Fluorogenic pH sensitive dyes and their method of use

Patent metadata

Field	Value
Publication number	US-9939454-B2
Application number	US-201514937736-A
Country	US
Kind code	B2
Filing date	Nov 10, 2015
Priority date	Oct 27, 2006
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

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 detecting phagocytosis of a carrier molecule in a solution, the method comprising:
(a) conjugating a carrier molecule to a pH-sensitive compound of Formula I

 wherein,
R 1 , R 2 , R 3 and R 6 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 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;

or a stereoisomer, tautomer, or salt thereof to form a carrier conjugate, wherein the pH-sensitive compound has a pKa value of about 6-7;
(b) contacting the carrier conjugate with a cell to form a contacted cell;
(c) incubating the contacted cell to form an incubated solution;
(d) illuminating the incubated solution to form an illuminated solution; and
(e) detecting fluorescent emissions from the illuminated solution;
wherein fluorescent emissions indicate phagocytosis of the carrier molecule, and wherein no quenching step is needed before the detecting step.

2. The method of claim 1 , wherein the carrier molecule is an E. coli bioparticle.

3. The method according to claim 1 , wherein X is a xanthene, an indole or a borapolyazaindacine.

4. The method according to claim 3 , 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, hydroxy, 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 10 to form a fused ring.

5. The method of claim 4 , wherein R 7 , R 8 , R 9 and R 10 are alkyl.

6. The method of claim 5 , wherein R 7 , R 8 , R 9 and R 10 are methyl.

7. The method of claim 6 , wherein R 11 , R 12 , R 13 and R 14 are H.

8. The method of claim 1 , wherein the EDG is selected from the group consisting of alkoxy, substituted alkoxy, amino, substituted amino, thiol, alkylthio, hydroxy, acylamino, and (carboxyl ester)oxy.

Assignees

• Life Technologies Corp

Inventors

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

Classifications

• C09K11/06

  containing organic luminescent materials · CPC title

• C07K1/13

  Labelling of peptides · CPC title

• G01N33/5058

  Neurological cells · CPC title

• C07D311/82

  Xanthenes · CPC title

• C09B11/28

  Pyronines {; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes} · CPC title