US 11,867,698 B2
Fluorogenic pH sensitive dyes and their method of use
Jeffrey Dzubay, Bend, OR (US); Kyle Gee, Springfield, OR (US); Vladimir Martin, Bothell, WA (US); Aleksey Rukavishnikov, Eugene, OR (US); and Daniel Beacham, Eugene, OR (US)
Assigned to Life Technologies Corporation, Carlsbad, CA (US)
Filed by Life Technologies Corporation, Carlsbad, CA (US)
Filed on Sep. 21, 2020, as Appl. No. 17/027,391.
Application 15/899,021 is a division of application No. 14/937,736, filed on Nov. 10, 2015, granted, now 9,939,454, issued on Apr. 10, 2018.
Application 17/027,391 is a continuation of application No. 15/899,021, filed on Feb. 19, 2018, granted, now 10,845,373.
Application 14/937,736 is a continuation of application No. 13/614,995, filed on Sep. 13, 2012, abandoned.
Application 13/614,995 is a continuation of application No. 11/927,588, filed on Oct. 29, 2007, abandoned.
Claims priority of provisional application 60/940,323, filed on May 25, 2007.
Claims priority of provisional application 60/863,318, filed on Oct. 27, 2006.
Prior Publication US 2021/0041465 A1, Feb. 11, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/58 (2006.01); G01N 33/50 (2006.01); G01N 33/84 (2006.01); C09B 11/24 (2006.01); C09B 11/28 (2006.01); C07D 311/82 (2006.01); C07D 311/90 (2006.01); C07D 405/12 (2006.01); C07D 413/04 (2006.01); C07D 491/22 (2006.01); C07D 209/14 (2006.01); C09K 11/06 (2006.01); C07F 5/02 (2006.01); C07K 1/13 (2006.01); C09B 7/00 (2006.01); C09B 11/12 (2006.01); C09B 57/00 (2006.01); C09B 69/00 (2006.01); G01N 31/22 (2006.01); G01N 33/80 (2006.01)
CPC G01N 33/582 (2013.01) [C07D 209/14 (2013.01); C07D 311/82 (2013.01); C07D 311/90 (2013.01); C07D 405/12 (2013.01); C07D 413/04 (2013.01); C07D 491/22 (2013.01); C07F 5/02 (2013.01); C07K 1/13 (2013.01); C09B 7/00 (2013.01); C09B 11/12 (2013.01); C09B 11/24 (2013.01); C09B 11/28 (2013.01); C09B 57/00 (2013.01); C09B 69/00 (2013.01); C09K 11/06 (2013.01); G01N 31/22 (2013.01); G01N 33/5005 (2013.01); G01N 33/5058 (2013.01); G01N 33/5091 (2013.01); G01N 33/80 (2013.01); G01N 33/84 (2013.01); G01N 2333/245 (2013.01); Y10T 436/143333 (2015.01)] 20 Claims
 
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:

OG Complex Work Unit Chemistry
wherein,
R1, R2, R3 and R6 are each independently H, Z, or an electron donating group (EDG), provided that R1 and R2 are not hydroxyl or thiol or their deprotonated forms;
R4 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;
R5 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 ═CRbRc or ═CRbRd;
Z is —ORc, —SRc, —NRbRc;
Rb is H, alkyl, or substituted alkyl;
Rc is alkyl or substituted alkyl; and
Rd 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.