Benzocyanine compounds
Compounds useful as labels with properties comparable to known fluorescent compounds. The compounds are conjugated to proteins and nucleic acids for biological imaging and analysis. Synthesis of the compounds, formation and use of the conjugated compounds, and specific non-limiting examples of each are provided.
1. A method of detecting at least one biomolecule, the method comprising combining at least one biomolecule with a composition comprising at least one excipient and a compound in an effective concentration to detect at least one biomolecule under conditions sufficient for binding the compound to the biomolecule, and detecting the biomolecule-bound compound, where the compound is selected from the group consisting of
where
each of R 1 , R 2 , R 5 , and R 6 is the same or different and is independently selected from the group consisting of an aliphatic, heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO 3 —, a PEG group P—Z where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH 2 CH 2 O) s , where s is an integer from 3-6 inclusive, a sulfonamide group -L-SO 2 NH—P—Z, and a carboxamide group -LCONH—P—Z, where Z is selected from H, CH 3 , a CH 3 group, an alkyl group, or a heteroalkyl group;
each of R 7 , R 8 , R 11 , R 12 , R 13 , and R 14 is the same or different and is independently selected from the group consisting of H, SO 3 —, a PEG group P—Z where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH 2 CH 2 O)s, where s is an integer from 3-6 inclusive, a sulfonamide-containing group -L-SO 2 NH—P—Z, and a carboxamide-containing group —CONH—PZ, where Z is selected from H, CH 3 , a CH 3 group, an alkyl group, or a heteroalkyl group;
X is selected from —OH, —SH, —NH 2 , —NH—NH 2 , —F, —Cl, —Br, —I, —NHS (hydroxysuccinimidyl-/sulfosuccinimidyl), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, imidazole, azide, —O-carbodiimide, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH 2 , —NR-L-NH—NH 2 , —NR-L-CO 2 H, —NR-L-CO—NHS, —NR-L-CO-STP, NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NR-L-NH—CO—CH 2 —I, or —NR-LNH—CO—CH 2 —Br wherein R is —H or an aliphatic or heteroaliphatic group;
L is selected from a divalent linear (—(CH 2 ) t —, t=0 to 15), crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and/or sulfur atom;
Kat is a number of Na + , K + , Ca 2+ , ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;
each of R 3 and R 4 is the same or different and is independently hydrogen, an aliphatic group, a heteroaliphatic group, or a PEG group P—Z where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH 2 CH 2 O) s , where s is an integer from 3-6 inclusive, and Z is selected from H, CH 3 , a CH 3 group, an alkyl group, or a heteroalkyl group; or R 3 and R 4 together form a cyclic structure where R 3 and R 4 are joined using a divalent structural element selected from the group consisting of —(CH 2 ) q —, —(CH 2 ) q O(CH 2 ) q —, —(CH 2 ) q S(CH 2 ) q′ —, —(CH 2 ) g CH═CH—, —OCH═CH— where each of q and q′ is the same or different and is a integer from 2 to 6 inclusive; and
Y is selected from the group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine, bromine, a substituted or unsubstituted aryl, phenylmercapto function, and a PEG group P—Z where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH 2 CH 2 O) s , where s is an integer from 3-6 inclusive, and Z is selected from H, CH 3 , a CH 3 group, an alkyl group, or a heteroalkyl group.
2. The method of claim 1 wherein the biomolecule is selected from a protein, antibody, enzyme, nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor, lectin, antibody binding protein, carotenoid, carbohydrate, hormone, neurotransmitter, growth factors, toxin, biological cell, lipid, receptor binding drug, fluorescent proteins, organic polymer carrier material, inorganic polymeric carrier material, and combinations thereof.
3. The method of claim 1 wherein the at least one biomolecule is detected in an assay selected from fluorescence microscopy, flow cytometry, immunoassay, hybridization, chromatographic assay, electrophoretic assay, microwell plate based assay, fluorescence resonance energy transfer (FRET) system, high throughput screening, or microarray.
4. The method of claim 1 wherein the biomolecule is detected by in vivo imaging comprising providing the biomolecule-bound compound to at least one of a biological sample, tissue, or organism, and detecting the biomolecule within the at least one of a biological sample, tissue, or organism.
5. A method of labeling at least one biomolecule, the method comprising combining at least one biomolecule with a composition comprising at least one excipient and a compound in an effective concentration to label the at least one biomolecule under conditions sufficient for labeling the biomolecule with the compound, where the compound is selected from the group consisting of
X is selected from —OH, —SH, —NH 2 , —NH—NH 2 , —F, —Cl, —Br, —I, -NETS (hydroxysuccinimidyl-/sulfosuccinimidyl), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, imidazole, azide, —O-carbodiimide, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH 2 , —NR-L-NH—NH 2 , —NR-L-CO 2 H, —NR-L-CO—NHS, —NR-L-CO-STP, NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NR-L-NH—CO—CH 2 —I, or —NR-LNH—CO—CH 2 —Br wherein R is —H or an aliphatic or heteroaliphatic group;
L is selected from a divalent linear (—(CH 2 ) t —, t=0 to 15), crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and/or sulfur atom;
Kat is a number of Na + , K + , Ca 2+ , ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine; m is an integer from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;
each of R 3 and R 4 is the same or different and is independently hydrogen, an aliphatic group, a heteroaliphatic group, or a PEG group P—Z where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH 2 CH 2 O) s , where s is an integer from 3-6 inclusive, and Z is selected from H, CH 3 , a CH 3 group, an alkyl group, or a heteroalkyl group; or R 3 and R 4 together form a cyclic structure where R 3 and R 4 are joined using a divalent structural element selected from the group consisting of —(CH 2 ) q —, —(CH 2 ) q O(CH 2 ) q —, —(CH 2 ) q S(CH 2 ) q′ —, —(CH 2 ) g CH═CH—, —OCH═CH— where each of q and q′ is the same or different and is an integer from 2 to 6 inclusive; and
Y is selected from the group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine, bromine, a substituted or unsubstituted aryl, phenylmercapto function, and a PEG group P—Z where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH 2 CH 2 O) s , where s is an integer from 3-6 inclusive, and Z is selected from H, CH 3 , a CH 3 group, an alkyl group, or a heteroalkyl group.
6. The method of claim 5 wherein the biomolecule is selected from the group consisting of a protein, antibody, enzyme, nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor, lectin, antibody binding protein, carotenoid,
where
each of R 1 , R 2 , R 5 , and R 6 is the same or different and is independently selected from the group consisting of an aliphatic, heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO 3 —, a PEG group P—Z where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH 2 CH 2 O) s , where s is an integer from 3-6 inclusive, a sulfonamide group -L-SO 2 NH—P—Z, and a carboxamide group -LCONH—P—Z, where Z is selected from H, CH 3 , a CH 3 group, an alkyl group, or a heteroalkyl group;
each of R 7 , R 8 , R 11 , R 12 , R 13 , and R 14 is the same or different and is independently selected from the group consisting of H, SO 3 —, a PEG group P—Z where P is selected from an ethylene glycol group, a diethylene glycol group, and a polyethylene glycol group, where the polyethylene glycol group is (CH 2 CH 2 O) s , where s is an integer from 3-6 inclusive, a sulfonamide-containing group -L-SO 2 NH—P—Z, and a carboxamide-containing group —CONH—PZ, where Z is selected from H, CH 3 , a CH 3 group, an alkyl group, or a heteroalkyl group;
carbohydrate, hormone, neurotransmitter, growth factors, toxin, biological cell, lipid, receptor binding drug, fluorescent proteins, organic polymer carrier material, inorganic polymeric carrier material, and combinations thereof.