Temperature-sensitive fluorescent probe for introduction into cell

Patent metadata

Field	Value
Publication number	US-11199537-B2
Application number	US-201816219121-A
Country	US
Kind code	B2
Filing date	Dec 13, 2018
Priority date	Dec 21, 2011
Publication date	Dec 14, 2021
Grant date	Dec 14, 2021

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

The invention claimed is:

1. A temperature-sensitive probe comprising a copolymer, the copolymer comprising at least one thermoresponsive unit, a first fluorescent unit and a second fluorescent unit, and a cationic unit,
wherein the first fluorescent unit and the second fluorescent unit each have a mutually different maximum fluorescence wavelength,
wherein the copolymer comprises repeating units represented by formula (I), formula (II), formula (III), and formula (XIII):

wherein R 1 is selected from hydrogen atom and C 1-3 alkyl;
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; and
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 the alkylene may be independently inserted into O, S, —O—P(═O)(—OH)—O—, or phenylene 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; and
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 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; and
wherein R 55 is selected from hydrogen atom and C 1-3 alkyl;
R 51 , R 52 , R 53 , and R 54 are independently selected from hydrogen atom and C 1-6 alkyl;
X 4 is direct bond, phenylene, -Q 4 -O—C(═O)— (in which Q 4 is directly bonded to a boron dipyrromethene skeleton), or -Q 4 -N(—R 61 )—C(═O)— (in which Q 4 is directly bonded to a boron dipyrromethene skeleton);
R 61 is selected from hydrogen atom and C 1-6 alkyl;
Q 4 is selected from C 1-20 alkylene, phenylene, and naphthylene, and the phenylene and naphthylene may be optionally substituted by one or more substituent(s) selected from halogen atom, C 1-6 alkoxy, hydroxy, amino, and carboxy, and
wherein a, b, c, and d are numerals representing a ratio of each repeating unit in formula of more than 0, the sum of a and b being 100, b being 2 to 10.

2. The temperature-sensitive probe according to claim 1 , wherein the first fluorescent unit and the second fluorescent unit generate fluorescence with mutually different maximum fluorescence wavelengths by excitation light irradiation at an identical wavelength.

3. The temperature

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

• C08F220/382

  and containing oxygen, e.g. 2-sulfoethyl (meth)acrylate · CPC title