Coumarin-based fluorogenic agents and uses thereof for specific protein labelling

Patent metadata

Field	Value
Publication number	US-9701667-B2
Application number	US-201514704289-A
Country	US
Kind code	B2
Filing date	May 5, 2015
Priority date	May 5, 2014
Publication date	Jul 11, 2017
Grant date	Jul 11, 2017

Abstract

There are provided fluorescent labelling agents comprising a dimaleimide core connected to a fluorophore, processes for preparation thereof, and uses thereof for labelling and/or detection of specific protein targets. Fluorescent labelling agents comprising a compound having the structure of Formula I, and salts thereof, are described.

First claim

What is claimed is:

1. A fluorogenic labelling agent which is a compound of Formula I, or a salt thereof:

wherein:
R 1 is OR 1 ′ or NR 2 ′R 3 ′, wherein R 1 ′, R 2 ′ and R 3 ′ are independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, and carboxy alkyl, alkyl being optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl; or R 1 ′ and R 2 or R 1 ′ and R 3 come together to form a 5, 6 or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl, and heteroaromatic; or R 2 ′, R 2 , R 3 ′, and R 3 come together independently to form at least one 5, 6 or 7-membered ring which is selected from aryl, heterocyclic, heteroaryl and heteroaromatic;
R 2 , R 3 and R 4 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carboxy alkyl, aryl, heterocyclic, heteroaryl, and heteroaromatic, alkyl being optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl;
R 5 and R 6 are independently selected from hydrogen, halogen, alkyl, alkoxy, keto, carboxylate, carboxylic ester, carbamate, amide, amino, alkylamino, polyether, alkylthio, cyano, nitro, sulfonyl, X 1 , and X 2 , wherein both R5 and R6 are X1, both R5 and R6 are X2 or only one of R 5 and R 6 is X 1 or X 2 , and alkyl is optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl;
X 1 is

X 2 is

 and
R 7 and R 8 are independently R 9 or OR 10 , wherein R 9 is selected from hydrogen, halogen, and alkyl, alkyl being optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl, and R 10 is alkyl, alkyl being optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl.

2. The fluorogenic labelling agent of claim 1 , wherein: at least one of R 7 and R 8 is OR 10 ; R 7 and R 8 are the same; R 9 and R 10 are the same; when R 7 is OR 10 , R 8 is R 9 ; or, when R 7 is R 9 , R 8 is OR 10 .

3. The fluorogenic labelling agent of claim 1 , wherein: R 5 is X 1 or X 2 and R 6 is hydrogen; R 5 is hydrogen and R 6 is X 1 or X 2 ; R 5 and R 6 are both X 1 or X 2 ; one of R 5 and R 6 is X 1 and the other is X 2 ; or, wherein R 5 is hydrogen; R 6 is X 2 ; R 7 is methyl; and R 8 is methoxy; or, wherein R 5 is hydrogen; R 6 is X 2 ; and R 7 and R 8 are methoxy; or, wherein R 2 , R 3 , and R 4 are hydrogen.

4. The fluorogenic labelling agent of claim 1 , wherein R 1 is selected from:

or
wherein R 1 is selected from:

where n 1 and n 2 are independently 1 or higher than 1.

5. The fluorogenic labelling agent of claim 1 , wherein said fluorogenic labelling agent is a compound of Formula II, or a salt thereof:

wherein:
R 1 , R 2 , R 3 , R 4 , R 7 , and R 8 are as defined in claim 1 ; and
R 5 is selected from hydrogen, halogen, alkyl, alkoxy, keto, carboxylate, carboxylic ester, carbamate, amide, amino, alkylamino, polyether, alkylthio, cyano, nitro, and sulfonyl, wherein alkyl is optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl.

6. The fluorogenic labelling agent of claim 1 , wherein said fluorogenic labelling agent is a compound of Formula III, or a salt thereof:

wherein:
R 1 , R 2 , R 3 , R 4 , R 7 , and R 8 are as defined in claim 1 ; and
R 5 is selected from hydrogen, halogen, alkyl, alkoxy, keto, carboxylate, carboxylic ester, carbamate, amide, amino, alkylamino, polyether, alkylthio, cyano, nitro, and sulfonyl, wherein alkyl is optionally substituted by hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl.

7. The fluorogenic labelling agent of claim 5 , wherein R 7 and R 8 are the same; R 9 and R 10 are the same; when R 7 is OR 10 , R 8 is R 9 ; when R 7 is R 9 , R 8 is OR 10 ; at least one of R 7 and R 8 is OR 10 ; R 7 is methyl and R 8 is methoxy; or, both R 7 and R 8 are methoxy.

8. The fluorogenic labelling agent of claim 5 , wherein R 2 , R 3 , and R 4 are hydrogen; R 5 is hydrogen; or, R 1 is selected from:

or
wherein R 1 is selected from:

where n 1 and n 2 are independently 1 or higher than 1.

9. The fluorogenic labelling agent of claim 5 , wherein R 2 , R 3 , and R 4 are hydrogen, and R 1 is selected from:

10. The fluorogenic labelling agent of claim 1 , wherein R 2 , R 3 , and R 4 are hydrogen, and R 1 is selected from:

where n 1 and n 2 are independently 1 or higher than 1.

11. The fluorogenic labelling agent of claim 5 , wherein R 2 , R 3 , R 4 , and R 5 are hydrogen; R 7 is methyl or methoxy; R 8 is hydrogen, methyl or methoxy; and R 1 is selected from:

or
wherein R 1 is selected from:

where n 1 and n 2 are independently 1 or higher than 1.

12. The fluorogenic labelling agent of claim 1 , wherein said fluorogenic labelling agent is selected from:

and salts thereof.

13. A method for labelling and/or detecting a target protein, comprising:
a) contacting the target protein with the fluorogenic labelling agent of claim 1 , under conditions where the fluorogenic labelling agent reacts with sterically unhindered sulfhydryl groups on the target protein; and
b) detecting a fluorescent signal from the fluorogenic labelling agent,
wherein the fluorescence of the fluorogenic labelling agent is quenched in the absence of reaction with the target protein, and detection of the fluorescent signal indicates that reaction of the fluorogenic labelling agent with the target protein; or, wherein the fluorescence of the fluorogenic labelling agent increases after reaction with the target protein.

14. The method of claim 13 , wherein said contacting occurs in vivo, ex vivo, or in vi

Assignees

• Univ Ottawa

Inventors

• Keillor Jeffrey
• Chen Yingche

Classifications

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• C07D405/14 Primary

  containing three or more hetero rings · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title

• C09B57/02

  Coumarine dyes · CPC title