Cyanine compounds and their application as quenching compounds

Patent metadata

Field	Value
Publication number	US-9150922-B2
Application number	US-201314109475-A
Country	US
Kind code	B2
Filing date	Dec 17, 2013
Priority date	Aug 11, 2003
Publication date	Oct 6, 2015
Grant date	Oct 6, 2015

Abstract

The present invention provides methods and non-fluorescent carbocyanine quencher compounds having the general formula:

Wherein the A moiety is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or substituted indolinium. The invention further provides luminescent donor molecule-quencher pairs and luminescent donor molecule-quencher-luminescent acceptor molecule conjugates wherein the quencher is a cyanine compound of the present invention. The energy transfer pairs are used to detect an analyte of interest in a sample.

First claim

What is claimed is:

1. A compound having the formula:

wherein R 2 is alkyl, substituted, alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
R 3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
R 4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support; or
R 3 and R 4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
each R 5 is independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, fused benzene, substituted fused benzene, trifluoromethyl, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, halogen, reactive group, carrier molecule, or solid support;
R 22 , R 23 , R 24 , R 25 and R 26 are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, aminoalkyl, substituted aminoalkyl, hydroxyl, halogen, thioether, carbonyl, substituted carbonyl, sulfo, substituted sulfo, sulfoalkyl, reactive group, carrier molecule, or solid support; or
a member independently selected from
R 22 in combination with R 23 ;
R 23 in combination with R 24 ;
R 24 in combination with R 25 ; and
R 25 in combination with R 26 ;
together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; and

A is

R 12 is alkyl, substituted, alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, a substituted or unsubstituted aromatic, heteroaromatic, cyclic or heterocyclic moiety, reactive group, carrier molecule, or solid support;

R 10 , R 11 , R 13 , R 14 , R 15 , R 16 , R 17 and R 18 are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfo, substituted sulfo, sulfoalkyl, substituted sulfoalkyl, amino, substituted amino, aminoalkyl, substituted aminoalkyl, trifluoromethyl, halogen, reactive group, carrier molecule, or solid support; or
a member independently selected from
R 10 in combination with R 11 ;
R 11 in combination with R 13 ;
R 13 in combination with R 14 ;
R 14 in combination with R 15 ;
R 15 in combination with R 16 ;
R 11 in combination with R 17 ; and
R 17 in combination with R 18 ;
together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; or
a member independently selected from
R 12 in combination with R 16 ;
R 12 in combination with R 18 ,
R 12 in combination with R 11 ;
R 10 in combination with R′;
R 11 in combination with R″; or
R′ in combination with R″;
together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl; and
R′ and R″ are hydrogen, alkyl or aminoalkyl.

2. The compound according to claim 1 , wherein the reactive group, solid support and carrier molecule independently comprise a linker that is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S; and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

3. The compound according to claim 1 , wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide, a thiol or a photoactivatable group.

4. The compound according to claim 1 , wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

5. The compound according to claim 1 , wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a biotin-binding protein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

6. The compound according to claim 1 , wherein the carrier molecule is an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

7. The compound according to claim 1 , wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

8. The compound according to claim 1 , wherein the solid support is Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

9. A composition comprising;
a) a luminescent donor molecule;
b) a luminescent acceptor molecule; and
c) a compound that is non-fluorescent acceptor molecule, wherein the compound has the formula according to claim 1 .

10. A method for absorbing residual energy from an energy donor molecule during energy transfer between a luminescent donor molecule and a luminescent acceptor molecule, wherein the method comprises:
a) covalently bonding a luminescent donor molecule to a luminescent acceptor molecule to prepare an energy transfer pair;
b) covalently bonding a compound to the energy transfer pair to prepare a quencher labeled energy transfer pair, wherein the compound has the formula:

wherein R 2 is alkyl, substituted, alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
R 3 is

Assignees

• Life Technologies Corp

Inventors

• Haugland Rosaria
• Cheung Ching-Ying
• Yue Stephen

Classifications

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C07D209/24

  with an alkyl or cycloalkyl radical attached to the ring nitrogen atom · CPC title

• C07D209/12 Primary

  Radicals substituted by oxygen atoms · CPC title

• C07D401/04

  directly linked by a ring-member-to-ring-member bond · CPC title

• C09B23/12

  the polymethine chain being branched {"branched" means that the substituent on the polymethine chain forms a new conjugated system, e.g. most trinuclear cyanine dyes} · CPC title