Temperature-sensitive fluorescent probe for introduction into cell

Patent metadata

Field	Value
Publication number	US-2019113505-A1
Application number	US-201816219035-A
Country	US
Kind code	A1
Filing date	Dec 13, 2018
Priority date	Dec 21, 2011
Publication date	Apr 18, 2019
Grant date	—

Abstract

There is provided a method for introducing a temperature-sensitive probe comprising a copolymer, which comprises a thermoresponsive unit and a fluorescent unit, into a cell, and the method using the copolymer further comprising a cationic unit as the temperature-sensitive probe, and the method comprising the step of mixing the copolymer with the cell in a solvent. The copolymer can be preferably used as a fluorescence temperature sensor which measures intracellular temperature since the copolymer has a cationic group and thus enters into a cell without using a special method.

First claim

1 . A method for introducing a temperature-sensitive probe comprising a copolymer, which comprises a thermoresponsive unit and a fluorescent unit, into a cell, the method using the copolymer further comprising a cationic unit as a temperature-sensitive probe, and the method comprising the step of mixing the copolymer with cells in a solvent.

2 . The method according to claim 1 , wherein the cationic unit is a repeating structure derived from a monomer which contains an ionic functional group having one or more positive charges.

3 . The method according to claim 1 , wherein the cationic unit is a repeating structure derived from a monomer represented by the following formula (b):

wherein R 2 is selected from hydrogen atom and C 1-3 alkyl;
W is aromatic ring or —X 1 —C(═O)— (in which X 1 is directly bonded to Q 1 );
X 1 is O, S, or N—R 11 ;
R 11 is hydrogen atom, C 1-6 alkyl, or -Q 1 -Y, in which the alkyl may be optionally substituted by one or more substituent(s) selected from hydroxy, halogen atom, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylsulfinyl, and C 1-6 alkylsulfonyl;
Q 1 is independently selected from C 1-20 alkylene, C 3-20 alkenylene, or C 3-20 alkynylene, in which O, S, —O—P(═O)(—OH)—O—, or phenylene may be independently inserted into the alkylene at one or more position(s);
Y is independently an ionic functional group capable of having one or more positive charges, and is selected from —N + R 21 R 22 R 23 X e − , —C(═NR 41 )—NR 42 R 43 , and a group represented by the following formula:

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 may be combined with the nitrogen atom to which they are attached to form a 5- to 7-membered nitrogen-containing hetero ring;
R 24 is C 1-10 alkyl or C 7-14 aralkyl; and
R 41 , R 42 , and R 43 are independently selected from hydrogen atom and C 1-10 alkyl, or R 41 and R 42 may be combined with the nitrogen and carbon atoms to which they are attached to form a 5- to 7-membered hetero ring containing two nitrogen atoms, or R 42 and R 43 may be combined with the nitrogen atom to which they are attached to form a 5- to 7-membered nitrogen-containing hetero ring.

4 . The method according to claim 1 , wherein the thermoresponsive unit is a repeating structure derived from a monomer comprising acrylamide as a basic skeleton.

5 . The method according to claim 1 , wherein the thermoresponsive unit is a repeating structure derived from a monomer represented by the following formula (a):

wherein R 1 is selected from hydrogen atom and C 1-3 alkyl; and
R 4 and R 5 are independently selected from hydrogen atom and C 1-20 alkyl, in which the alkyl may be optionally substituted by one or more substituent(s) selected from hydroxy, C 1-6 alkoxy, and aryl, or R 4 and R 5 may be combined with the nitrogen atom to which they are attached to form a 4- to 8-membered nitrogen-containing hetero ring, in which the hetero ring may be optionally substituted by one or more substituent(s) selected from C 1-6 alkyl, C 1-6 alkoxy, nitro, halogen atom, C 1-10 alkylcarbonylamino, and arylcarbonylamino.

6 . The method according to claim 1 , wherein the fluorescent unit is a repeating structure derived from a monomer represented by the following formula (c):

wherein R 3 is selected from hydrogen atom and C 1-3 alkyl;
X 2 is O, S, or N—R 12 ;
X 3 is 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, in which O, S, or phenylene may be independently inserted into the alkylene at one or more position(s);
Ar is selected from a 6- to 18-membered aromatic carbocyclic group or a 5- to 18-membered aromatic heterocyclic group, in which the aromatic carbocyclic group and aromatic heterocyclic group may contain a fused ring, one or more ring(s) contained therein being aromatic ring(s), —CH 2 — existing in the aromatic carbocyclic group and the aromatic heterocyclic group as a ring atom may be optionally replaced with —C(O)—, and also the aromatic carbocyclic group and the aromatic heterocyclic group may be optionally substituted by one or more substituent(s) selected from halogen atom, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, nitro, cyano, C 1-6 alkylcarbonyl, C 1-6 alkoxycarbonyl, carboxy, formyl, —NR 6 R 7 , and —SO 2 NR 14 R 15 (in which alkyl contained in the C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylsulfinyl, C 1-6 alkylsulfonyl, C 1-6 alkylcarbonyl, and C 1-6 alkoxycarbonyl may be optionally substituted by one or more substituent(s) selected from halogen atom, 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 selected from 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, in which alkyl contained in the C 1-10 alkyl, C 1-10 alkylcarbonyl, C 1-10 alkylsulfonyl, N—(C 1-10 alkyl)carbamoyl, and N,N-di(C 1-10 alkyl)carbamoyl may be optionally substituted by one or more substituent(s) selected from halogen atom, C 1-6 alkoxy, hydroxy, amino, C 1-6 alkylamino, di(C 1-6 alkyl)amino, aryl, and carboxy, and also aryl contained in the aryl, arylcarbonyl, and arylsulfonyl may be optionally substituted by one or more substituent(s) selected from halogen atom, C 1-6 alkyl, C 1-6 alkoxy, and carboxy; or
R 6 and R 7 may be combined with the nitrogen atom to which they are attached to form a 4- to 8-membered nitrogen-containing hetero ring, in which the hetero ring may be optionally substituted by one or more substituent(s) selected from C 1-6 alkyl, C 1-6 alkoxy, nitro, halogen atom, C 1-10 alkylcarbonylamino, and arylcarbonylamino;
R 12 is hydrogen atom, C 1-6 alkyl, or -Q 2 -X 3 —Ar, in which the alkyl may be optionally substituted by one or more substituent(s) selected from hydroxy, halogen atom, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylsulfinyl, and C 1-6 alkylsulfonyl;
R 13 is hydrogen atom or C 1-6 alkyl, in which the alkyl may be optionally substituted by one or more substituent(s) selected from hydroxy, halogen atom, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylsulfinyl, and C 1-6 alkylsulfonyl;
R 14 and R 15 are independently selected from hydrogen atom and C 1-6 alkyl; or R 14 and R 15 may be combined with the nitrogen atom to which they are attached to form a 4- to 8-membered nitrogen-containing hetero ring; and
R 16 , R 17 , R 18 , and R 19 are independently selected from hydrogen atom and C 1-6 alkyl, in which the alkyl may be optionally substituted by one or more substituent(s) selected from hydroxy, halogen atom, C 1-6 alkoxy, C 1-6 alkylthio, C 1-6 alkylsulfinyl, and C 1-6 alkylsulfonyl.

7 . The method according to claim 1 , wherein the copolymer is represented by formula (IV):

wherein R 1 , R 4 , and R 5 are as defined in claim 5 , R 2 , Y, W, and Q 1 are as defined in claim 3 , R 3 , X

Assignees

• Kirin Holdings Kk
• Univ Tokyo

Inventors

• Tsuji Toshikazu
• Ikado Kumiko
• Uchiyama Seiichi
• Kawamoto Kyoko

Classifications

• C08F228/06

  by a heterocyclic ring containing sulfur · CPC title

• C08F220/60 Primary

  containing nitrogen in addition to the carbonamido nitrogen · CPC title

• C08F220/56 Primary

  Acrylamide; Methacrylamide · CPC title

• C08F28/06

  by a heterocyclic ring containing sulfur · CPC title

• C08F220/38

  Esters containing sulfur · CPC title