: Δ. Nanogel 1 (0.01 w / v% in 150 mM potassium chloride aqueous solution, fluorescence wavelength 550 nm; when temperature rises: ●, when temperature falls: shows ○) and nanogel 3 (0.01 w / v% in 150 mM potassium chloride aqueous solution, fluorescence) It is an example of the result of the thermal sensitivity test of the fluorescence intensity at a wavelength of 554 nm; temperature rise: ▲, temperature drop: Δ. Nanogel 2 (0.01 w / v% in pure water, fluorescence wavelength 555 nm; when temperature rises: ●, when temperature falls: indicates ○) and Nanogel 4 (0.01 w / v% in pure water, fluorescence wavelength 558 nm; temperature) It is an example of the result of a heat-sensitive response test of fluorescence intensity when rising: ▲, when temperature falls: Δ. Nanogel 2 (0.01 w / v% in 150 mM potassium chloride aqueous solution, fluorescence wavelength 550 nm; when temperature rises: ●, when temperature falls: shows ○) and nanogel 4 (0.01 w / v% in 150 mM potassium chloride aqueous solution, fluorescence) It is an example of the result of a thermal response test of fluorescence intensity at a wavelength of 558 nm; temperature rise: ▲, temperature drop: △. It is an example of the temperature resolution evaluation result of the fluorescence intensity of Nanogel 2 (0.01 w / v% in pure water, fluorescence wavelength 555 nm) and Nanogel 4 (0.01 w / v% in pure water, fluorescence wavelength 558 nm). It is an example of the temperature resolution evaluation result of Nanogel 2 (0.01 w / v% in 150 mM potassium chloride aqueous solution, fluorescence wavelength 550 nm) and Nanogel 4 (0.01 w / v% in 150 mM potassium chloride aqueous solution, fluorescence wavelength 558 nm). It is an example of the result of the thermal sensitivity test of the fluorescence intensity of the nanogel 5 (pure water 0.01 w / v%, fluorescence wavelength 550 nm) (when temperature rises: ●, when temperature falls: ◯). It is an example of the temperature resolution evaluation result of the nanogel 5 (0.01 w / v% in pure water, fluorescence wavelength 550 nm). Results of thermal response test of nanogel 3 (all 0.001 w / v%, excitation wavelength 456 nm, fluorescence wavelength 560 nm) in 0, 50, 100, 150, and 200 mM potassium chloride aqueous solution (● in water, ▲ 50 mM KCl , ◆ in 100 mM KCl, ■ in 150 mM KCl, in ■ 200 mM KCl). It is an example of the photograph of the fluorescence image (Excitation wavelength 488nm, Fluorescence wavelength 515-550nm, Medium temperature about 37 degreeC) of the living cell containing the nanogel 3. FIG. It is an example of the photograph which shows the change of the fluorescence image (excitation wavelength 488nm, fluorescence wavelength 515-550nm) of the living cell containing the nanogel 3 at the time of changing culture medium temperature. It is an example of the result of the thermosensitive response test (n = 4) of the fluorescence intensity of the nanogel 3 (excitation wavelength 488 nm, fluorescence wavelength 515-550 nm) in a living cell, and the fluorescence intensity in each temperature has a value of 37 degreeC as 1. The relative intensity is shown. It is an example of the result of the reversibility confirmation test of the fluorescence response of the nanogel 3 (excitation wavelength 488 nm, fluorescence wavelength 515-550 nm) in a living cell, and the fluorescence intensity in each temperature is relative to the first value as 1. Shown as strength. It is an example of the result of the thermal response test of the fluorescence lifetime of the compound 3 (0.02 w / v% in pure water, excitation wavelength 275 nm, fluorescence wavelength 560 nm).

BEST MODE FOR CARRYING OUT THE INVENTION

  Hereinafter, the present invention will be described more specifically.

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

In the present specification, “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, the previously defined C _1-6 Alkyl and C _1-3 alkyl and the like are included.

The "C _1-20 alkyl" as used herein, refers to a linear, branched, cyclic or partially cyclic alkyl group having 1 to 20 carbon atoms, e.g., C _1-10 previously defined 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 a C _1-6 alkyl group which has already been defined as an alkyl moiety, and includes, 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. And further include C _1-10 alkylene and C _1-6 alkylene.

As used herein, “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 and C _3-6 alkynylene are included.

In the present specification, “C _1-6 alkylthio” means an alkylthio group having an alkyl group having 1 to 6 carbon atoms already defined as an alkyl moiety, such as methylthio, ethylthio, n-propylthio, i-propylthio, n-butylthio, s-butylthio, i-butylthio, t-butylthio and the like are included.

As used herein, “C _1-6 alkylsulfinyl” means an alkylsulfinyl group having an alkyl group having 1 to 6 carbon atoms already defined as the alkyl moiety, and examples thereof include methylsulfinyl, ethylsulfinyl, n-propylsulfinyl. I-propylsulfinyl, n-butylsulfinyl, s-butylsulfinyl, i-butylsulfinyl, t-butylsulfinyl and the like.

In the present specification, “C _1-6 alkylsulfonyl” means an alkylsulfonyl group having an alkyl group having 1 to 6 carbon atoms already defined as an alkyl moiety, such as 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.

In the present specification, “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 that 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 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 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 easily understand that the process is performed so as not to form the structure of O— and —O—CH ₂ —O—. The above also applies to the insertion of S into alkylene.

In the present specification, the “counter cation” is not particularly limited as long as it is a cation usually used as a counter cation of an organic compound in the technical field of organic chemistry. For example, ammonium ion, alkali metal ion (for example, sodium, lithium and Potassium ions), alkaline earth metal ions (eg, calcium ions, barium ions), substituted ammonium cations (eg, N- (C _1-6 alkyl) pyridinium cations, tetra (C _1-10 alkyl) ammonium cations), and the like. included. Preferred counter cations in the present invention include, for example, sodium ion, potassium ion, tetramethylammonium ion and the like.

  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 anions (eg, acetic acid, trifluoroacetic acid), nitrate ions, sulfate ions, carbonate ions, and the like. Preferred counter anions in the present invention include, for example, chloride ions and nitrate ions.

  In addition, when the counter cation or the counter anion is divalent or more, as will be easily understood by those skilled in the art, an ionic bond is formed with the corresponding number of ionic functional groups.

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

In formula (a), formula (I) and (IV), —NR ⁴ R ⁵ is not particularly limited. For example, R ⁴ may be a hydrogen atom, and 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.

-X ¹ -Q ^1- in Formula (b), Formula (II) and (IV) is preferably X ¹ is O, NH or N (C _1-6 alkyl), and Q ¹ is C _{2 -10} alkylene.

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

  -Ar in the formulas (c), (III) and (IV) is preferably the following formulas (V) to (XII):

［式中、Ｒ³¹は水素原子、ハロゲン原子、ニトロ、シアノ、および−ＳＯ₂ＮＲ¹⁴Ｒ¹⁵から選択され；Ｒ³²はＣ_1-6アルキルであり；Ｘ¹¹は、Ｎ−Ｒ³³、ＯまたはＳであり；Ｒ³³は水素原子またはＣ_1-6アルキルであり；Ｘ¹⁰、Ｒ¹⁴およびＲ¹⁵は、既に定義したとおりである］
から選択される基である。

[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) is, 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 (preferable 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) is, 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 the 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 as the temperature increases. 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.

  The amount of the monomer of the formula (b) to be used is not particularly limited. For example, the amount is 0.01 to 20 mol% based on the amount of the monomer of the formulas (a), (b) and (c). Can be used.

  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 that case is not particularly limited, and examples thereof include α, α′-azobisisobutyronitrile, benzoyl peroxide, and ammonium persulfate.

However, when the copolymer of the present invention does not contain a repeating structure derived from the monomer represented by the formula (b), the copolymer starts reaction in an amount of 7 mol% or more based on the monomer used. Prepared by a copolymerization reaction using an agent, and the reaction initiator is represented by the formula (d):
Y ^1- (C _1-10 alkylene) -N═N— (C _1-10 alkylene) -Y ¹
{Wherein Y ¹ is independently an ionic functional group that may have one or more positive or negative charges, and the C _1-10 alkylene is independently selected from a halogen atom and cyano. Are optionally substituted with the above substituents} or a persulfate. Specific examples of the reaction initiator include persulfates such as ammonium persulfate, sodium persulfate, and potassium persulfate; 2,2′-azobis (2-methylpropionamidine) dihydrochloride, 2,2′-azobis [ And azo compounds such as 2- (2-imidazolin-2-yl) propane] dihydrochloride and 4,4′-azobis (4-cyanovaleric acid). By using these reaction initiators, a polymer into which an ionic functional group derived from the reaction initiator is introduced can be obtained.

  The amount of the reaction initiator used may be an amount of 7 mol% or more with respect to the monomer to be used (including the crosslinking agent monomer), for example, 14 mol% or more, preferably 25 mol% or more of the reaction initiator. 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.

  The crosslinking agent monomer used in the present invention is not particularly limited as long as it is a monomer containing two or more vinyl groups in the molecule and is usually used as a crosslinking agent. Examples of the crosslinking agent include the formula (e):

［式中、Ｒ⁹およびＲ¹⁰は、独立に、水素原子およびＣ_1-3アルキルから選択され；
Ｘ⁴およびＸ⁵は、独立に、Ｏ、Ｓ、またはＮ−Ｒ¹²であり；
Ｑ³は、Ｃ_1-20アルキレン、Ｃ_3-20アルケニレン、またはＣ_3-20アルキニレンから選択され、ここで前記アルキレンは、１以上の個所において、Ｏ、Ｓまたはフェニレンが独立に挿入されていてもよい］
で表される化合物、ジビニルスルホンおよびＣ_1-10アルキレンビスマレイミドから選択される化合物を使用することができ、具体的には、式（ｅ）｛式中、Ｘ⁴およびＸ⁵は、独立に、ＮＨまたはＮ（Ｃ_1-6アルキル）であり、Ｑ³は、Ｃ_1-6アルキレンである｝の化合物が挙げられる。架橋剤モノマーのより具体的な例としては、Ｎ，Ｎ'−メチレンビスアクリルアミド、Ｎ，Ｎ'−エチレンビスアクリルアミド、Ｎ，Ｎ'−メチレンビスメタクリルアミド、Ｎ，Ｎ'−エチレンビスメタクリルアミド、エチレングリコールジアクリレート、エチレングリコールジメタクリレートなどが挙げられる。

Wherein R ⁹ and R ¹⁰ are independently selected from a hydrogen atom and C _1-3 alkyl;
X ⁴ and X ⁵ are independently O, S, or N—R ¹² ;
Q ³ 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. It is good]
Or a compound selected from divinylsulfone and C _1-10 alkylene bismaleimide, specifically, the compound represented by formula (e) {wherein X ⁴ and X ⁵ are independently , NH or N (C _1-6 alkyl), and Q ³ is C _1-6 alkylene}. More specific examples of the crosslinking agent monomer include N, N′-methylenebisacrylamide, N, N′-ethylenebisacrylamide, N, N′-methylenebismethacrylamide, N, N′-ethylenebismethacrylamide, Examples include ethylene glycol diacrylate and ethylene glycol dimethacrylate.

  The amount of the crosslinking agent monomer to be used is not particularly limited. For example, an amount of 0.1 to 20 mol% can be used with respect to the monomers of the formulas (a), (b) and (c). .

  The monomer used in the production of the copolymer of the present invention can be obtained by purchase, the synthesis method is known from the literature, can be easily prepared based on the known synthesis method, or the following It can be synthesized based on the description of the examples.

  The copolymerization reaction in the case of using a crosslinking agent monomer can be performed by a method commonly used in the technical field. The reaction solvent used for the copolymerization reaction is not particularly limited. For example, a surfactant (for example, sodium dodecyl sulfate, sodium dodecylbenzene sulfate, sodium pentadecane sulfate, N-dodecyl-N, N, N-trimethylammonium). Water containing bromide, N-cetyl-N, N, N-trimethylammonium bromide, Triton X-100, etc.) can be used. The size of the copolymer nanogel obtained using the crosslinker monomer is controlled by the stirring efficiency, reaction temperature, amount of surfactant used, amount of initiator used, and amount of crosslinker monomer used in the copolymerization reaction. can do. For example, a nanogel having a small size can be obtained by increasing the amount of surfactant and / or initiator used. A person skilled in the art to which the present invention belongs can appropriately adjust the size of the obtained nanogel, and the size of the nanogel of the copolymer of the present invention is, for example, 5 to 100 nm. The copolymerization reaction 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 for example, a reaction time of 1 to 48 hours, preferably 2 to 16 hours.

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 ionic functional group contained in the copolymer maintains ionicity in a wide range of pH. Is preferable. From such a viewpoint, Y is preferably —SO ₃ X _a or —N ⁺ R ²¹ R ²² R ²³ X _e ^— . Furthermore, an ionic functional group can also be introduced by using a specific reaction initiator in the copolymerization reaction. Thus the same reason, initiators, persulfate may be introduced -SO ₃ X _a is preferable.

  In one aspect of the present invention, a copolymer including a repeating structure derived from each of the monomer represented by the formulas (a), (b), and (c) and the crosslinking agent monomer is provided. Here, the amount of the monomer represented by the formula (b) used for obtaining the copolymer is the total amount of the monomers represented by the formulas (a), (b) and (c). On the other hand, it is 1-20 mol%, for example, Preferably it is 2-15 mol%, More preferably, it is 2.5-10 mol%. Further, the amount of the monomer represented by the formula (c) used is, for example, 0.01 to about the total amount of the monomers represented by the formulas (a), (b) and (c). It is 20 mol%, specifically 0.01 to 10 mol%, preferably 0.05 to 2 mol%, more preferably 0.1 to 1 mol%. Moreover, the amount of the crosslinking agent monomer used is, for example, 0.1 to 20 mol% with respect to the total amount of the monomers represented by the formulas (a), (b) and (c), Preferably it is 0.5-10 mol%, More preferably, it is 1-5 mol%.

  In another aspect of the present invention, there is provided a copolymer comprising a repeating structure derived from each of the monomers represented by the formulas (a), (b) and (c). Here, the amount of the monomer represented by the formula (b) used for obtaining the copolymer is the total amount of the monomers represented by the formulas (a), (b) and (c). On the other hand, it is 1-20 mol%, for example, Preferably it is 2-15 mol%, More preferably, it is 2.5-10 mol%. Further, the amount of the monomer represented by the formula (c) used is, for example, 0.01 to about the total amount of the monomers represented by the formulas (a), (b) and (c). It is 20 mol%, specifically 0.01 to 10 mol%, preferably 0.05 to 2 mol%, more preferably 0.1 to 1 mol%.

  In still another aspect of the present invention, the copolymer includes a repeating structure derived from each of the monomer represented by the formulas (a) and (c) and the crosslinking agent monomer, and the copolymer is 7 to the monomer used. The copolymer prepared by a copolymerization reaction using an initiator in an amount of mol% or more, wherein the initiator is selected from the azo compound represented by the formula (d) or a persulfate salt. Provided. Here, the amount of the monomer represented by the formula (c) used for obtaining the copolymer is, for example, with respect to the total amount of the monomers represented by the formulas (a) and (c). 0.01 to 20 mol%, specifically 0.01 to 10 mol%, preferably 0.05 to 2 mol%, more preferably 0.1 to 1 mol%. Moreover, the amount of the crosslinking agent monomer used is, for example, 0.1 to 20 mol% with respect to the total amount of the monomers represented by the formulas (a), (b) and (c), Preferably it is 0.5-10 mol%, More preferably, it is 1-5 mol%.

  In one aspect of the present invention, a, b and c in formula (IV) are numbers greater than 0 representing the ratio of each repeating unit in the formula, and are not particularly limited. For example, a + b + c = 100 , C is 0.01-20, specifically 0.01-10, preferably 0.05-2, more preferably 0.1-1. For example, b is 1-20, Preferably it is 2-15, More preferably, it is 2.5-10. Moreover, the weight average molecular weight of the polymer of this invention is although it does not specifically limit, For example, it is 5000-1 million, Preferably it is 10000-200000.

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

  The change in fluorescence lifetime due to the thermal sensitivity of the copolymer of the present invention is measured by a general fluorescence lifetime measurement method, for example, a single photon counting method, a phase modulation method, a pulse sampling method, an excitation probe method, or the like. be able to. Among these, the single photon counting method is a method for measuring the fluorescence lifetime by utilizing the fact that the emission intensity distribution on the time axis and the emission probability of one photon are in correlation, and the fluorophore is about 1 ns. After excitation with very short (pulsed) light, the generation time of the detected light is measured, and the fluorescence lifetime is determined by approximating the histogram obtained by repeating excitation many times as a fluorescence decay curve with the sum of exponential functions. . The fluorescence lifetime is measured by the single photon counting method. A commercially available time-correlated single photon counting fluorescence lifetime measuring apparatus (for example, Edinburgh Analytical Instruments FL-900CDT, see Examples) and accompanying measurement and analysis are used. Can be done using a program.

  The copolymer of the present invention can be used for temperature measurement inside cells. The method for introducing the copolymer into the cell is not particularly limited. For example, it may be possible to introduce the copolymer by culturing the cell in a medium to which the copolymer is added. Other methods such as methods using positively charged lipids (such as lipofection methods), electrical methods (such as electroporation methods), chemical methods (such as calcium phosphate methods), and physical methods (such as microinjection methods) are used. May be.

  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 N-isopropylacrylamide was purified by recrystallization using n-hexane. Nn-propyl acrylamide was synthesized and purified according to a general synthesis procedure of N-alkyl acrylamide. α, α′-Azobisisobutyronitrile was used after being purified by recrystallization using methanol. 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. Mass spectrometry was performed using a JEOL MStation JMS-700 spectrometer, and elemental analysis was performed using a Yanaco MT-6 CHN CORDER spectrometer. The melting point was measured using a Yanagimoto Micro Melting Point Apparatus and no correction was made. Number average molecular weight and 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]
Synthesis of N- {2-[(7-N, N-dimethylaminosulfonyl) -2,1,3-benzooxadiazol-4-yl] (methyl) amino} ethyl-N-methylacrylamide

４−Ｎ，Ｎ−ジメチルアミノスルホニル−７−フルオロ−２，１，３−ベンゾオキサジアゾール（９３ｍｇ）をアセトニトリル（５ｍｌ）に溶解し、この溶液をＮ，Ｎ'−ジメチルエチレンジアミン（１．２９ｍｌ）に加えて１５分間室温にて撹拌した。反応終了後、反応溶液を減圧乾固させた後、シリカゲルカラムクロマトグラフィー（展開溶媒としてジクロロメタン−メタノール＝１０：１→５：１を使用）にて精製を行い、Ｎ，Ｎ−ジメチル−７−［メチル｛２−（メチルアミノ）エチル｝アミノ］−２，１，３−ベンゾオキサジアゾール−４−スルホンアミドを橙色液体（１１８ｍｇ、＞９９％）として得た。

4-N, N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzooxadiazole (93 mg) was dissolved in acetonitrile (5 ml), and this solution was dissolved in N, N′-dimethylethylenediamine (1.29 ml). ) And stirred at room temperature for 15 minutes. After completion of the reaction, the reaction solution was dried under reduced pressure and purified by silica gel column chromatography (dichloromethane-methanol = 10: 1 → 5: 1 used as a developing solvent), and N, N-dimethyl-7- [Methyl {2- (methylamino) ethyl} amino] -2,1,3-benzooxadiazole-4-sulfonamide was obtained as an orange liquid (118 mg,> 99%).

¹ H-NMR (CDCl ₃ ) δ 7.87 (1H, d, J = 8.3 Hz), 6.09 (1H, d, J = 8.3 Hz), 4.15 (2H, t, J = 13. 4 Hz), 3.35 (3H, s), 2.94 (2H, t, J = 13.4 Hz) 2.87 (6H, s), 2.48 (3H, s); HR-FAB-MS: m / z calcd _{(C 12 H 20 N 5 O} 3 S) [M + H] + 314.1287; measured 314.1271.

N, N-dimethyl-7- [methyl {2- (methylamino) ethyl} amino] -2,1,3-benzooxadiazole-4-sulfonamide (130 mg) was dissolved in acetonitrile (13 ml) and triethylamine was dissolved. (57.8 μL, 1 eq) and acrylic acid chloride (43.8 μL, 1.3 eq) were added at 0 ° C., and the mixture was stirred at room temperature for 1 hour and a half. Na ₂ CO ₃ (1 g) was added to the reaction solution, filtered, and then dried under reduced pressure, and purified by silica gel column chromatography (using ethyl acetate-n-hexane = 3: 1 as a developing solvent). The compound was obtained as orange crystals (88 mg, 58%).

Melting point 144-146 ° C .; ¹ H-NMR (CDCl ₃ ) δ 7.88 (1H, d, J = 8.3 Hz), 6.48 (1H, dd, J = 16.7 Hz, 10.4 Hz), 6. 28 (1H, d, J = 16.7 Hz), 6.14 (1 H, d, J = 8.3 Hz), 5.68 (1 H, d, J = 10.4 Hz) 4.27 (2H, t, J = 13.9 Hz), 3.74 (2H, t, J = 13.9 Hz), 3.35, 3.23 (3H, s), 3.13, 3.12 (3H, s), 2. 90,2.87 (6H, s); HR -FAB-MS: m / z calcd _{(C 15 H 22 N 5 O} 3 S) [M + H] + 368.1393; measured 368.1422. Calcd _{(C 15 H 21 N 5 O} 3 S) C: 49.03, H: 5.76, N: 19.06; measurement C: 49.19, H: 5.78, N: 18.52.
[Example 2]
Nt-butylacrylamide / 3-sulfopropyl acrylate / N- {2-[(7-N, N-dimethylaminosulfonyl) -2,1,3-benzooxadiazol-4-yl] (methyl) amino } Production of ethyl-N-methylacrylamide copolymer

Ｎ−ｔ−ブチルアクリルアミド（５７２ｍｇ、４．５ｍｍｏｌ、以下、ＮＴＢＡＭとも称する）、３−スルホプロピル アクリレートカリウム塩（１１６ｍｇ、５００μｍｏｌ、以下、ＳＰＡカリウム塩とも称する）、Ｎ−｛２−［（７−Ｎ，Ｎ−ジメチルアミノスルホニル）−２，１，３−ベンゾオキサジアゾール−４−イル］（メチル）アミノ｝エチル−Ｎ−メチルアクリルアミド（１．８４ｍｇ、５μｍｏｌ、以下、ＤＢＤ−ＡＡとも称する）、α,α'−アゾビスイソブチロニトリル（８．２１ｍｇ、５０μｍｏｌ、以下、ＡＩＢＮとも称する）をＤＭＦ（１０ｍｌ）に溶解し、３０分間窒素ガスを通じることにより溶存酸素を除去した。その後、６０℃にて１２時間反応させ、反応溶液から減圧下ＤＭＦを一部留去したのち、ジエチルエーテル（３００ｍｌ）に注いだ。得られた結晶を濾取し、再沈操作（アセトン３ｍｌ−ｎ−ヘキサン２００ｍｌ）にて精製を行い、表題の共重合体を淡黄色粉末（２６８ｍｇ、３８％）として得た。

Nt-butylacrylamide (572 mg, 4.5 mmol, hereinafter also referred to as NTBAM), 3-sulfopropyl acrylate potassium salt (116 mg, 500 μmol, hereinafter also referred to as SPA potassium salt), N- {2-[(7- N, N-dimethylaminosulfonyl) -2,1,3-benzooxadiazol-4-yl] (methyl) amino} ethyl-N-methylacrylamide (1.84 mg, 5 μmol, hereinafter also referred to as DBD-AA) , Α, α′-azobisisobutyronitrile (8.21 mg, 50 μmol, hereinafter also referred to as AIBN) was dissolved in DMF (10 ml), and dissolved oxygen was removed by passing nitrogen gas for 30 minutes. Then, it was made to react at 60 degreeC for 12 hours, and DMF was partially distilled off from the reaction solution under reduced pressure, Then, it poured into diethyl ether (300 ml). The obtained crystals were collected by filtration and purified by reprecipitation (acetone 3 ml-n-hexane 200 ml) to obtain the title copolymer as a pale yellow powder (268 mg, 38%).

Number average molecular weight 27200, weight average molecular weight 51400, composition ratio of each unit in the copolymer NTBAM: SPA: DBD-AA = 88.8: 11.2: 0.12. In addition, the ratio of NTBAM unit and SPA unit in the copolymer is the integrated value in ¹ H-NMR measurement, and the ratio of DBD-AA unit is the absorbance in methanol 4-N, N- (dimethylamino) -7-N. , N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (molar extinction coefficient at 442 nm, 10600 M ⁻¹ cm ⁻¹ ).
[Example 3]
Nn-propylacrylamide / 3-sulfopropyl acrylate / N- {2-[(7-N, N-dimethylaminosulfonyl) -2,1,3-benzooxadiazol-4-yl] (methyl) amino } Production of ethyl-N-methylacrylamide copolymer

Ｎ−ｎ−プロピルアクリルアミド（５５２ｍｇ、４．８７５ｍｍｏｌ、以下、ＮＮＰＡＭとも称する）、ＳＰＡカリウム塩（２９．０ｍｇ、１２５μｍｏｌ）、ＤＢＤ−ＡＡ（１．８４ｍｇ、５μｍｏｌ）、ＡＩＢＮ（８．２１ｍｇ、５０μｍｏｌ）をＤＭＦ（１０ｍｌ）に溶解し、３０分間窒素ガスを通じることにより溶存酸素を除去した。その後、６０℃にて１２時間反応させ、反応溶液から減圧下ＤＭＦを一部留去したのち、ジエチルエーテル（２００ｍｌ）に注いだ。得られた結晶を濾取し、再沈操作（メタノール３ｍｌ−ジエチルエーテル２００ｍｌ）にて精製を行い、表題の共重合体を淡黄色粉末（３５２ｍｇ、６０％）として得た。

Nn-propylacrylamide (552 mg, 4.875 mmol, hereinafter also referred to as NNPAM), SPA potassium salt (29.0 mg, 125 μmol), DBD-AA (1.84 mg, 5 μmol), AIBN (8.21 mg, 50 μmol) Was dissolved in DMF (10 ml), and dissolved oxygen was removed by passing nitrogen gas for 30 minutes. Then, it was made to react at 60 degreeC for 12 hours, and DMF was partially distilled off from the reaction solution under reduced pressure, Then, it poured into diethyl ether (200 ml). The obtained crystals were collected by filtration and purified by reprecipitation (methanol 3 ml-diethyl ether 200 ml) to give the title copolymer as a pale yellow powder (352 mg, 60%).

Number average molecular weight 16100, weight average molecular weight 29400, composition ratio of each unit in the copolymer NNPAM: SPA: DBD-AA = 96.2: 3.8: 0.094. The ratio of NNPAM unit and SPA unit in the copolymer is based on the integral value in ¹ H-NMR measurement, and the ratio of DBD-AA unit is the absorbance in methanol of 4-N, N- (dimethylamino) -7-N. , N-dimethylaminosulfonyl-2,1,3-benzoxadiazole.
[Example 4]
N-isopropylacrylamide / 3-sulfopropyl acrylate / N- {2-[(7-N, N-dimethylaminosulfonyl) -2,1,3-benzooxadiazol-4-yl] (methyl) amino} ethyl -N-methylacrylamide copolymer production

Ｎ−イソプロピルアクリルアミド（５５２ｍｇ、４．８７５ｍｍｏｌ、以下、ＮＩＰＡＭとも称する）、ＳＰＡカリウム塩（２９．０ｍｇ、１２５μｍｏｌ）、ＤＢＤ−ＡＡ（１．８４ｍｇ、５μｍｏｌ）、ＡＩＢＮ（８．２１ｍｇ、５０μｍｏｌ）をＤＭＦ（１０ｍｌ）に溶解し、３０分間窒素ガスを通じることにより溶存酸素を除去した。その後、６０℃にて１２時間反応させ、反応溶液から減圧下ＤＭＦを一部留去したのち、ジエチルエーテル（２００ｍｌ）に注いだ。得られた結晶を濾取し、再沈操作（メタノール４ｍｌ−ジエチルエーテル２００ｍｌ）にて精製を行い、表題の共重合体を淡黄色粉末（２２８ｍｇ、３９％）として得た。

N-isopropylacrylamide (552 mg, 4.875 mmol, hereinafter also referred to as NIPAM), SPA potassium salt (29.0 mg, 125 μmol), DBD-AA (1.84 mg, 5 μmol), AIBN (8.21 mg, 50 μmol) in DMF Dissolved in (10 ml) and dissolved nitrogen was removed by passing nitrogen gas for 30 minutes. Then, it was made to react at 60 degreeC for 12 hours, and DMF was partially distilled off from the reaction solution under reduced pressure, Then, it poured into diethyl ether (200 ml). The obtained crystals were collected by filtration and purified by reprecipitation (methanol 4 ml-diethyl ether 200 ml) to give the title copolymer as a pale yellow powder (228 mg, 39%).

Number average molecular weight 30000, weight average molecular weight 48700, composition ratio of each unit in the copolymer NIPAM: SPA: DBD-AA = 95.9: 4.1: 0.13. The ratio of NIPAM unit and SPA unit in the copolymer is based on the integrated value in ¹ H-NMR measurement, and the ratio of DBD-AA unit is the absorbance in methanol 4-N, N- (dimethylamino) -7-N. , N-dimethylaminosulfonyl-2,1,3-benzoxadiazole.
[Example 5]
Nt-butylacrylamide / (3-acrylamidopropyl) trimethylammonium / N- {2-[(7-N, N-dimethylaminosulfonyl) -2,1,3-benzooxadiazol-4-yl] ( Production of methyl) amino} ethyl-N-methylacrylamide copolymer

ＮＴＢＡＭ（５７２ｍｇ、４．５ｍｍｏｌ）、（３−アクリルアミドプロピル）トリメチルアンモニウムクロリド（１０３ｍｇ、５００μｍｏｌ、以下、ＡＰＴＭＡクロリドとも称する）、ＤＢＤ−ＡＡ（１．８４ｍｇ、５μｍｏｌ）、ＡＩＢＮ（８．２１ｍｇ、５０μｍｏｌ）をＤＭＦ（１０ｍｌ）に溶解し、３０分間窒素ガスを通じることにより溶存酸素を除去した。その後、６０℃にて１２時間反応させ、反応溶液から減圧下ＤＭＦを一部留去したのち、ジエチルエーテル（３００ｍｌ）に注いだ。得られた結晶を濾取し、再沈操作（アセトン４ｍｌ−ヘキサン３００ｍｌ）にて精製を行い、淡黄色粉末（２１８ｍｇ）を得た。この粉末９７．８ｍｇを透析によりさらに精製し、表題の化合物（２５．９ｍｇ、３．８％）を得た。なお、上記透析は、直径２８．６ｍｍのヴィスキングチューブ（透析用セルローズチューブ）を使用し、得られた粉末を１０ｍｌの純水に溶解してチューブに入れ、透析外液を１０００ｍｌとして冷蔵庫内で４８時間透析、その間、約１２時間ごとに透析外液を純水に交換して行った。

NTBAM (572 mg, 4.5 mmol), (3-acrylamidopropyl) trimethylammonium chloride (103 mg, 500 μmol, hereinafter also referred to as APTMA chloride), DBD-AA (1.84 mg, 5 μmol), AIBN (8.21 mg, 50 μmol) Was dissolved in DMF (10 ml), and dissolved oxygen was removed by passing nitrogen gas for 30 minutes. Then, it was made to react at 60 degreeC for 12 hours, and DMF was partially distilled off from the reaction solution under reduced pressure, Then, it poured into diethyl ether (300 ml). The obtained crystals were collected by filtration and purified by reprecipitation (acetone 4 ml-hexane 300 ml) to obtain a pale yellow powder (218 mg). 97.8 mg of this powder was further purified by dialysis to give the title compound (25.9 mg, 3.8%). The above dialysis uses a Visking tube (dialysis cellulose tube) with a diameter of 28.6 mm. The obtained powder is dissolved in 10 ml of pure water and placed in the tube. During dialysis for 48 hours, the dialysis solution was replaced with pure water every 12 hours.

Number average molecular weight 4300, weight average molecular weight 5450, composition ratio of each unit in the copolymer NTBAM: APTMA: DBD-AA = 85.3: 14.7: 0.086. The ratio of NTBAM unit and APTMA unit in the copolymer is the integrated value in ¹ H-NMR measurement, and the ratio of DBD-AA unit is the absorbance in methanol 4-N, N- (dimethylamino) -7-N. , N-dimethylaminosulfonyl-2,1,3-benzoxadiazole.

Thermal Sensitivity Test A thermal responsiveness test of the copolymers produced in Examples 2, 3, 4 and 5 (hereinafter referred to as compounds 1, 2, 3 and 4) was carried out by the following procedure. A JASCO FP-6500 spectrofluorometer was used, and ultrapure water obtained from Milli-Q reagent system of Millipore was used as a solvent. The initial concentrations of 1, 2, 3, and 4 were 0.01 w / v%, and the excitation wavelength was 456 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. For compounds 1 to 3, the solution temperature was raised in 1 ° C increments, then lowered to the measurement start temperature in 1 ° C increments, and the fluorescence intensity at each temperature was measured. For Compound 4, the solution temperature was raised in increments of 2 degrees, then lowered to the measurement start temperature in increments of 2 ° C., and the fluorescence intensity at each temperature was measured.

  The test results are shown in FIG. All of the compounds showed strong fluorescence in the high temperature region, and it was confirmed that the fluorescence intensity changed in response to the change in temperature. It was also confirmed that the fluorescence intensities at the time of temperature rise and fall were in good agreement at each temperature. The low reproducibility of the fluorescence response is due to the intermolecular aggregation of polymers observed at high temperatures, but the ionic functional group newly introduced by the present invention effectively suppresses this by electrostatic repulsion. The reproducibility is considered to have improved.

Evaluation of Temperature Resolution FIG. 2 shows a schematic diagram of the relationship between temperature and fluorescence intensity. ∂ represents a minute amount and δ represents an error. From FIG. 2A, it can be seen that there is the following relationship between ∂ and δ.

ここで、∂が微小量を表すことから、

Here, since ∂ represents a minute amount,

は図２の直線の傾きを示している。
Ｔ〜Ｔ＋１℃においては、傾きが

Indicates the slope of the straight line in FIG.
From T to T + 1 ° C, the slope is

で表されるので、

Is represented by

となる。（図２（ｂ）参照）
δは誤差を表しているので、δＦＩは蛍光強度の誤差である。ここでは標準偏差を誤差値として用いた。Ｔ〜Ｔ＋１℃における蛍光強度の標準偏差は、

It becomes. (See Fig. 2 (b))
Since δ represents an error, δFI is an error in fluorescence intensity. Here, the standard deviation was used as the error value. The standard deviation of the fluorescence intensity from T to T + 1 ° C. is

で表される。したがって、温度の誤差、すなわち温度分解能は、

It is represented by Therefore, the temperature error, ie the temperature resolution, is

により算出される。図３に化合物１，２，３における、温度と（１）式により算出された温度分解能の関係を示す。化合物１、２、３において、広い温度範囲にわたり高い温度分解能が保たれていることがわかる。具体的には、化合物１では９℃から３３℃の間、化合物２では３０℃から５１℃の間、化合物３では４９℃から６６℃の間で、温度分解能が０．２℃以下に保たれている。したがって、この３種類の蛍光性温度センサーにより、約１０℃から６５℃の広範囲で、０．２℃の温度分解能が達成された。高い温度分解能は、本発明により新たに導入されたイオン性官能基の効果により、蛍光応答の再現性が向上したため達成されたと考えられる。

Is calculated by FIG. 3 shows the relationship between the temperature and the temperature resolution calculated by the equation (1) in the compounds 1, 2, and 3. It can be seen that compounds 1, 2, and 3 maintain high temperature resolution over a wide temperature range. Specifically, the temperature resolution is maintained at 0.2 ° C. or less between 9 ° C. and 33 ° C. for Compound 1, between 30 ° C. and 51 ° C. for Compound 2, and between 49 ° C. and 66 ° C. for Compound 3. ing. Therefore, with these three types of fluorescent temperature sensors, a temperature resolution of 0.2 ° C. was achieved in a wide range of about 10 ° C. to 65 ° C. It is considered that high temperature resolution was achieved because the reproducibility of the fluorescence response was improved by the effect of the ionic functional group newly introduced according to the present invention.

  FIG. 4 shows the relationship between the temperature and the calculated temperature resolution in Compound 4. In Compound 4, since the temperature is changed in increments of 2 ° C., the calculation formula for the temperature resolution is as shown in Equation (2).

化合物４においても、広い温度範囲にわたり高い温度分解能が保たれていることがわかる。したがって、イオン性官能基としてカチオン性のものを用いても高い温度分解能が達成された。

It can be seen that also in Compound 4, high temperature resolution is maintained over a wide temperature range. Therefore, even when a cationic group is used as the ionic functional group, high temperature resolution is achieved.

  [Comparative Example 1]

比較例として、上記の化合物５、６および７を以下の方法により合成した。

As a comparative example, the above compounds 5, 6 and 7 were synthesized by the following method.

NNPAM, NIPAM, or N-isopropylmethacrylamide (5 mmol), AIBN (50 μmol), DBD-AA (5 μmol) was dissolved in dioxane (10 ml), and dissolved oxygen was removed by passing nitrogen gas for 30 minutes. Thereafter, the mixture was reacted at 60 ° C. for 12 hours (compounds 5 and 6) or 16 hours (compound 7), and the reaction solution was poured into 200 ml of diethyl ether (compound 6) or 300 ml (compounds 5 and 7). The obtained crystals were collected by filtration and the desired compound was obtained by reprecipitation. Reprecipitation solvents include acetone (3 ml) and diethyl ether (200 ml) (compound 5), dioxane (10 ml) and diethyl ether (200 ml) (compound 6), acetone (3 ml) and hexane (200 ml) (compound 7), respectively. Use. Each unit ratio and molecular weight of the obtained temperature sensor were determined by ¹ H-NMR, absorbance, and GPC measurement in the same manner as in the examples.

FIG. 5 shows the results of the thermal sensitivity test of the fluorescence intensities of the compounds 5 to 7, and FIG. 6 shows the results of the evaluation of the temperature resolution of the compounds 5 to 7, respectively. Although the maximum temperature resolution of each compound was about 0.2 ° C., the temperature range was very narrow, and in most temperature ranges, the temperature resolution greatly exceeded 0.2 ° C.
[Examples 6 to 9]
Production of copolymer nanogel particles N-isopropylacrylamide (NIPAM), N, N'-methylenebisacrylamide (MBAM), 3-sulfopropyl acrylate (SPA), N- {2-[(7-N, N- Dimethylaminosulfonyl) -2,1,3-benzooxadiazol-4-yl] (methyl) amino} ethyl-N-methylacrylamide (DBD-AA), sodium persulfate (APS), and N, N, N Copolymers of Examples 6-9 were prepared using each of ', N'-tetramethylethylenediamine (TMEDA) in the amounts shown in the table below.

反応は以下の方法により行った。ＮＩＰＡＭ、ＭＢＡＭ、ＳＰＡ（実施例７および９のみ）、ＤＢＤ−ＡＡ、およびＴＭＥＤＡを、ドデシル硫酸ナトリウム（ＳＤＳ）水溶液（１２．６ｍＭ、２０ｍＬ、但し、実施例９では１６．６ｍＬを使用）に溶解させ、メカニカルスターラーを用いて約２５０ｒｐｍで撹拌し、７０℃に加熱した。溶存する酸素を除去するために、窒素ガスを３０分間撹拌中の溶液に吹き込み、ＡＰＳを加えた。７０℃、２５０ｒｐｍで４時間撹拌し、その後、反応液を冷水（４００ｍＬ）に注いだ。析出物が生じるまでＮａＣｌを加え、グラスフィルターで回収した。透析により精製した後に、凍結乾燥し、共重合体のナノゲル粒子を得た。各実施例におけるナノゲル粒子の収量、０．０１ｗ／ｖ％溶液に含まれる蛍光団の濃度、水中で膨潤状態のサイズ（直径）を以下の表に示す。なお、上記の透析は、直径２８．６ｍｍのヴィスキングチューブ（透析用セルローズチューブ）を使用し、サンプルを１０〜２５ｍｌの純水に溶解してチューブに入れ、透析外液を２００ｍｌとして攪拌しながら常温で５〜６日間透析し、その間、約１２時間ごとに透析外液を純水に交換して行った。透析外液のＵＶ測定または凍結乾燥後のサンプルのＮＭＲ測定によりモノマー由来のピークが検出されないことを確認した。

The reaction was carried out by the following method. NIPAM, MBAM, SPA (Examples 7 and 9 only), DBD-AA, and TMEDA are dissolved in aqueous sodium dodecyl sulfate (SDS) solution (12.6 mM, 20 mL, but 16.6 mL is used in Example 9). The mixture was stirred at about 250 rpm using a mechanical stirrer and heated to 70 ° C. In order to remove dissolved oxygen, nitrogen gas was blown into the stirring solution for 30 minutes and APS was added. The mixture was stirred at 70 ° C. and 250 rpm for 4 hours, and then the reaction solution was poured into cold water (400 mL). NaCl was added until a precipitate was formed, and collected with a glass filter. After purification by dialysis, freeze-drying was performed to obtain copolymer nanogel particles. The following table shows the yield of nanogel particles in each example, the concentration of the fluorophore contained in the 0.01 w / v% solution, and the size (diameter) of the swollen state in water. The above dialysis uses a Visking tube (dialysis cellulose tube) having a diameter of 28.6 mm, dissolves the sample in 10 to 25 ml of pure water, puts it in the tube, and stirs the dialysis solution as 200 ml. Dialysis was performed at room temperature for 5 to 6 days, and during this period, the dialyzed external solution was replaced with pure water every 12 hours. It was confirmed that no monomer-derived peak was detected by UV measurement of the dialyzed external solution or NMR measurement of the sample after lyophilization.

なお、表２における膨潤状態のナノゲルのサイズは、０．０１ｗ／ｖ％（実施例７および８）または０．００１ｗ／ｖ％（実施例６）のナノゲル水溶液を用いて、ＭＡＬＶＥＲＮ社 ゼーターサイザーナノ ＺＳを使用し動的光散乱法により測定した。

In addition, the size of the swollen nanogel in Table 2 is 0.01% w / v (Examples 7 and 8) or 0.001 w / v% (Example 6) using a nanogel aqueous solution. Measurement was performed by a dynamic light scattering method using ZS.

Thermal Sensitivity Tests The thermal responsiveness tests of the copolymers prepared in Examples 6-9 (hereinafter referred to as nanogels 1, 2, 3, and 4) were obtained from Millipore's Milli-Q reagent system as solvents. The procedure was the same as that described above except that measurement was performed using ultrapure water and 150 mM potassium chloride aqueous solution. After increasing the solution temperature in increments of 1 ° C. with compounds 1 to 4, the solution intensity was decreased in increments of 1 ° C. to the measurement start temperature, and the fluorescence intensity at each temperature was measured.

  The results of thermal sensitivity test are shown in FIGS. 7 to 10 and the temperature resolution evaluation results of Nanogel 2 and Nanogel 4 are shown in FIGS. All nanogels were confirmed to function as temperature sensors, and it was also confirmed that the fluorescence intensity at the time of temperature rise and that of nanogels 2 and 4 well matched at each temperature.

[Comparative Example 2]
Using the starting materials shown in Table 3 below, Nanogel of Comparative Example 2 (hereinafter referred to as Nanogel 5) was obtained by the same operation as in Examples 6-9. Yields and physical properties are shown in Table 4 below.

様々な塩濃度下における蛍光強度の感熱応答試験
ナノゲル３を０，５０，１００，１５０，２００ｍＭ 塩化カリウム水溶液にそれぞれ０．００１ｗ／ｖ％になるよう溶解し、上述の手順と同様の手順で蛍光強度の感熱応答試験を行った。各溶液について、溶液温度を１℃刻みで上昇させたあと、測定開始温度まで１℃刻みで下降させ、各温度における蛍光強度を測定した。

Thermal response test of fluorescence intensity under various salt concentrations Nanogel 3 was dissolved in 0, 50, 100, 150, and 200 mM potassium chloride aqueous solution to 0.001 w / v%, respectively, and fluorescence was obtained in the same procedure as described above. An intense thermal response test was conducted. For each solution, the solution temperature was raised in 1 ° C increments, then lowered to the measurement start temperature in 1 ° C increments, and the fluorescence intensity at each temperature was measured.

  The results of the thermal sensitivity test are shown in FIG. It was found that as the concentration of potassium chloride in the solution increased, the temperature at which the fluorescence intensity increased decreased, and the relative fluorescence intensity increment increased. It was also revealed that the response was almost constant when the potassium chloride concentration was 100 mM or more.

Intracellular fluorescence intensity thermosensitive response test Nanogel 3 was added to an aqueous injection solution (80 mM potassium chloride, 10 mM dipotassium hydrogen phosphate, 4 mM sodium chloride, pH 7.2) to prepare an injection solution containing nanogel 3 at 1 w / v%. . The injection solution was placed in an Eppendorf tube equipped with a filter (Ultrafree-MC, Millipore), centrifuged at 10,000 rpm for 7 minutes, and filtered to remove suspended matters having a diameter of 220 nm or more.

  Cos7 cells (cynomolgus monkey kidney cell culture cell / African Green Monkey SV40-transf'd kidney fibroblast cell line, transferred from Tokyo Metropolitan Institute of Medical Science) are cultured in DMEM medium under normal conditions, and cultured in HEPES medium only for observation did. Place petri dish containing cultured cells on stage with heater (MI-IBC, Olympus), epi-illuminated inverted fluorescence microscope (IX70, Olympus), 60x oil immersion objective (UplanApo NA 1.40, Olympus), cooled CCD camera ( ORCA-ER, Hamamatsu Photonics). The injection solution containing the prepared nanogel was injected into a glass needle for injection (Femtotips II, Eppendorf), and several fL injection was performed on Cos7 cells using an injector (FemtoJet, Eppendorf). Then, with the above CCD camera, apply a 488 nm laser (Saphhire488, Coherent) to the cells and pass through a filter (DM505, BA515-550) for cutting excitation light and scattered light. (Fluorescence wavelength: 515-550 nm) was observed by changing the medium temperature. FIG. 17 shows a fluorescence image at each temperature, FIG. 18 shows a change in fluorescence intensity, and FIG. 19 shows a reversibility test result. It was confirmed that the copolymer of the present invention showed thermosensitive response even in an intracellular environment and reversibly changed the fluorescence intensity.

Thermal Response Test of Fluorescence Lifetime Change A thermal response test of the fluorescence lifetime of Compound 3 produced in Example 4 was performed according to the following procedure.

  The Edinburgh Analytical Instruments FL-900CDT time-correlated single photon counting fluorescence lifetime measurement apparatus was used, and ultrapure water obtained from Millipore's Milli-Q reagent system was used as a solvent. The initial concentration of Compound 3 was 0.02 w / v%, and the excitation wavelength was 275 nm. For excitation of the solution, a nanosecond pulse hydrogen discharge tube (pulse width 1 nanosecond, pulse repetition rate 40 kHz) was used. A thermostat was used for temperature control of the solution. After confirming that the temperature of the solution was stabilized by a thermocouple before measurement, the fluorescence lifetime at each temperature was measured with the fluorescence wavelength set at 560 nm. The obtained fluorescence decay curve was approximated by the following formula to obtain a two-component fluorescence lifetime.

得られた蛍光寿命から、以下に示す式を用いて各温度における平均蛍光寿命を算出した。

From the obtained fluorescence lifetime, the average fluorescence lifetime at each temperature was calculated using the following formula.

試験結果を表５および図２０に示す。高温領域で化合物３の平均蛍光寿命が延長し、温度の変化に鋭敏に反応して平均蛍光寿命が変化することが確認された。

The test results are shown in Table 5 and FIG. It was confirmed that the average fluorescence lifetime of Compound 3 was extended in the high temperature region, and the average fluorescence lifetime was changed in response to the change in temperature.

Claims (14)

Formula (a):

[Wherein R ¹ is selected from a hydrogen atom and methyl;
R ⁴ and R ⁵ are independently selected from a hydrogen atom and C _1-20 alkyl, or R ⁴ and R ⁵ together with the nitrogen atom to which they are attached, form a pyrrolidine ring, piperidine ring, or homopiperidine Form a ring ]
And a monomer represented by formula (b):

[Wherein R ² is selected from a hydrogen atom and methyl;
X ¹ is O or N—R ¹¹ ;
R ¹¹ is a hydrogen atom or methyl;
Q ¹ is C _2-5 alkylene;
Y is independently an ionic functional group that may have one or more positive or negative charges, and is —SO ₃ X _a , —PO (OX _b ) (OX _c ), —COOX _d , and —N ⁺ R ²¹ R ²² R ²³ X _e ⁻
Selected from the group represented by
Where X _a , X _b , X _c and X _d are independently selected from a hydrogen atom or a counter cation, and X _e ⁻ is a counter anion;
R ²¹ , R ²² , and R ²³ are methyl, or R ²¹ and R ²² together with the nitrogen atom to which they are attached may form a pyrrolidine, piperidine, or homopiperidine ring ]
And / or a crosslinker monomer containing two or more double bonds in the molecule: and formula (c):

Wherein R ³ is selected from a hydrogen atom and methyl;
X ² is O or N—R ¹² ;
X ³ is a direct bond, N (—R ¹³ ), CON (—R ¹⁶ ) (where the nitrogen atom is linked to Q ² ), or SO ₂ N (—R ¹⁹ ) (where the nitrogen atom is Q ² Connected to);
Q ² is C _2-4 alkylene;
Ar is the following formula:

An aromatic heterocyclic group selected from the group represented by: wherein these groups are from C _1-6 alkyl, C _1-6 alkoxy, nitro, cyano, and —SO ₂ NR ¹⁴ R ¹⁵ on the ring. It is optionally substituted with one or more substituents selected;
X ¹⁰ is selected from O, S or Se;
R ¹² is a hydrogen atom or methyl;
R ¹³ is a hydrogen atom or methyl;
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 pyrrolidine ring, piperidine ring, or homo Forming a piperidine ring ;
R ¹⁶ and R ¹⁹ are independently selected from a hydrogen atom or methyl]
A copolymer comprising a repeating structure derived from each of the monomers represented by:
Here, when the copolymer does not contain a repeating structure derived from the monomer represented by the formula (b), the copolymer is a reaction initiator in an amount of 7 mol% or more based on the monomer used. Wherein the initiator is represented by the formula (d):
Y ^1- (C _1-10 alkylene) -N═N— (C _1-10 alkylene) -Y ¹
{ _Wherein C _1-10 alkylene may be independently substituted with one or more substituents selected from halogen atoms and cyano; Y ¹ is independently one or more positive or negative charges. Which is an ionic functional group that may have a azo compound, or a persulfate. The crosslinking agent monomer is represented by the formula (e):

Wherein R ⁹ and R ¹⁰ are independently selected from a hydrogen atom and methyl;
X ⁴ and X ⁵ are N—R ^12, where R ¹² is independently as defined in claim 1;
Q ³ is C _1-6 alkylene]
The copolymer of Claim 1 which is a compound represented by these.   The copolymer according to claim 1 or 2, wherein the reaction initiator is selected from persulfates.   The copolymer according to any one of claims 1 to 3, which is prepared by a copolymerization reaction using a reaction initiator in an amount of 14 mol% or more based on a monomer to be used. Formulas (I), (II) and (III):

[Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , Y, X ¹ , X ² , X ³ , Q ¹ , Q ² and Ar are as defined in claim 1]
The copolymer of Claim 1 containing the repeating unit represented by these. Formula (IV):

[Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , Y, X ¹ , X ² , X ³ , Q ¹ , Q ² , and Ar are as defined in claim 1;
a, b and c are numbers greater than 0 representing the ratio of each repeating unit in the formula]
The copolymer according to claim 5, wherein a + b + c = 100, and c is 0.01-20.   The copolymer according to claim 5 or 6, wherein b is 1 to 20. Y is, _-SO 3 _{X a} or ^{-N + R 21 R 22 R 23} X e - a copolymer according to any one of claims 1-7.   The copolymer according to any one of claims 1 to 8, which is used for measuring temperature.   A fluorescent temperature sensor comprising the copolymer according to any one of claims 1 to 8.   The temperature measuring method which uses the copolymer of any one of Claims 1-8.   The temperature measuring method of Claim 11 including the process of melt | dissolving the copolymer of any one of Claims 1-8 in a liquid; and the process of measuring fluorescence intensity under excitation light irradiation.   The temperature measuring method of Claim 11 including the process of melt | dissolving the copolymer of any one of Claims 1-8 in a liquid; and the process of measuring an average fluorescence lifetime under excitation light irradiation.   The temperature measuring method according to any one of claims 11 to 13, for measuring an intracellular temperature.

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