Use of novel coumarins as glutathione and thiol labels

• US 8,981,100 B2
• Filed: 07/24/2008
• Issued: 03/17/2015
• Est. Priority Date: 07/27/2007
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Kang, Hee Chol, Gee, Kyle, Johnson, Iain, Janes, Michael
• Primary Examiner(s): Mabry, John
• Assistant Examiner(s): CARCANAGUE, DANIEL R
• Application Number:
  US12/666,256
• Publication Number:
  US 20110045503A1
• Time in Patent Office: 2,427 Days
• Field of Search: 546/66, 514/288
• US Class Current: 546/66
• CPC Class Codes: C07D 491/06 Peri-condensed systems
• CPC Class Codes: C07D 491/16 Peri-condensed systems
• CPC Class Codes: C07F 9/5442 Aromatic phosphonium compounds (P-C aromatic linkage)
• CPC Class Codes: C07K 5/0215 containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
• CPC Class Codes: G01N 2333/4701 Details
• CPC Class Codes: G01N 33/52 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 Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10T 436/17 Nitrogen containing
• CPC Class Codes: Y10T 436/182 Organic or sulfhydryl containing [e.g., mercaptan, hydrogen, sulfide, etc.]

Abstract

Fluorescent quinolizinocoumarin compounds substituted with electrophilic reactive groups that bind thiol compounds are described. The compounds are useful in detecting oxidative stress and processes associated therewith in live cells.

Claims

1. A compound selected from the group consisting of 2,3,6,7-tetrahydro-9-bromomethyl-1H,5H-quinolizino(9,1-gh)coumarin and 2,3,6,7-tetrahydro-9-chloromethyl-1H,5H-quinolizino(9,1-gh)coumarin.

2. A coumarin compound of Formula (I) or a salt or tautomer thereof:

3. The compound of claim 2, wherein X is halo.

4. The compound of claim 2, wherein X is bromo.

5. The compound of claim 2, wherein X is chloro.

6. The compound of claim 2, wherein X is a tosyl, brosyl or mesyl group.

8. A method for detecting or quantifying a thiol compound in a sample, the method comprising:

(a) contacting the sample with the coumarin compound of claim 2, to form a contacted sample;

(b) incubating the contacted sample for an appropriate amount of time to form an incubated sample;

(c) illuminating the incubated sample with an appropriate wavelength to form an illuminated sample; and

(d) detecting fluorescent emissions from the illuminated sample;

wherein the fluorescent emissions are used to detect or quantify the thiol compound in the sample.

9. The method of claim 8, wherein the sample comprises cells.

10. The method of claim 9, wherein the contacted sample is incubated for a sufficient amount of time for the coumarin compound to enter the cell.

11. The method of claim 8, wherein the sample comprises live cells, intracellular fluids, extracellular fluids, sera, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions biological fluids or chemical reactors, 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.

12. The method of claim 8, wherein the thiol compound reacts with the coumarin compound.

13. The method of claim 8, wherein the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, and on a microfluidic chip.

14. The method of claim 9, wherein the coumarin compound is substituted with a cationic group and the glutathione that is detected or quantified is present in the mitochondria of the cell.

15. The method of claim 14, wherein the cationic group comprises a triphenylphosphonium ion.

16. The method of claim 8, wherein the thiol compound comprises a cysteine residue.

17. The method of claim 8, wherein the thiol compound is glutathione.

18. The method of claim 8, further comprising a step of fixing the sample.

19. The method of claim 8, further comprising a step of permeablizing the sample.

20. The method of claim 8, further comprising a step of separating unbound coumarin compound.

21. The method of claim 8, further comprising a step of adding an antibody specific for the thiol compound after the incubating step.

22. A method of synthesizing the coumarin compound of claim 2 comprising:

contacting 8-hydroxyjulolidine with ethyl 4-haloacetoacetate to form a reaction mixture;

incubating the reaction mixture for a sufficient amount of time for the coumarin compound to form.

23. The method of claim 22, wherein the incubating step comprises heating the reaction mixture to at least 100°

C.

24. The method of claim 22, wherein the reaction mixture further comprises a Lewis acid.

25. The method of claim 24, wherein the Lewis acid is aluminum chloride (AlCl₃).

7. A compound of Formula (I) or a salt or tautomer thereof:

26. A coumarin compound, selected from the group consisting of the following structures: