Cyanine compounds and their application as quenching compounds

A compound having the formula:

• wherein Z is S, O, NR12 or CR3R4;
• wherein R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl or substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R12 is substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl or substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R22 is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, aminoalkyl, substituted aminoalkyl, halogen, thioether, carbonyl, substituted carbonyl, sulfo, substituted sulfo, sulfoalkyl, carrier molecule, or solid support;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• and
• R26 is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, aminoalkyl, substituted aminoalkyl, halogen, thioether, carbonyl, substituted carbonyl, sulfo, substituted sulfo, sulfoalkyl, carrier molecule, or solid support;
• or
• a member independently selected from
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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;
• with the proviso that if a carrier molecule comprising a biological cell or virus is present, the biological cell or virus is bound by a linker that is not a single covalent bond.

A composition comprising:

• a. a luminescent donor molecule;
• b. a luminescent acceptor molecule;
• and,
• c. a compound that is a non-fluorescent acceptor molecule, having the formula:
• wherein R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R22 is 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;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• R26 is 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
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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 substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium;
• with the proviso that if a carrier molecule comprising a biological cell or virus is covalently attached to the compound, the biological cell or virus is attached by a linker that is not a single covalent bond.

A composition comprising:

• a. a luminescent donor molecule;
• b. a luminescent acceptor molecule;
• c. a compound that is a non-fluorescent acceptor molecule, wherein the compound has the formula
• wherein R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R22 is 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;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• R26 is 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
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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 substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium;
• and,
• d. a carrier molecule that is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a biotin-binding protein, a polymeric microparticle, a biological cell or a virus
• with the proviso that if a carrier molecule comprising a biological cell or virus is covalently attached to the compound, the biological cell or virus is attached by a linker that is not a single covalent bond.

A composition comprising:

• a) a compound that is a non-fluorescent acceptor molecule, wherein the compound has the formula:
• wherein R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R22 is 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;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• R26 is 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
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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 substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium;
• and,
• b) a carrier molecule with the proviso that if a carrier molecule comprising a biological cell or virus is covalently attached to the compound, the biological cell or virus is attached by a linker that is not a single covalent bond.

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 R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R22 is 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;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• R26 is 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
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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 substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium;
• c) covalently bonding the quencher labeled energy transfer pair to a carrier molecule to prepare a ternary labeled carrier molecule;
• d) illuminating the ternary labeled carrier molecule with an appropriate wavelength wherein fluorescence from the luminescent donor molecule is accepted by the luminescent acceptor molecule and the residual fluorescence from the luminescent donor molecule is accepted by the quenching compound whereby the residual energy is absorbed during energy transfer between a luminescent donor molecule and a luminescent acceptor molecule;
• with the proviso that if a carrier molecule comprising a biological cell or virus is covalently attached to the compound, the biological cell or virus is attached by a linker that is not a single covalent bond.

A method for detecting presence or absence of an analyte in a sample, wherein the method comprises:

• a. contacting the sample with a composition to prepare a labeled sample, wherein the composition comprises a carrier molecule, a luminescent donor molecule, a luminescent acceptor molecule and a compound having the formula
• wherein R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R22 is 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;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• R26 is 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
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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 substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium;
• b. incubating the labeled sample for a sufficient amount of time for the carrier molecule to associate with the ligand to form an incubated sample;
• c. illuminating the incubated sample with an appropriate wavelength to form an illuminated sample;
• and
• d. observing the illuminated sample whereby the presence or absence of an analyte in the sample is detected;
• with the proviso that if a carrier molecule comprising a biological cell or virus is covalently attached to the compound, the biological cell or virus is attached by a linker that is not a single covalent bond.

A method for detecting the presence of an analyte in a sample, wherein the method comprises:

• a. contacting the sample with a composition to prepare a labeled sample, wherein the composition comprises a carrier molecule and a compound having the formula
• wherein R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R22 is 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;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• R26 is 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
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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 substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium;
• b. incubating the labeled sample for a sufficient amount of time for the carrier molecule to associate with the ligand to form an incubated sample;
• c. illuminating the incubated sample with an appropriate wavelength to form an illuminated sample;
• and
• d. observing the illuminated sample whereby the presence or absence of the analyte in the sample is detected;
• with the proviso that if a carrier molecule comprising a biological cell or virus is covalently attached to the compound, the biological cell or virus is attached by a linker that is not a single covalent bond.

A kit for detecting a ligand in a sample, wherein the kit comprises:

• a composition comprising a carrier molecule, a luminescent donor molecule, a luminescent acceptor molecule and a non-fluorescent acceptor molecule having the formula
• wherein R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R22 is 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;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• R26 is 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
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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 substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium;
• with the proviso that if a carrier molecule comprising a biological cell or virus is covalently attached to the compound, the biological cell or virus is attached by a linker that is not a single covalent bond.

A kit for labeling a carrier molecule, wherein the kit comprises:

• a non-fluorescent compound, wherein the compound has the formula
• wherein R2 is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support;
• R3 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• R4 is alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support;
• or
• R3 and R4 taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring;
• each R5 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;
• R22 is 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;
• R23 is 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;
• R24 is 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;
• R25 is 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;
• R26 is 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
• R22 in combination with R23;
• R23 in combination with R24;
• R24 in combination with R25;
• and
• R25 in combination with R26;
• 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 substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium;
• with the proviso that if a carrier molecule comprising a biological cell or virus is covalently attached to the compound, the biological cell or virus is attached by a linker that is not a single covalent bond.