Fluorogenic pH sensitive dyes and their method of use

Patent metadata

Field	Value
Publication number	US-10845373-B2
Application number	US-201815899021-A
Country	US
Kind code	B2
Filing date	Feb 19, 2018
Priority date	Oct 27, 2006
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

Abstract

A new class of pH sensitive fluorescent dyes and assays relating thereto are described. The dyes and assays are particularly suited for biological applications including phagocytosis and monitoring intracellular processes. The pH sensitive fluorescent dyes of the present invention include compounds of Formula I:

wherein the variables are described throughout the application.

First claim

What is claimed is:

1. A method for determining the pH of a sample, the method comprising:
(a) contacting the sample with a compound of Formula I:

wherein,
R 1 and R 2 are each independently H, Z, or an electron donating group (EDG), provided that R 1 and R 2 are not hydroxyl or thiol or their deprotonated forms;
R 3 and R 6 are each independently H, Z, or an electron donating group (EDG);
EDG is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted alkyl, heterocyclyl, substituted heterocyclyl, heteroaryl, and substituted heteroaryl;
R 4 is selected from the group consisting of H, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
R 5 is independently selected from the group consisting of Y, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, a reactive group, a carrier molecule, heterocyclyl, and substituted heterocyclyl;
X is a fluorophore;
Y is ═CR b R c or ═CR b R d ;
Z is —OR c , —SR c , —NR b R c ;
R b is H, alkyl, or substituted alkyl;
R c is alkyl or substituted alkyl; and
R d is amino or substituted amino, to form a contacted sample;

(b) incubating the contacted sample to form an incubated sample;
(c) illuminating the incubated sample to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated sample;
wherein the fluorescent emissions are used to determine the pH of the sample.

2. The method of claim 1 , wherein the sample comprises cells, intracellular fluids, extracellular fluids, sera, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions, 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.

3. The method of claim 2 , 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, or on a microfluidic chip.

4. The method of claim 3 , wherein the contacting step further comprises contacting the sample with a second dye.

5. The method of claim 4 , wherein the second dye is a pH sensitive dye having a different fluorescent emission spectrum from the dye or compound and a different pKa from the pKa of the compound.

6. The method of claim 2 , wherein the cells are live cells and the incubating comprises the compound entering the cell to form a labeled cell.

7. The method of claim 1 , wherein X is a xanthene, an indole or a borapolyazaindacine.

8. The method of claim 1 , wherein X is:

wherein,
R 7 , R 8 , R 9 and R 10 are each independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and
R 11 , R 12 , R 13 and R 14 are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxyl, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or
R 11 and R 14 are taken together with R 7 and R 8 to form a fused ring; and R 12 and R 13 are taken together with R 9 and R 19 to form a fused ring.

Assignees

• Life Technologies Corp

Inventors

• Dzubay Jeffrey
• Gee Kyle
• Martin Vladimir
• Rukavishnikov Aleksey
• Beacham Daniel

Classifications

• G01N33/84 Primary

  involving inorganic compounds or pH · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title

• G01N33/5091

  for testing the pathological state of an organism · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title

• G01N33/5058

  Neurological cells · CPC title