Highly fluorogenic protein labelling agents

Patent metadata

Field	Value
Publication number	US-9958452-B2
Application number	US-201615378400-A
Country	US
Kind code	B2
Filing date	Dec 14, 2016
Priority date	Dec 14, 2015
Publication date	May 1, 2018
Grant date	May 1, 2018

Abstract

There are provided fluorogenic labelling agents comprising a dimaleimide core connected to a BODIPY-based fluorophore, processes for preparation thereof, and uses thereof for labelling and/or detection of specific protein targets. Fluorogenic labelling agents having an excitation wavelength that can be visualized in the green or red channels of fluorescence microscopes are provided. In some embodiments, fluorogenic labelling agents comprising a compound having the structure of Formula I or II, and salts thereof, are described.

First claim

What is claimed is:

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

wherein:
R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carboxy alkyl, heterocyclic, and heteroaryl; and
X and Y are independently R 7 or OR 8 , wherein R 7 is selected from hydrogen, halogen, and substituted or unsubstituted alkyl and R 8 is substituted or unsubstituted alkyl.

2. The fluorogenic labelling agent of claim 1 , wherein:
at least one of X and Y is OR 8 ;
when one of X and Y is OR 8 , then the other is R 7 ;
X and Y are the same;
X and Y are both OR 8 , optionally wherein R 8 is alkyl, and optionally wherein X and Y are both methoxy; or
R 7 and R 8 are the same.

3. The fluorogenic labelling agent of claim 1 , wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen and alkyl.

4. The fluorogenic labelling agent of claim 3 , wherein:
R 2 and R 5 are hydrogen and R 1 , R 3 , R 4 , and R 6 are alkyl, optionally wherein said alkyl is methyl; or
R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are hydrogen.

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

wherein:
R 1 is hydrogen, R 1 ′, SR 1 ′, OR 1 ′ or NR 2 ′R 3 ′, wherein R 1 ′, R 2 ′ and R 3 ′ are independently selected from hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, and carboxy alkyl; 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, and heteroaryl; 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, and heteroaryl;
R 2 , R 3 , R 4 , R 5 and R 6 are independently selected from hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carboxy alkyl, heterocyclic, and heteroaryl;
X and Y are independently R 7 or OR 8 , wherein R 7 is selected from hydrogen, halogen, and substituted or unsubstituted alkyl, and R 8 is substituted or unsubstituted alkyl; and
Ar is substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic, or substituted or unsubstituted heteroaryl.

6. The fluorogenic labelling agent of claim 5 , wherein:
at least one of X and Y is OR 8 ;
when one of X and Y is OR 8 , then the other is R 7 ;
X and Y are the same;
X and Y are both OR 8 , optionally wherein R 8 is alkyl; or
R 7 and R 8 are the same, optionally wherein both X and Y are methoxy.

7. The fluorogenic labelling agent of claim 5 , wherein:
R 2 , R 3 , R 4 , R 5 and R 6 are hydrogen;
R 1 is R 1 ′, SR 1 ′, OR 1 ′ or NR 2 ′R 3 ′;
R 1 is an amino substituent, an oxygen substituent, a sulfur substituent, a thiol ether, or an ester; or
R 1 is selected from hydrogen, MeO 2 CCH 2 S, and NMePh.

8. The fluorogenic labelling agent of claim 5 , wherein Ar is phenyl, pyridine, pyrimidine or triazine and is optionally substituted by alkyl, cycloalkyl or halogen, alkyl being optionally substituted with hydroxyl, amino, carboxyl, sulfonate, carboxylic ester, amide, carbamate, or aminoalkyl.

9. The fluorogenic labelling agent of claim 1 , wherein said fluorogenic labelling agent comprises a compound selected from YC23, YC28, and YC29:

or a salt thereof.

10. The fluorogenic labelling agent of claim 1 , wherein the fluorogenic labelling agent is not toxic to animal cells.

11. The fluorogenic labelling agent of claim 10 , wherein the animal cells are mammalian cells, invertebrate cells, vertebrate cells, human cells, rodent cells, mouse cells, rat cells, insect cells, nematode cells, or fish cells.

12. The fluorogenic labelling agent of claim 1 , wherein the fluorogenic labelling agent's fluorescence is quenched when the fluorogenic labelling agent is in its conjugated form, and not quenched in the form of a thiol adduct, or wherein the fluorescence of the fluorogenic labelling agent increases after reaction with sulfhydryl groups on a protein.

13. The fluorogenic labelling agent of claim 1 , wherein the fluorogenic labelling agent specifically reacts with two Cys residues separated by about 10 Å or a dC10α tag, or wherein the fluorogenic labelling agent does not react appreciably with cellular proteins or with glutathione (GSH).

14. The fluorogenic labelling agent of claim 1 , wherein the fluorogenic labelling agent has one or more of the following characteristics: aqueous solubility; non-toxic to animal cells; low background fluorescence before reaction with a target protein; increased fluorescence after reaction with a target protein; bright fluorescence after reaction with a target protein; cell permeability; non-reactivity with GSH; and specific binding to two sulfhydryl residues separated by about 10 Å or a dC10α tag.

15. The fluorogenic labelling agent of claim 1 , wherein the fluorogenic labelling agent is visible by fluorescent microscope using the green or red channel.

16. 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.

17. The method of claim 16 , wherein said contacting occurs in vivo, ex vivo, or in vitro.

18. The method of claim 16 , wherein the target protein comprises two Cys residues separated by about 10 Å or comprises a dC10α tag.

19. The method of claim 16 , wherein the target protein has been genetically engineered to comprise two Cys residues separated by about 10 Å or a dC10α tag.

20. The method of claim 16 , wherein said contacting occurs in a cultured cell expressing the target protein, wherein said target protein is an intracellular protein, an extracellular protein, or a cell-surface protein, optionally wherein said contacting occurs intracellularly.

21. A method for live imaging of 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 increases after reaction with the target protein, or is detectable only after reaction with the

Assignees

• Univ Ottawa

Inventors

• Keillor Jeffrey
• Chen Yingche

Classifications

• C09K2211/1029

  containing one nitrogen atom as the heteroatom · CPC title

• C07K1/13

  Labelling of peptides · CPC title

• C09K2211/1022

  bridged by heteroatoms, e.g. N, P, Si or B · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title

• C09K11/06

  containing organic luminescent materials · CPC title