Excimer forming compounds

Patent metadata

Field	Value
Publication number	US-2016304473-A1
Application number	US-201415102953-A
Country	US
Kind code	A1
Filing date	Dec 19, 2014
Priority date	Dec 19, 2013
Publication date	Oct 20, 2016
Grant date	—

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

1 .- 45 . (canceled)

46 . A method of detecting pyrophosphates, 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; Y is a metal ion coordinating moiety; 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 pyrophosphates, RNA or a proximally phosphorylated polypeptide.

47 . The method of claim 46 , 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.

48 . The method of claim 47 , 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.

49 . The method of claim 48 , wherein the wherein the excimer forming fluorophore is substituted or unsubstituted

50 . The method of claim 46 , wherein the linker moiety is
i) C 1-40 -alkylene, C 2-40 -alkenylene, C 2-40 -alkynylene, or C 3-20 -cycloalkyl each of which is optionally oxo-substituted (═O) 1-3 times, and in which 1-3 carbon atoms are optionally replaced with a heteroatom selected from N, O, S or Si; ii) C 6-10 -aryl, or C 5-10 -heteroaryl, each of which is optionally substituted with 1-4 R groups, wherein R is simultaneously or independently C 1-20 -alkylene, C 2-20 -alkenylene or C 2-20 -alkynylene, each of which is optionally oxo-substituted (═O) between 1-3 times, and in which 1-3 carbon atoms are optionally replaced with a heteroatom selected from N, O, S or S.

51 . The method of claim 50 , wherein the linker moiety is C 1-20 -alkylene, C 2-20 -alkenylene, or C 2-20 -alkynylene, each of which is optionally oxo-substituted (═O) 1-3 times, and in which 1-3 carbon atoms are optionally replaced with a heteroatom selected from N, O, S or Si.

52 . The method of claim 51 , wherein the linker moiety is C 1-6 -alkylene, which is optionally oxo-substituted (═O) 1-3 times, and in which 1-3 carbon atoms are optionally replaced with a heteroatom selected from N, O, S or Si.

53 . The method of claim 50 , wherein the linker moiety is methylene, butylene,

54 . The method of claim 46 , wherein the metal ion coordinating moiety is a multi-dentate moiety comprising one or more of amino, carboxyl or hydroxyl groups.

55 . The method of claim 54 , wherein the metal ion coordinating moiety is a tetra-dentate amino group.

56 . The method of claim 55 , wherein the tetra-dentate amino compound is optionally substituted

57 . The method of claim 54 , wherein the metal ion coordinating moiety is

58 . The method of claim 46 , wherein the compound is

59 . The method of claim 46 , wherein the metal ion is a transition metal ion, a lanthanide metal ion or a post-transition metal ion.

60 . The method of claim 59 , 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), Al(III), and the lanthanide metal ion is Tb (III).

61 . The method of claim 46 , wherein the sample is a bodily sample.

62 . The method of claim 61 , wherein the bodily sample is urine, synovial fluid or blood.

63 . The method of claim 46 , wherein the proximally phosphorylated polypeptide comprises amino acids that are proximally phosphorylated within 1-6 amino acid residues of each other.

64 . A method of quantifying pyrophosphates, RNA or proximally phosphorylated polypeptides, comprising:
(a) contacting a sample with an excimer forming compound of the Formula I as defined in claim 46 , 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 pyrophosphates, RNA or proximally phosphorylated polypeptides; wherein detection of a signal having a fluorescence intensity similar to the control sample indicates that the sample contains pyrophosphates, RNA or proximally phosphorylated polypeptides.

65 . 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 as defined in claim 46 , 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 unactivated protein sample indicates that the protein sample is activated.

Assignees

• Governing Council Univ Toronto

Inventors

• Gunning Patrick Thomas
• Kraskouskaya Dziyana

Classifications

• G01N2440/14

  phosphorylation · CPC title

• C07C2103/50

  Chemistry & Metallurgy · mapped topic

• G01N33/52

  Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} · CPC title

• C07D257/02 Primary

  not condensed with other rings · CPC title

• C07C309/65

  of a saturated carbon skeleton · CPC title