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WO2008029770A1 - Thermomètre moléculaire fluorescent - Google Patents

Thermomètre moléculaire fluorescent Download PDF

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Publication number
WO2008029770A1
WO2008029770A1 PCT/JP2007/067147 JP2007067147W WO2008029770A1 WO 2008029770 A1 WO2008029770 A1 WO 2008029770A1 JP 2007067147 W JP2007067147 W JP 2007067147W WO 2008029770 A1 WO2008029770 A1 WO 2008029770A1
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Prior art keywords
alkyl
alkoxy
temperature
formula
atom
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English (en)
Japanese (ja)
Inventor
Seiichi Uchiyama
Chie Gota
Tomohiko Ohwada
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University of Tokyo NUC
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University of Tokyo NUC
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Priority to JP2008533148A priority Critical patent/JP5441098B2/ja
Publication of WO2008029770A1 publication Critical patent/WO2008029770A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/38—Esters containing sulfur
    • C08F220/382—Esters containing sulfur and containing oxygen, e.g. 2-sulfoethyl (meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
    • C08F220/603—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen and containing oxygen in addition to the carbonamido oxygen and nitrogen

Definitions

  • the present invention relates to a novel fluorescent polymer and a temperature measurement method using the polymer.
  • Non-patent documents The use of molecules whose physical properties change with changes in temperature as a temperature sensor has been studied (Non-patent documents;! To 5).
  • a fluorescent temperature sensor based on the principle of incorporating 2,1,3-benzoxaziazolyl, an environmentally responsive fluorophore, into polyacrylamide, a heat-sensitive polymer, has already been developed. It has been reported (Non-Patent Document 6). Also reported is the use of a polyacrylolamide microgel incorporating a fluorophore obtained by adding a cross-linking agent during polymer production as a fluorescent temperature sensor! / Non-patent document 7).
  • This fluorescent temperature sensor has the property that the fluorescence intensity increases as the temperature rises in an aqueous solution due to the heat phase transition of polyacrylamide, the main chain. Changes in temperature inside can be observed.
  • a temperature sensor has attracted attention in recent years because its minimum functional unit is a single molecule and enables temperature measurement in a minute space such as the inside of a microreactor.
  • Non-patent Document 9 The use of polyacrylamide as a logic gate that responds to ambient temperature and pH is also being studied.
  • Non-patent literature l Photochem. Photobiol. 1995, 62, 416-425
  • Non-patent literature 2 J. Phys. D: Appl. Phys. 2004, 37, 2938-2943
  • Non-patent Reference 3 Anal. Chem. 2006, 78, 5094-5 dishes
  • Non-Patent Document 4 Appl. Phys. Lett. 2005, Vol. 87, 201102
  • Non-Patent Document 5 Biophys. J. 1998, 74, 82-89
  • Non-Patent Document 6 Anal. Chem. 2003, 75, 5926-5935
  • Non-Patent Document 7 J. Mater. Chem. 2005, Vol. 15, pp. 2796-2800
  • Non-Patent Document 8 Anal. Chem. 2004, 76, 1793-; 1798
  • Non-Patent Document 9 J. Am. Chem. Soc. 2004, Vol. 126, pp. 3032-3033 Disclosure of the Invention
  • the present inventor has intensively studied to solve the above-mentioned problems. As a result, by introducing a ionic unit into the polymer, the present inventor can be used as a more practical fluorescent temperature sensor. The present invention has been completed.
  • R 1 is selected from a hydrogen atom and C alkyl
  • R 4 and R 5 are independently selected from a hydrogen atom and C alkyl, wherein
  • Rualkyl is one or more substituents selected from hydroxy, C alkoxy, and aryl
  • 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 a C alkyl , And optionally substituted with one or more substituents selected from C alkoxy, nitro, halogen atoms, C alkylcarbonylamino and arylenocarbonylcarbonyl;
  • R 2 is selected from a hydrogen atom and a C alkyl force
  • X 1 is ⁇ , S, or N—R 11 ;
  • R U is a hydrogen atom, C alkyl, or G ⁇ —Y, where the alkyl is selected from hydroxy, halogen atom, c alkoxy, C alkylthio, C alkylsulfinyl, and C alkylsulfonyl May be substituted by one or more substituents;
  • Q 1 is independently selected from C alkylene, C alkenylene, or C alkynylene, wherein the alkylene may be independently inserted with o, S, or phenylene in one or more locations. ;
  • X, X, X, and X are independently from a hydrogen atom or counter force thione. Selected, X— and X— are counteranions;
  • R 21 , R 22 , and R 23 are independently selected from C alkyl and C aralkyl.
  • R 21 and R 22 together with the binding nitrogen atom may form a 5-7 membered nitrogen-containing heterocycle
  • R 24 is C alkyl or C aralkyl
  • R 41 , R 42 and R 43 are independently selected from a hydrogen atom and C alkyl, or
  • R 41 and R 42 together with the nitrogen and carbon atoms to which they are attached may form a 5-7 membered heterocycle containing two nitrogen atoms or R 42 and R 43 are A 5- to 7-membered nitrogen-containing heterocycle may be formed together with the nitrogen atom to be bonded! /,] And / or a bridge containing two or more double bonds in the molecule Agent monomer: and
  • R 3 is selected from a hydrogen atom and a C alkyl force
  • X 2 is ⁇ , S, or N—R 12 ;
  • X 3 is a direct bond, ⁇ , S, SO, SO, N (—R 13 ), CON (—R 16 ), N (—R 16 ) CO, N (R 17 ) CON (—R 18 ), SO N (—R 19 ) or N (—R 19 ) SO;
  • Q 2 is selected from C alkylene, C alkenylene, or C alkynylene,
  • alkylene may be independently inserted with o, S or phenylene at one or more points;
  • Ar is selected from 6 to; 18-membered aromatic carbocyclic group, or 5 to; 18-membered aromatic heterocyclic group, wherein the aromatic carbocyclic group and aromatic heterocyclic group are included in the ring
  • the aromatic carbocyclic group and aromatic heterocyclic group include a halogen atom, C alkyl, C
  • Alkyl in alkoxycarbonyl is a halogen atom, c alkoxy, hydroxy
  • R 6 and R 7 are independently a hydrogen atom, C alkyl, aryl, C alkyl carbo
  • N— (C alkynole) rubamoyl N, N di (C alkynole) carbamo
  • Alkyl contained in rubamoyl contains halogen atom, C alkoxy, hydroxy, amino
  • the aryls contained in the aryl, arylcarbonyl, and arylylsulfonyl, which may be substituted with one or more substituents, are a halogen atom, C alkyl.
  • R 6 and R 7 together with the nitrogen atom to which they are attached form a 4-8 membered nitrogen-containing heterocycle, where the heterocycle is a C alkyl, C alkoxy, nitro, halogen atom.
  • R 12 is a hydrogen atom, C alkyl, or -0 2 — 3 — eight, where the alkyl
  • R is a hydrogen atom or C alkyl, where the alkyl is hydroxy
  • R 14 and R 15 are independently selected from a hydrogen atom and C alkyl; or R 14
  • R 15 together with the nitrogen atom to which they are attached form a 4-8 membered nitrogen-containing heterocycle
  • R 16 , R 17 , R 18 and R 19 are independently selected from a hydrogen atom and C alkyl
  • alkyl is hydroxy, halogen atom, C alkoxy, c alkylthio,
  • C alkylene is independently one or more selected from a halogen atom and cyan
  • R 41 , R 42 , and R 43 are as defined above ⁇
  • 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.
  • Alkyl is a halogen atom, C alkoxy, hydroxy, amino, canolenoquinamino,
  • X 1 "is ⁇ is selected from S or Se;
  • R 8 is selected from a hydrogen atom, C alkyl, and aryl, where the alkyl is
  • Halogen atom C alkoxy, hydroxy, amino, C alkylamino, di (C alkyl
  • the aryl may be substituted with one or more substituents selected from amino, aryl, and carboxy. Further, the aryl may be a halogen atom, C alkyl, C alkoxy,
  • Ar represents the following formula:
  • a temperature measurement method using the above copolymer including a step of dissolving the above copolymer in a liquid; and a step of measuring fluorescence intensity under excitation light irradiation. A step of dissolving the above-mentioned copolymer in a liquid; and a step of measuring an average fluorescence lifetime under excitation light irradiation.
  • 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.
  • 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.
  • FIG. 2 is a schematic diagram showing the relationship between the temperature of a fluorescent temperature sensor and the fluorescence intensity.
  • FIG. 4 is an example of a temperature resolution evaluation result of Compound 4.
  • FIG. 5 is an example of a thermal sensitivity test result (0.01 w / v%, excitation wavelength 456 nm) of fluorescence intensity of compounds 5 to 7 ( ⁇ Compound 5, ⁇ Compound 6, ⁇ Compound 7) as comparative examples. .
  • FIG. 6 is an example of the results of temperature resolution evaluation of compounds 5-7.
  • Nanogel 1 (0.01 w / v% in pure water, fluorescence wavelength 550 nm; temperature rise: ⁇ , temperature drop: ⁇ ) and Nanogel 3 (0.01 w / v% in pure water) It is an example of the result of a thermal response test of fluorescence intensity at a fluorescence wavelength of 554 nm; temperature rise: ⁇ , temperature drop: ⁇ .
  • Nanogel l (0.01 w / v% in 150 mM potassium chloride aqueous solution, fluorescence wavelength 550 nm; when temperature rises: ⁇ , when temperature falls: indicates ⁇ ) and nanogel 3 (0.01 w / in 150 mM potassium chloride aqueous solution) It is an example of the result of a thermal response test of the fluorescence intensity of v%, fluorescence wavelength 554 nm; temperature rise: A, temperature fall: ⁇ .
  • Nanogel 2 (0.01w / v% in pure water, fluorescence wavelength 555nm; temperature rise: haze, under temperature Results of the thermal response test of fluorescence intensity of nanogel 4 (0.01 w / v% in pure water, fluorescence wavelength 558 nm; temperature rise: A, temperature fall: ⁇ ) It is an example.
  • Nanogel 2 (0.01 w / v% in 150 mM potassium chloride aqueous solution, fluorescence wavelength 550 nm; when temperature rises: ⁇ , when temperature falls: indicates ⁇ ) and nanogel 4 (0.01 w / 150 mM potassium chloride aqueous solution) It is an example of the result of a thermal response test of the fluorescence intensity of v%, fluorescence wavelength 558 nm; temperature rise: A, temperature fall: ⁇ .
  • FIG. 13 An example of the results of a thermal sensitivity test of the fluorescence intensity of Nanogel 5 (0.01 w / v% in pure water, fluorescence wavelength 550 nm) (when temperature rises: ⁇ , when temperature falls: indicates ⁇ ) .
  • FIG. 14 An example of temperature resolution evaluation results for Nanogel 5 (0.01 w / v% in pure water, fluorescence wavelength 550 nm).
  • FIG. 16 is an example of a photograph of a fluorescence image of a living cell containing nanogel 3 (excitation wavelength: 488 nm, fluorescence wavelength: 515-550 ⁇ m, medium temperature: about 37 ° C.).
  • FIG. 19 An example of the results of a reversibility confirmation test of the fluorescence response of nanogel 3 (excitation wavelength: 488 nm, fluorescence wavelength: 515-550 nm) in living cells, showing the fluorescence intensity at each temperature. Is shown as relative intensity with 1 as the first value.
  • FIG. 20 shows an example of the result of a thermal response test of the fluorescence lifetime of Compound 3 (0.02 w / v% in pure water, excitation wavelength 275 nm, fluorescence wavelength 560 nm).
  • C alkyl refers to a straight chain, branched chain, or carbon number
  • C alkyl means a straight chain, branched chain, or cyclic group having a carbon number;! -6.
  • a partially cyclic alkyl group such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 3-methylol-butyl, 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, and cyclopropylmethyl ,
  • C alkyl means a straight chain, branched chain, or cyclic group having a carbon number
  • a partially cyclic alkyl group e.g. a c alkyl group as defined above.
  • C alkyl means a straight chain, branched chain, or cyclic group having a carbon number
  • Alkyl and C alkyl etc. are included.
  • C alkoxy means the number of carbon atoms already defined as an alkyl moiety.
  • aryl means a 6- to 10-membered aromatic carbocyclic group, and includes, for example, phenyl, 1-naphthyl, 2-naphthyl and the like.
  • C aralkyl refers to an alkyl having 7 to 14 carbon atoms including an aryl group.
  • An alkyl group includes, for example, benzyl, 1-phenethyl, 2-phenethyl, 1-naphthylmethyl, 2-naphthylmethyl and the like.
  • examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • C alkylene is a straight chain, branched chain, or ring having 1 to 20 carbon atoms.
  • Means a partially or partially cyclic alkylene group and includes, for example, methylene, ethylene, propylene, butylene and the like, and C alkylene and C alkylene.
  • C alkenylene means a linear or branched chain having 3 to 20 carbon atoms
  • a cyclic or partially cyclic alkenylene group means, for example, propenylene, butenylene and the like, and C alkenylene and C alkenylene are included.
  • C alkynylene is a straight chain or branched chain having 3 to 20 carbon atoms
  • C alkylthio refers to a carbon that has already been defined as an alkyl moiety.
  • alkylthio group having an alkyl group of 1 to 6, and includes, for example, methylthio, ethinoretio, n propylthio, i propylthio, n butylthio, s butylthio, i-butyl thio, t-butylthio and the like.
  • C alkylsulfiel is already defined as an alkyl moiety.
  • alkyl sulfinyl group having an alkyl group having 1 to 6 carbon atoms for example, main Chino less Honoré Fini Honoré, E Chino less Honoré Fini Honoré, n pro Pinot less Honoré Fini Honoré, i Puropinoresunore Fininore, n Buchinoresu Norefininore, s Butinolesnorefininore, i-butinoresnorefininore, t-butylsulfinyl and the like.
  • C alkylsulfonyl is defined as an alkyl moiety.
  • an alkyl sulfonyl Le group having an alkyl group having 1 to 6 carbon atoms for example, methylcarbamoyl Roh less Honoré Honi Honoré, E Chino less Honoré Honi Honoré, n pro Pinot less Honoré Honi Honoré, i pro Pinot less Honoré ho Ninore, n butinolesnorehoninore, s butinoresnorehoninore, i-butinoresnorehoninore, t-butinoresnorehonil, etc. are included.
  • 6- to 18-membered aromatic carbocyclic group includes, for example, phenyl, naphthyl, anthracenyl, pyrenyl, indanyl, tetralinyl and the like.
  • the “5- to 18-membered aromatic heterocyclic group” is an aromatic heterocyclic group having one or more heteroatoms selected from oxygen, nitrogen and sulfur.
  • examples include pyrrolyl, pyrazolyl, imidazolyl, pyridyl, indolyl, quinolyl, quinoxalinyl, quinazolinyl, benzofuranyl, benzocenyl, benzopyranyl, benzochromenyl and the like.
  • C alkenylsulfonyl is already defined as an alkenyl moiety.
  • Alkenylsulfonyl group having a defined C alkenyl group such as vinyls
  • Examples include sulfonyl, arylsulfonyl and the like.
  • alkenylcarbonyl is already defined as an alkenyl moiety.
  • alkynylcarbonyl is already defined as an alkynyl moiety.
  • C alkylcarbonyl represents a group CO (C alkyl).
  • c alkoxycarboninole refers to the group cO (c alkoxy).
  • C alkylcarbonylamino refers to the group NHC0 (C alkyloxy).
  • C arylcarbonylamino refers to the group NHC ⁇ (arinore).
  • the “5- to 7-membered nitrogen-containing heterocycle” includes, for example, a pyrrole ring, pyrrolidine Saturated hetero rings such as a ring, piperidine ring, homopiperidine ring, piperazine ring, homopiperazine ring, morpholine ring and thiomorpholine ring are included.
  • the "4- to 8-membered nitrogen-containing heterocycle” includes, for example, a pyrrole ring, azetidin ring, pyrrolidine ring, piperidine ring, homopiperidine ring, piperazine ring, homopipete ring. Includes azine ring, morpholine ring, thiomorpholine ring, and 5- to 7-membered nitrogen-containing heterocycle
  • the "5- to 7-membered heterocycle containing two nitrogen atoms" in the present specification includes, for example, imidazolidine, tetrahydropyrimidine and the like.
  • the alkylene chain contains an ether bond in the main chain, and the insertion has a stable structure. Therefore, those skilled in the art should easily understand that the process is performed so as not to have the structure of 0—0—i′—0—CH—O. The above also applies to the purchase of S into alkylene!
  • the “counter force thione” is not particularly limited as long as it is a cation that is usually used as a counter force thione of an organic compound in the technical field of organic chemistry.
  • ammonium ion alkali ion Metal ions (for example, sodium, lithium and potassium ions), alkaline earth metal ions (for example, calcium ions, norlium ions), substituted ammonium cations (for example, N— (C alkyl) pyridinium cations, tetra
  • Examples of the counter cation include sodium ion, potassium ion, tetramethylammonium ion and the like.
  • the "counteryuon” is not particularly limited as long as it is an anion that is usually used as a counteranion of an organic compound in the technical field of organic chemistry.
  • a halide anion chloride ion
  • organic acid anions eg, acetic acid, trifluoroacetic acid
  • nitrate ions e.g. acetic acid, trifluoroacetic acid
  • sulfate ions e.g, carbonate ions, and the like.
  • Preferred counter anions in the present invention include, for example, chloride ions and nitrate ions.
  • R 1 , R 2 , and R 3 are preferably selected from a hydrogen atom and methyl.
  • the NR 4 R 5 in the formulas (a), (I) and (IV) is not particularly limited.
  • R 4 is a hydrogen atom, and R 5 may be C alkyl.
  • R 4 and R 5 are
  • pyrrolidine ring When combined with a nitrogen atom to form a 4- to 8-membered nitrogen-containing heterocycle, for example, pyrrolidine ring, piperidine ring, homopiperidine ring, piperazine ring, homopiperazine ring, A ruphorin ring, a thiomorpholine ring or the like may be formed.
  • X 2 Q 2 in formula (c), formula (III) and (IV) is preferably X 2 is ⁇ , NH or N (C alkyl), and Q 2 is C alkylene .
  • Ar is preferably represented by the following formulas (V) to (XII):
  • R is selected from a hydrogen atom, a halogen atom, nitro, cyan, and —SO NR 14 R 15
  • R 32 is C alkyl; X 11 is N—R 33 , O or S; R 33 is a hydrogen atom
  • Preferred X 3 for formula (V) is, for example, a direct bond, CON (—R 16 ), N (— CO, SO N (—R 19 ) or N (—R 19 ) SO may be mentioned.
  • Preferred X 3 for formula (VI) includes, for example, N—R 13 (wherein preferred R 13 includes C alkyl such as methyl) or S.
  • X 3 for the formula (VII) for example, a direct bond, CON (—R 16 ), N (—R 16
  • Preferred X 3 for [0068] for example, a direct bond, CON (- R 16), N (- R 1 6) CO, SO N (- R 19) or N (- R 19) SO Is mentioned.
  • Preferred X 3 for formula (IX) includes, for example, a direct bond.
  • Preferred X 3 for the formula (X) includes, for example, a direct bond.
  • Preferred X 3 for formula (XI) is, for example, CO, SO, SO N (—R 19 ) or CO
  • Preferred X 3 for formula (XII) is, for example, CO, SO, SO N (—R 19 ) or C
  • the group —X 3 —Ar functions as an environmentally responsive fluorophore, for example, the formula (V
  • the amount of the monomer of the formula (b) used is not particularly limited.
  • the amount of the monomer of the formula (a), (b) and (c) is 0 ⁇ 0; Amount of ⁇ 20 mol% can be used
  • the copolymer according to the present invention can be synthesized based on general 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, and examples thereof include ⁇ , ⁇ ′-azobisisobutyronitrile, benzoyl peroxide, and ammonium persulfate.
  • the copolymer of the present invention is a repeating structure derived from the monomer represented by the formula (b). Is not prepared, the copolymer is 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 is independently an ionic functional group that can have one or more positive or negative charges
  • the C alkylene is independently selected from one or more halogen atoms and cyan
  • reaction initiator include ammonium persulfate and sodium persulfate.
  • Persulfates such as potassium persulfate; 2, 2'-azobis (2-methylpropionamidine) dihydrochloride, 2, 2 'azobis [2- (2 imidazoline 2-yl) propane] dihydrochloride, 4, 4 Azobis (4-cyananovaleric acid) and other azo compounds.
  • the amount of the reaction initiator used may be 7 mol% or more with respect to the monomer used (including the crosslinking agent monomer), for example, 14 mol% or more, preferably 25 mol% or more. Reaction initiators 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 148 hours, preferably 2 to 16 hours, for example. it can.
  • the crosslinking agent monomer used in the present invention is not particularly limited as long as it is a monomer containing two or more bur groups in the molecule and is usually used as a crosslinking agent.
  • Examples of the crosslinking agent include formula (e):
  • R 9 and R 1Q are independently selected from a hydrogen atom and C alkyl; X 4 and X 5 are independently 0, S, or N—R 12 ;
  • Q 3 is selected from C alkylene, C alkenylene, or C alkynylene,
  • alkylene o, S or phenylene may be independently inserted at one or more points.
  • crosslinker monomers include N, ⁇ '-methylene bisacrylamide, ⁇ , ⁇ '-ethylene bisacrylamide, ⁇ , ⁇ 'methylene bismethacrylamide, ⁇ , N' ethylene bismethacrylolamide. , Ethylene glycol diatalylate, ethylene glycol dimethacrylate, and the like.
  • the amount of the crosslinker monomer used is not particularly limited, but is, for example, an amount of 0 ⁇ ;! to 20 mol% with respect to the monomers of the formulas (a), (b) and (c) Can be used.
  • the copolymerization reaction in the case of using a crosslinking agent monomer can be carried out by a method commonly used in the art.
  • the reaction solvent used in the copolymerization reaction is not particularly limited.
  • a surfactant for example, sodium dodecyl sulfate, sodium dodecylbenzene sulfate, sodium pentadecane sulfate, N dodecyl mono N, N, N trimethylan Water containing monum bromide, N cetyl N, N, N—trimethylammonium bromide, triton X-100, etc.
  • monum bromide for example, sodium dodecyl sulfate, sodium dodecylbenzene sulfate, sodium pentadecane sulfate, N dodecyl mono N, N, N trimethylan Water containing monum bromide, N cetyl N, N, N—trimethylammonium bromide, triton X
  • the size of the copolymer nanogel obtained using the crosslinker monomer is determined by the stirring efficiency in the copolymerization reaction, reaction temperature, amount of surfactant used, amount of initiator used, amount of crosslinker monomer used. Can be adjusted. For example, a nanogel with a small size can be obtained by increasing the amount of surfactant and / or initiator used.
  • the size of the obtained nanogel can be appropriately adjusted by those skilled in the art to which the present invention belongs, and the size of the copolymer of the present invention can be adjusted.
  • the size of the nanogel is, for example, 5 to! OOnm.
  • the copolymerization reaction is not particularly limited, but may be, for example, 0 to; 100 ° C, preferably 50 to 70 ° C, and for example;! To 48 hours, preferably 2 to 16 hours.
  • the reaction time can be carried out.
  • the ionic functional group contained in the copolymer has an ionic function in a wide range of pH. It is better to maintain sex. From this point of view, Y is —SO X or —N + R 21 R
  • reaction initiator is preferably a persulfate capable of introducing SO X.
  • a copolymer including a repeating structure derived from each of the monomers represented by the formulas (a), (b) and (c) and the crosslinking agent monomer.
  • 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). in contrast, for example,;! a ⁇ 20 Monore 0/0, preferably (or 2; a 15 Monore 0/0, more preferably (or 2. 5;. 10 Monore 0/0 the use
  • the amount of the monomer represented by the formula (c) is, for example, 0.0;!
  • the amount of the crosslinking agent monomer used is, for example, 0.;! To 20 mol% with respect to the total amount of monomers represented by the formulas (a), (b) and (c), preferably 0. 5; a 10 mole 0/0, Ri is preferably 1 to 5 mol 0/0.
  • a copolymer including a repeating structure derived from each of the monomers represented by the formulas (a), (b) and (c).
  • 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). in contrast, for example,;! a ⁇ 20 mol%, preferably rather is 2; a 15 mole 0/0, more preferably 2. 5; 10 mol 0/0.
  • the amount of the monomer represented by the formula (c) used is, for example, 0.0 with respect to the total amount of the monomers represented by the formulas (a), (b) and (c); !
  • a monomer comprising a repeating structure derived from each of the monomers represented by the formulas (a) and (c) and the crosslinking agent monomer, the monomer used Prepared by a copolymerization reaction using an initiator in an amount of 7 mol% or more, and the initiator is selected from the azo compound represented by the formula (d) or the persulfate power.
  • the copolymer is provided.
  • the amount of the monomer represented by the formula (c) used to obtain the copolymer is based on the total amount of the monomers represented by the formulas (a) and (c). For example, 0.0;! a ⁇ 20 mol%, particularly 0.0;! a ⁇ 1 0 mole 0/0, preferably from 0.05 to 2 Monore 0/0, more preferably 0.5;! is to 1 mol 0/0.
  • the amount of the crosslinking agent monomer used, the formula (a), the total amount of monomers is Ru represented by (b) and (c), for example, 0.1;! ⁇ 20 mol 0/0 and it is preferably is from 0.5 to 10 mol 0/0, and more preferably, is to 5 mol 0/0!.
  • a, b and c in the formula (IV) represent a ratio of each repeating unit in the formula and are a number greater than 0, and are not particularly limited.
  • a + b + c 100, ci ci 0.0;! ⁇ 20, and more specifically white birch (preferably 0.001 ⁇ ; 10, preferably (preferably 0.05. (Ma 0.;! ⁇ 1.
  • the weight-average molecular weight of the polymer of the present invention is particularly (not limited to a range of 5,000 to 1000000, preferably ⁇ (10000 to 200000).
  • the change in 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.
  • the maximum excitation wavelength when the excitation spectrum of the measurement sample is measured or a wavelength in the vicinity thereof can be used.
  • 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 determined by a general fluorescence lifetime measurement method such as a single photon counting method, a phase modulation method, a noise sampling method, or an excitation probe. It can be measured by a method such as a method.
  • the single photon counting method uses the fact that the emission intensity distribution on the time axis and the emission probability of one photon are correlated with each other. After the excitation of the fluorophore with very short (pulsed) light such as Ins, the generation time of the detected light is measured, and a histogram obtained by repeating excitation many times is used as a fluorescence decay curve.
  • the fluorescence lifetime is determined by approximation with the sum of exponential functions.
  • Single photon counting fluorescence lifetime measurements can be made using commercially available time-correlated single photon counting fluorescence lifetime measuring instruments (eg, Edinburgh Analytical Instruments FL-900CDT, see Examples) and accompanying measurements. This can be done using an analysis 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, but it can be introduced, for example, by culturing the cell in a medium to which the copolymer is added.
  • Other methods such as methods using positively charged lipids (such as ribofection method), electrical methods (such as eletroporation method), chemical methods (such as calcium phosphate method), and physical methods (such as microinjection method). You can use it.
  • N-isopropylacrylamide was used after being purified by recrystallization using n-hexane.
  • N-n-propyl acrylamide was synthesized and purified according to the general synthesis procedure of N-alkyl acrylamide.
  • ⁇ , ⁇ '-azobisisobutyronitrile was used after being purified by recrystallization using methanol.
  • Other reagents were purchased without further purification.
  • Number average molecular weight, weight Average molecular weight is JASCO GPC system (JAS CO PU— 2080 pump, JASCO RI — 2031 differential refractometer, JASCO CO— 2060 column oven, Shodex GPC KD — 806M column) and calculated using a calibration curve obtained with polystyrene standards.
  • JASCO GPC system JS CO PU— 2080 pump, JASCO RI — 2031 differential refractometer, JASCO CO— 2060 column oven, Shodex GPC KD — 806M column
  • silica gel column chromatography Kanto Chemical silica gel 60N (40-540) was used.
  • a JASCO V-550 ultraviolet-visible light spectrophotometer was used for measuring the absorbance.
  • N, N dimethyl-7 [methyl ⁇ 2 (methylamino) ethyl ⁇ amino] 2, 1,3-benzoxaziazole 4 sulfonamide 130 mg was dissolved in acetonitrile (13 ml) to give triethenoleamine 0 (57.8 ⁇ L, leq) and Atalinoleic acid chloride (43.8 ⁇ L, 1.3eq). After adding in C, the mixture was stirred at room temperature for 1.5 hours. Na CO (lg) was added to the reaction solution and filtered.
  • N-t butylacrylamide (572 mg, 4.5 mmol, hereinafter also referred to as NTBAM), 3 snolefopropinore attalylate potassium salt (116 mg, 500 ⁇ 11101, hereinafter also referred to as SPA potassium salt), N— ⁇ 2— [ (7—N, N dimethylaminosulfonyl) -2,1,3-benzoxaziazole-4-yl] (methyl) amino ⁇ ethyl-N methylacrylamide (1 • 84 mg, 5 mol, hereinafter also referred to as DBD—AA), a, ⁇ 'azobisisobutyronitrinole (8.21 mg, 50 mol, hereinafter also referred to as AIBN) was rapidly dissolved in DMF (10 ml).
  • the dissolved oxygen was removed by passing nitrogen gas for 30 minutes. Thereafter, the mixture was reacted at 60 ° C for 12 hours. After partially distilling off DMF under reduced pressure, the reaction solution was poured into jetyl 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%).
  • N—n-propylacrylamide (552 mg, 4.875 mmol, also referred to as NNPAM), SPA potassium salt (29. Omg, 125 ⁇ mol), DBD—AA (1.84 mg, 5 ⁇ mol), AI BN ( 8. 21 mg, 50 mol) was dissolved in DMF (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, and DMF was partially distilled off from the reaction solution under reduced pressure, and then poured into jetyl ether (200 ml). The obtained crystals were collected by filtration and purified by reprecipitation (methanol 3 ml-jetyl ether 200 ml) to give the title copolymer as a pale yellow powder (352 mg, 60%).
  • N Isopropylacrylamide (552 mg, 4. 875 mmol, hereinafter also referred to as NIPAM), SPA potassium salt (29. Omg, 125 mol), DBD—AA (1.84 mg, 5 ⁇ mol), ⁇ 8 (8 ⁇ 21 mg, 50 mol) was dissolved in DMF (10 ml) and dissolved oxygen was removed by passing nitrogen gas for 30 minutes. Thereafter, the reaction was carried out at 60 ° C. for 12 hours, and DMF was partially distilled off from the reaction solution under reduced pressure, and then poured into jetyl ether (200 ml). The obtained crystals were collected by filtration and purified by reprecipitation (methanol 4 ml-jetyl ether 200 ml) to obtain the title copolymer as a pale yellow powder (228 mg, 39%).
  • 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 28.6 mm diameter whisking tube (dialysis cellulose tube). Dissolve the resulting powder in 10 ml of pure water and place it in the tube. At 48 hours, the dialysis solution was replaced with pure water every 12 hours.
  • the thermal responsiveness test of the copolymers produced in Examples 2, 3, 4 and 5 was conducted according to the following procedure.
  • a JASCO FP-6500 spectrofluorimeter was used, and ultrapure water obtained from Millipore's Milli-Q reagent system was used as the 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 to control the temperature of the solution, and the temperature was measured with the attached thermocouple.
  • the solution temperature was increased in 1 ° C increments, then decreased to the measurement start temperature in 1 ° C increments, and the fluorescence intensity at each temperature was measured.
  • the solution temperature was raised in increments of 2 degrees, then lowered to the measurement start temperature in 2 ° C increments, and the fluorescence intensity at each temperature was measured.
  • Fig. 1 shows the test results. Both compounds show strong fluorescence at high temperatures, It was confirmed that the fluorescence intensity changed in response to the change. 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 force caused by the intermolecular aggregation of macromolecules observed at high temperatures. The ionic functional group force S newly introduced by the present invention is effectively suppressed by electrostatic repulsion. The reproducibility is considered to have improved.
  • Figure 2 shows a schematic diagram of the relationship between temperature and fluorescence intensity. 3 represents a minute amount, and ⁇ represents an error. From Fig. 2 (a), it can be seen that there is the following relationship between 3 and ⁇ .
  • ⁇ FI is an error in fluorescence intensity.
  • the standard deviation is used as the error straight.
  • the standard deviation of the fluorescence intensity from T to T + c is
  • Is calculated by Figure 3 shows the relationship between the temperature and the temperature resolution calculated by Eq. (1) for 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, compound 1 has a temperature resolution between 9 ° C and 33 ° C, compound 2 between 30 ° C and 51 ° C, and compound 3 between 49 ° C and 66 ° C with a temperature resolution of 0. It is kept below 2 ° C. Therefore, with these three types of fluorescent temperature sensors, a temperature resolution of 0.2 ° C was achieved over a wide range of about 10 ° C to 65 ° C. It is considered that high temperature resolving ability was achieved because the reproducibility of the fluorescence response was improved by the effect of the ionic functional group newly introduced by the present invention.
  • FIG. 4 shows the relationship between the temperature and the calculated temperature resolution of Compound 4.
  • the temperature is changed in increments of 2 ° C, so the formula for calculating the temperature resolution is (2). It becomes like this.
  • NNPAM, NIP AM, or N-isopropylmethacrylamide (5 mmol), AIBN (50 11101), DBD-AA ⁇ mol) 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 (compound 6) or 300 ml (compounds 5 and 7) of jetyl ether. The obtained crystals were collected by filtration and the desired compound was obtained by reprecipitation.
  • a reprecipitation solvent includes acetone (3 ml) and jetyl ether (200 ml).
  • Fig. 5 shows the results of thermal response tests of the fluorescence intensities of compounds 5-7
  • Fig. 6 shows the results of evaluation of the temperature resolution of compounds 5-7.
  • the maximum temperature resolution of each compound was about 0.2 ° C, but the temperature resolution was much higher than 0.2 ° C in most temperature ranges where the temperature range was very narrow.
  • N Isopropylacrylamide N, N, Monomethylenebisacrylamide (MB AM), 3-Sulfopropyl Atalylate (SPA), N— ⁇ 2— [(7— N, N Dimethylaminosunoleonyl) 2, 1, 3 Benzoxadiazol-4-yl] (methyl) amino ⁇ ethyl-N-methylacrylamide (DBD—AA), sodium persulfate (APS), and N, N, ⁇ ', N' tetramethylethylene Copolymers of Examples 6-9 were prepared using each of diamine (TMEDA) in the amounts shown in the table below.
  • the reaction was carried out by the following method. NIPAM, MBAM, SPA (Examples 7 and 9 only), DBD—AA, and TMEDA in sodium dodecyl sulfate (SDS) aqueous solution (12.6 mM, 20 mL, but Example 9 uses 16.6 mL) Dissolved and 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. 4 hours at 70 ° C, 250rpm The mixture was stirred for a while and then the reaction was poured into cold water (400 mL). NaCI was added until precipitates were formed, and collected with a glass filter.
  • SDS sodium dodecyl sulfate
  • copolymer nanogel particles 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 28.6 mm diameter whisking tube (dialysis cellulose tube). Dissolve the sample in 10 to 25 ml of pure water and place it in the tube. The mixture was dialyzed at room temperature for 5 to 6 days with stirring, and the dialysis 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.
  • the size of the nanogenole in the S-rich state is MALVERN using an aqueous nanogel solution of 0.01 w / v% (Examples 7 and 8) or 0.001 w / v% (Example 6). It was measured by dynamic light scattering method using Zeta Sizer Nano ZS.
  • the ultrapure obtained from Milli-Q reagent system of Millipore was used as a solvent for the thermal sensitivity test of the copolymers produced in Examples 6-9 (hereinafter referred to as nanogels 1, 2, 3, and 4).
  • the procedure was the same as described above except that the measurement was performed using water and a 150 mM potassium chloride aqueous solution. After increasing the solution temperature in increments of 1 ° C with compounds:! To 4, the solution temperature was decreased in increments of 1 ° C to the measurement start temperature, and the fluorescence intensity at each temperature was measured.
  • Nanogel 5 a nanogel of Comparative Example 2 (hereinafter referred to as Nanogel 5) was obtained in the same manner as in Examples 6 to 9. Yield and physical properties are shown in Table 4 below.
  • Nanogenole 3 was dissolved in 0, 50, 100, 150, 200 mM aqueous solution of sodium chloride and potassium at a concentration of 0.001 wZv% each night, and a thermal response test of fluorescence intensity was performed in the same procedure as described above. 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.
  • Fig. 15 shows the results of the thermal sensitivity test. It was found that as the potassium chloride concentration in the solution increased, the temperature at which the fluorescence intensity increased decreased, and the relative fluorescence intensity increment increased. It was also clarified that the response became almost constant when the potassium chloride concentration was over lOOmM.
  • Nanogel 3 was added to an injection aqueous solution (80 mM potassium chloride, 10 mM dibasic sodium hydrogen phosphate, 4 mM sodium chloride, pH 7.2), and an injection solution containing nanogel 3 at lw / v% was prepared.
  • the injection solution was put into an Eppendorf tube equipped with a filter (Ultrafree-MC, Millipore), centrifuged at 10,000 ⁇ m for 7 minutes, and filtered to remove suspended matters having a diameter of 220 nm or more.
  • Cos7 cells African Green Monkey SV40-transfdkid fibroblast cell line, transferred from Tokyo Metropolitan Institute of Medical Science
  • the cell is irradiated with a 488 nm laser (Saphhire488, Coherent) and passed through a filter (DM505, BA515-550) for cutting excitation light and scattered light.
  • a 488 nm laser (Saphhire488, Coherent)
  • a filter (DM505, BA515-550) for cutting excitation light and scattered light.
  • Fig. 17 shows the fluorescence image at each temperature
  • Fig. 18 shows the change in fluorescence intensity
  • Fig. 19 shows the reversibility test results. It was confirmed that the copolymer of the present invention showed thermosensitive response even in an intracellular environment and reversibly changed the fluorescence intensity.

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Abstract

Copolymère à unités répétitives de formules (I)-(III) ci-après, pouvant également comporter une unité répétitive dérivée de monomère agent de réticulation. Également, copolymère à unité de répétition de formule (I) ou (III) ci-après et unité de répétition dérivée d'un monomère agent de réticulation. Également, thermomètre moléculaire fluorescent contenant ce type de copolymère. [ formule chimique 1] (I) (II) (III) (Dans les formules, R1, R2, R3, R4, R5, Y, X1, X2, X3, Q1, Q2 et Ar sont tels que spécifiés dans la description.)
PCT/JP2007/067147 2006-09-05 2007-09-03 Thermomètre moléculaire fluorescent Ceased WO2008029770A1 (fr)

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Cited By (3)

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WO2013094748A1 (fr) * 2011-12-21 2013-06-27 キリンホールディングス株式会社 Sonde fluorescente sensible à la température destinée à être introduite dans une cellule
JP2015218304A (ja) * 2014-05-20 2015-12-07 キリン株式会社 細胞内の温度を測定するためのレシオ型蛍光性プローブ
CN113214422A (zh) * 2015-09-07 2021-08-06 麒麟控股株式会社 细胞内递送媒介物

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JP5514368B2 (ja) * 2011-12-21 2014-06-04 キリンホールディングス株式会社 細胞内に導入するための温度感受性蛍光プローブ
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CN113214422A (zh) * 2015-09-07 2021-08-06 麒麟控股株式会社 细胞内递送媒介物
CN113214422B (zh) * 2015-09-07 2023-10-10 麒麟控股株式会社 细胞内递送媒介物

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