Excimer forming compounds

Patent metadata

Field	Value
Publication number	US-10889554-B2
Application number	US-201415102953-A
Country	US
Kind code	B2
Filing date	Dec 19, 2014
Priority date	Dec 19, 2013
Publication date	Jan 12, 2021
Grant date	Jan 12, 2021

Abstract

The present application is directed to excimer forming compounds of Formula I: W—V—[Y]n wherein W is an excimer forming fluorophore, V is a linker moiety, Y is a metal ion coordinating moiety and n is 1, 2 or 3. In particular, the application is directed to excimer forming compounds for the detection of proximally phosphorylated sites including those found on polypeptides, proteins, pyrophosphate and RNA, for example in aqueous solution, polyacrylamide gels blotting membranes, solid-support assays and in cell culture samples.

First claim

The invention claimed is:

1. A method of detecting RNA or proximally phosphorylated polypeptides, comprising:
(a) contacting a sample with an excimer forming compound of the Formula I
W—V Y] n   (I)

wherein,
W is an excimer forming fluorophore;
V is a linker moiety, wherein the linker moiety is C 1-10 -alkylene;
Y is a metal ion coordinating moiety having the structure

and
n is 1, 2 or 3; and
a suitable metal ion;
(b) detecting a fluorescence signal at a wavelength specific for the excimer forming fluorophore of the compound;
(c) comparing the fluorescence signal of (b) with the fluorescence intensity of a control sample;
wherein detection of a signal in the sample having a fluorescence intensity greater than the control sample indicates that the sample contains RNA or a proximally phosphorylated polypeptide.

2. The method of claim 1 , wherein the excimer forming fluorophore is optionally substituted C 10-40 -aryl or optionally substituted C 9-40 -heteroaryl, wherein the optional substituents are selected from halo, carboxy, hydroxyl, C 1-20 -alkyl, C 2-20 -alkenyl, C 2-20 -alkynyl, C 3-20 cycloalkyl, C 1-20 alkoxy, —NR′R″, C 6-14 -aryl, and C 5-14 -heteroaryl, wherein R′ and R″ are simultaneously or independently H or C 1-6 alkyl.

3. The method of claim 2 , wherein the excimer forming fluorophore is optionally substituted

wherein the optional substituents are selected from halo, carboxy, hydroxyl, C 1-20 -alkyl, C 2-20 -alkenyl, C 2-20 -alkynyl, C 3-20 cycloalkyl, C 1-20 alkoxy, —NR′R″ C 6-14 -aryl, and C 5-14 -heteroaryl, wherein R′ and R″ are simultaneously or independently H or C 1-6 alkyl.

4. The method of claim 3 , wherein the wherein the excimer forming fluorophore is substituted or unsubstituted

5. The method of claim 1 , wherein the linker moiety is C 1-6 -alkylene.

6. The method of claim 1 , wherein the compound is

7. The method of claim 1 , wherein the metal ion is a transition metal ion, a lanthanide metal ion or a post-transition metal ion.

8. The method of claim 7 , wherein the transition metal ion is Zn(II), Cu(II), Mn(II), Fe(II), Fe(III) or Ni(II) and the post-transition metal ion is Ga(III), AI(III), and the lanthanide metal ion is Tb (III).

9. The method of claim 1 , wherein the sample is a bodily sample.

10. The method of claim 9 , wherein the bodily sample is urine, synovial fluid or blood.

11. The method of claim 1 , wherein the proximally phosphorylated polypeptide comprises amino acids that are proximally phosphorylated within 1-6 amino acid residues of each other.

12. A method of quantifying RNA or proximally phosphorylated polypeptides, comprising:
(a) contacting a sample with an excimer forming compound of the Formula I
W—V Y] n   (I)

wherein
W is an excimer forming fluorophore;
V is a linker moiety, wherein the linker moiety is C 1-10 -alkylene;
Y is a metal ion coordinating moiety having the structure

and
n is 1, 2 or 3; and
a suitable metal ion;
(b) detecting a fluorescence signal at a wavelength specific for the excimer forming fluorophore of the compound;
(c) comparing the fluorescence signal of (b) with the fluorescence intensity of a control sample containing known quantities of RNA or proximally phosphorylated polypeptides;
wherein detection of a signal having a fluorescence intensity similar to the control sample indicates that the sample contains RNA or proximally phosphorylated polypeptides.

13. A method of monitoring the activity of a kinase or phosphatase protein, the method comprising:
(a) contacting a sample of the kinase or phosphatase protein with an excimer forming compound of the Formula I
W—V Y] n   (I)

wherein
W is an excimer forming fluorophore;
V is a linker moiety, wherein the linker moiety is C 1-10 -alkylene;
Y is a metal ion coordinating moiety having the structure

and
n is 1 2 or 3; and
a suitable metal ion;
(b) detecting a fluorescence signal at a wavelength specific for the excimer forming fluorophore of the compound;
(c) comparing the fluorescence signal of (b) with the fluorescence intensity of an unactivated protein sample;

wherein detection of a signal having a fluorescence intensity greater than the inactivated protein sample indicates that the protein sample is activated.

14. The method of claim 12 , wherein the excimer forming fluorophore is optionally substituted

wherein the optional substituents are selected from halo, carboxy, hydroxyl, C 1-20 -alkyl, C 2-20 -alkenyl, C 2-20 -alkynyl, C 3-20 cycloalkyl, C 1-20 alkoxy, —NR′R″ C 6-14 -aryl, and C 5-14 -heteroaryl, wherein R’ and R″ are simultaneously or independently H or C 1-6 alkyl.

15. The method of claim 14 , wherein the wherein the excimer forming fluorophore is substituted or unsubstituted

16. The method of claim 12 , wherein the compound is

17. The method of claim 12 , wherein the wherein the metal ion is a transition metal ion, a lanthanide metal ion or a post-transition metal ion, and wherein the transition metal ion is Zn(II), Cu(II), Mn(II), Fe(II), Fe(III) or Ni(II) and the post-transition metal ion is Ga(III), AI(III), and the lanthanide metal ion is Tb (III).

18. The method of claim 13 , wherein the excimer forming fluorophore is optionally substituted

wherein the optional substituents are selected from halo, carboxy, hydroxyl, C 1-20 -alkyl, C 2-20 -alkenyl, C 2-20 -alkynyl, C 3-20 cycloalkyl, C 1-20 alkoxy, —NR′R″ C 6-14 -aryl, and C 5-14 -heteroaryl, wherein R′ and R″ are simultaneously or independently H or C 1-6 alkyl.

19. The method of claim 13 , wherein the compound is

20. The method of claim 13 , wherein the wherein the metal ion is a transition metal ion, a lanthanide metal ion or a post-transition metal ion, and wherein the transition metal ion is Zn(II), Cu(II), Mn(II), Fe(II), Fe(III) or Ni(II) and the post-transition metal ion is Ga(III), AI(III), and the lanthanide metal ion is Tb (III).

Assignees

• Governing Council Univ Toronto

Inventors

• Gunning Patrick Thomas
• Kraskouskaya Dziyana

Classifications

• C09B57/001

  Pyrene dyes · CPC title

• G01N33/6842

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title

• C09B1/00

  Dyes with anthracene nucleus not condensed with any other ring · CPC title

• C07D403/10

  linked by a carbon chain containing aromatic rings · CPC title

• C09B57/00

  Other synthetic dyes of known constitution · CPC title