Fluorogenic pH sensitive dyes and their method of use

Patent metadata

Field	Value
Publication number	US-11867698-B2
Application number	US-202017027391-A
Country	US
Kind code	B2
Filing date	Sep 21, 2020
Priority date	Oct 27, 2006
Publication date	Jan 9, 2024
Grant date	Jan 9, 2024

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 internalization of a carrier molecule, the method comprising:
(a) conjugating the carrier molecule to a fluorescent pH-sensitive dye 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 molecule conjugate;
(b) contacting the carrier molecule 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 internalization of the carrier molecule and wherein no quenching step is performed before the detecting step.

2. The method of claim 1 , wherein the carrier molecule is chosen from an antibody or fragment thereof, an antigen, an avidin or streptavidin, a biotin, a dextran, an IgG binding protein, a fluorescent protein, agarose, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, a synthetic polymer, a polymeric microparticle, a non-biological microparticle, a biological cell, a cellular component, an ion chelating moiety, an enzymatic substrate or a virus.

3. The method of claim 1 , wherein the internalization of the carrier molecule occurs via receptor-mediated endocytosis.

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

5. 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, 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.

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

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

R 15 , R 16 , R 17 , R 18 , R 19 and R 20 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.

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

R 21 is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and
R 22 , R 23 , R 24 , R 25 and R 26 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.

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

10. The method of claim 1 , wherein at least one Z moiety is present at R 1 .

11. The method of claim 10 , wherein Z is an alkoxy.

12. The method of claim 11 , wherein Z is —OCH 3 .

13. A method for detecting receptor-mediated endocytosis, the method comprising:
(a) conjugating a target molecule to a fluorescent pH-sensitive dye 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

Assignees

• Life Technologies Corp

Inventors

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

Classifications

• G01N33/582 Primary

  with fluorescent label · CPC title

• C07D209/14

  Radicals substituted by nitrogen atoms, not forming part of a nitro radical · CPC title

• C07D311/82

  Xanthenes · CPC title

• C07D311/90

  with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9 · CPC title

• C07D405/12

  linked by a chain containing hetero atoms as chain links · CPC title