US 12,360,106 B2
Photochromic xanthene fluorophores and their utility in live-cell imaging beyond the diffraction limit
Luke Lavis, Leesburg, VA (US); and Fadi M. Jradi, Arlington, VA (US)
Assigned to Howard Hughes Medical Institute, Chevy Chase, MD (US)
Filed by HOWARD HUGHES MEDICAL INSTITUTE, Chevy Chase, MD (US)
Filed on Jun. 16, 2021, as Appl. No. 17/300,404.
Application 17/300,404 is a division of application No. 15/932,629, filed on Mar. 28, 2018, granted, now 11,067,566.
Claims priority of provisional application 62/601,808, filed on Mar. 31, 2017.
Prior Publication US 2021/0318293 A1, Oct. 14, 2021
Int. Cl. G01N 33/533 (2006.01); C07D 405/14 (2006.01); C07D 473/18 (2006.01); C07D 491/107 (2006.01); C07D 491/147 (2006.01); C07D 491/22 (2006.01); C07D 493/10 (2006.01); C09B 57/02 (2006.01); C09K 11/06 (2006.01)
CPC G01N 33/533 (2013.01) [C07D 405/14 (2013.01); C07D 473/18 (2013.01); C07D 491/107 (2013.01); C07D 491/147 (2013.01); C07D 491/22 (2013.01); C07D 493/10 (2013.01); C09B 57/02 (2013.01); C09K 11/06 (2013.01); C09K 2211/1018 (2013.01)] 5 Claims
 
1. A method of imaging one or more cellular structures within one or more cells, wherein the method comprises the steps of:
a) labeling one or more cells with a compound of the following structure

OG Complex Work Unit Chemistry
wherein,
X is O, N-alkyl, S, Si(alkyl)2 or C(alkyl)2;
Y1 is O, OH, NH2, N(alkyl)2 or one of the following moieties

OG Complex Work Unit Chemistry
Y2 is O, OH, NH2, N(alkyl)2 or one of the following moieties

OG Complex Work Unit Chemistry
R1, which can be a substitution at either the 5′ position, the 6′ position or both, is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor, C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group, C(O)NH—(CH2CH2)nC(O)N(H)-Handle where “n” is an integer ranging from 1 to 100, C(O)NH—(CHCH)n-Acceptor where “n” is an integer ranging from 1 to 100, C(O)NH—(CH2CH2O)3—C(O)NH—(CH2CH2O)2-(CH2)6-Cl, or C(O)NH—(CH2CH2O)3—C(O)NH—(CH2CH2O)3—C(O)NH—(CH2CH2O)2—(CH2)6—Cl;
R2 is hydrogen, C(O)NH-Handle, C(O)-linker-Handle, C(O)NH-Acceptor, C(O)-linker-Acceptor, C(O)NH-linker-CH2-X where X is a leaving group, C(O)NH—(CH2CH2)nC(O)N(H)-Handle where “n” is an integer ranging from 1 to 100, C(O)NH—(CHCH)n-Acceptor where “n” is an integer ranging from 1 to 100, C(O)NH—(CH2CH2O)3—C(O)NH—(CH2CH2O)2-(CH2)6-Cl, or C(O)NH—(CH2CH2O)3—C(O)NH—(CH2CH2O)3—C(O)NH—(CH2CH2O)2—(CH2)6—Cl;
R3, R4, R5 and R6 are independently hydrogen, alkyl, —SO3H, halogen, or R3 and Y1 can form a ring, or R4 and Y1 can form a ring, or R5 and Y2 can form a ring, or R6 and Y2 can form a ring;
A1 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aromatic, heteroaromatic, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl, heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, or substituted heteroaromatic group;
A2 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aromatic, heteroaromatic, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heteroalkyl, substituted heteroalkenyl, heterocycloalkyl, heterocycloalkenyl, substituted aromatic group, or substituted heteroaromatic group,
to provide one or more labeled cells
b) directing at least one beam of light to the one or more labeled cells, such that a detectable signal is produced from the one or more labeled cells;
c) recording the detectable signal, thereby imaging one or more structures within the one or more cells.