Use of Novel Coumarins as Glutathione and Thiol Labels

Patent metadata

Field	Value
Publication number	US-2016334393-A1
Application number	US-201615221230-A
Country	US
Kind code	A1
Filing date	Jul 27, 2016
Priority date	Jul 27, 2007
Publication date	Nov 17, 2016
Grant date	—

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.

First claim

What is claimed is:

1 . A 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin.

2 . The 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin of claim 1 , wherein halo is bromo.

3 . The 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin of claim 1 , wherein halo is chloro.

4 . The 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin of claim 1 , further substituted with a cationic group.

5 . The 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin of claim 1 , wherein the cationic group comprises a triphenylphosphonium ion.

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

wherein,
X is an electrophilic reactive group that does not comprise a carbonyl group; or
X is a substituted thiol group.

7 . The compound of claim 6 , wherein X is halo.

8 . The compound of claim 6 , wherein X is bromo.

9 . The compound of claim 6 , wherein X is chloro.

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

11 . The compound of claim 6 , wherein X is a substituted thiol group.

12 . The compound of claim 6 , wherein X is glutathione or a derivative thereof.

13 . A 2,3,6,7-tetrahydro-9-bromomethyl-1H,5H-quinolizino(9,1-gh)coumarin.

14 . A method for detecting or quantifying a thiol compound in a sample, the method comprising:
(a) contacting the sample with 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin, 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.

15 . The method of claim 14 , wherein the sample comprises cells.

16 . The method of claim 15 , wherein the contacted sample is incubated for a sufficient amount of time for the 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin to enter the cell.

17 . The method of claim 14 , 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.

18 . The method of claim 14 , wherein the thiol compound reacts with 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin.

19 . The method of claim 14 , 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.

20 . The method of claim 15 , wherein the 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin is substituted with a cationic group and the glutathione that is detected or quantified is present in the mitochondria of the cell.

21 . The method of claim 20 , wherein the cationic group comprises a triphenylphosphonium ion.

22 . The method of claim 14 , wherein the thiol compound comprises a cysteine residue.

23 . The method of claim 14 , wherein the thiol compound is glutathione.

24 . The method of claim 14 , further comprising a step of fixing the sample.

25 . The method of claim 14 , further comprising a step of permeablizing the sample.

26 . The method of claim 14 , further comprising a step of separating unbound 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin.

27 . The method of claim 14 , further comprising a step of adding an antibody specific for the thiol compound after the incubating step.

28 . A method of synthesizing 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin comprising:
contacting 8-hydroxyjulolidine with ethyl 4-haloacetoacetate to form a reaction mixture; incubating the reaction mixture for a sufficient amount of time for 2,3,6,7-tetrahydro-9-halomethyl-1H,5H-quinolizino(9,1-gh)coumarin to form.

29 . The method of claim 28 , wherein the incubating step comprises heating the reaction mixture to at least 100° C.

30 . The method of claim 28 , wherein the reaction mixture further comprises a Lewis acid.

31 . The method of claim 30 , wherein the Lewis acid is aluminum chloride (AlCl 3 ).

32 . A pro-fluor of a compound of Formula (I) or a salt or tautomer thereof:

wherein,
X is a substituted thiol group.

Assignees

• Life Technologies Corp

Inventors

• Gee Kyle
• Janes Michael
• Johnson Iain
• Kang Hee Chol

Classifications

• Y10T436/182

  Organic or sulfhydryl containing [e.g., mercaptan, hydrogen, sulfide, etc.] · CPC title

• G01N2333/4701

  Details · CPC title

• Y10T436/17

  Nitrogen containing · CPC title

• C07D491/06

  Peri-condensed systems · CPC title

• G01N33/52 Primary

  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