Fluorescent metal ion indicators with large stokes shifts

• US 9,372,181 B2
• Filed: 06/17/2014
• Issued: 06/21/2016
• Est. Priority Date: 07/27/2004
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Gee, Kyle, Martin, Vladimir
• Primary Examiner(s): Xu, Robert
• Application Number:
  US14/307,445
• Publication Number:
  US 20140363824A1
• Time in Patent Office: 735 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07C 229/18 the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
• CPC Class Codes: C07C 311/44 having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 221/14 Aza-phenalenes, e.g. 1,8-naphthalimide
• CPC Class Codes: C07D 265/38 [b, e]-condensed with two six-membered rings
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 311/90 with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
• CPC Class Codes: C07D 413/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C09B 11/06 Hydroxy derivatives of triarylmethanes in which at least one OH group is bound to an aryl nucleus and their ethers or esters
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: C09B 19/00 Oxazine dyes
• CPC Class Codes: C09B 19/005 Gallocyanine dyes
• CPC Class Codes: C09B 5/60 Thiazines; Oxazines
• CPC Class Codes: C09B 5/62 Cyclic imides or amidines of peri-dicarboxylic acids of the anthracene, benzanthrene, or perylene series
• CPC Class Codes: C09B 57/08 Naphthalimide dyes; Phthalimide dyes
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/1014 bridged by heteroatoms, e.g. N, P, Si or B
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2458/00 : Labels used in chemical analysis of biological material
• CPC Class Codes: G01N 33/84 : involving inorganic compounds or pH

Abstract

The present invention provides fluorogenic compounds for the detection of target metal ions wherein the compounds exhibit a Stokes shift greater than 50 nm and the detectable signal is modulated by photoinduced electron transfer (PET). The present compounds consist of three functional elements, the ion sensing moiety (chelating moiety), the reporter moiety (fluorophore or fluorescent protein) and spacer or linker between the sensing and reporter moieties of the present compound that allows for PET upon binding of a metal ion and excitation by an appropriate wavelength.

Claims

1. A compound for the detection of metal ions, wherein the compound comprises:

a metal chelating BAPTA moiety; and

,

a fluorophore comprising a nitrogen atom covalently bonded to one or two C═

O groups or to one SO₂ group, wherein the nitrogen atom is optionally substituted with a H, alkyl, or substituted alkyl group, wherein the nitrogen atom in the fluorophore is covalently bonded to the metal chelating moiety by a linker, —

(CR₂)_n—

, wherein each R group is independently hydrogen, alkyl, or substituted alkyl; and

n is 1-10.

2. The compound of claim 1 wherein a detectable response is modulated by photoinduced electron transfer (PET), and wherein the metal ion is a calcium ion.

3. A method for binding a target metal ion in a sample, comprising:

a) contacting the sample with the compound of claim 1; and

b) incubating the contacted sample for sufficient time to allow the metal ion binding compound to chelate the target metal ion whereby the metal ion is bound.

4. The method of claim 3, wherein the sample comprises live cells, intracellular fluids, extracellular fluids, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions or biological fluids or chemical reactors.

5. The method according to claim 3, wherein the sample comprises blood cells, immune cells, cultured cells, muscle tissue, neurons, extracellular vesicles;

vascular tissue, blood fluids, saliva, urine;

water, soil, waste water, sea water;

pharmaceuticals, foodstuffs or beverages.

6. The method according to claim 3, wherein the method further comprises detecting a target metal ion wherein the sample is illuminated with an appropriate wavelength whereby the target metal ion is detected.

7. The method according to claim 3, wherein the metal ion is Ca²⁺, Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ba²⁺, or Sr²⁺.

8. A kit for binding a metal ion in a sample, comprising:

a) at least one compound of claim 1; and

b) instructions for use thereof.

9. The kit of claim 8, wherein the kit further comprises one or more components selected from the group consisting of a calibration standard of a metal ion, an ionophore, a metal ion indicator other than for calcium ions, a detectable signal standard, an aqueous buffer solution, an antibody or fragment thereof, a reference dye standard and an organic solvent.

10. The compound of claim 1, wherein the metal ion is Ca²⁺, Zn²⁺, Mg²⁺, Ga³⁺, Tb³⁺, La³⁺, Pb²⁺, Hg²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ba²⁺, or Sr²⁺.

11. The compound of claim 1, wherein the detectable response produced by the compound is a fluorescent response exhibiting a Stokes shift of greater than 50 nm.