Dyes, composite dyes, and oligonucleotides or polynucleotides comprising such dyes—useful or detection or quantification of desirable target molecules
The present invention provides dyes, reactive dyes and labeled reagents that may be used in the detection or quantification of desirable target molecules, such as proteins and nucleic acids. Dyes are provided that may be used free in solution where the binding of the dye to the target molecule provides signal generation. Dyes are also provided that comprise reactive groups that may be used to attach the dyes to probes that will bind to desirable target molecules. The novel dyes of the present invention have been modified by the addition of charged and polar groups to provide beneficial properties.
1. A dye having the structure:
wherein
at least one of R 8 and R 21 or R 7 and R 22 are taken together to form a five-membered or six-membered saturated or unsaturated heteroalkyl ring;
at least one of R 1 , R 2 , R 3 , R 6 , R 7 , R 8 R 9 , R 21 or R 22 is Q, wherein Q is selected from a sulfoxide (SOR 13 ), a sulfone (SO 2 CR 13 R 14 R 15 ), a sulfonamide (SO 2 NR 13 R 14 ), a phosphonate (PO 3 ═ ), a phosphonate monoester (PO 2 − ER 13 ) a phosphonate dioster (POER 13 ER 14 ), a thiophosphate (PSO 3 ═ ), a thiophosphate monoester (PSO 2 − ER 13 ) a thiophosphate diester (PSOER 13 ER 14 ), a thiophosphonate (PSO 2 ═ ), a thiophosphonate monoester (PSO − ER 13 ) a thiophosphonate diester (PSER 13 ER 14 ), a phosphonamide (PONR 13 R 14 NR 19 R 20 ), a thioanalogue of a phosphonamide, (PSNR 13 R 14 NR 19 R 20 ), a phosphoramide (PONR 13 R 14 NR 15 NR 19 R 20 ), a thioanalogue of a phosphoramide (PSNR 13 R 14 NR 15 NR 19 R 20 ), a phosphoramidite (PO 2 R 19 NR 13 R 14 ), a thioanalogue of a phosphoramidite (POSR 19 R 13 R 14 ), wherein E is O or S;
wherein Q is attached directly, or indirectly through a saturated or unsaturated, linear or branched, substituted or unsubstituted carbon linker arm, which may further comprise one or more heteroatoms selected from sulfur, oxygen and nitrogen, and wherein when Q is a sulfonamide, it does not comprise a terminal reactive group or a linker joining the dye to a target molecule;
R 13 , R 14 , R 15 , R 19 and R 20 are independently selected from hydrogen, halogen, an amino group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, or when taken together R 13 and R 14 form a five-membered or six-membered saturated or unsaturated heteroalkyl ring;
wherein the remaining R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 , R 21 and R 22 are independently selected from hydrogen, Z, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group alkoxy group, or when taken together, R 2 and R 3 , R 6 and R 7 , and R 8 and R 9 form a five-membered or six-membered ring;
Z is selected from a carboxyl group (CO 2 − ), a carbonate ester (COER 13 ), a sulfonate (SO 3 − ), a sulfonate ester (SO 2 ER 13 ), a sulfoxide (SOR 13 ), a sulfone (SO 2 CR 13 R 14 R 15 ), a sulfonamide (SO 2 NR 13 R 14 ), a phosphate (PO 4 ═ ), a phosphate monoester (PO 2 − ER 13 ), a phosphate diester (PO 2 ER 13 ER 14 ), a phosphonate (PO 3 ═ ) a phosphonate monoester (PSO 2 − ER 13 ) a phosphonate diester (POER 13 ER 14 ), a thiophosphate (PSO 3 ═ ), a thiophosphate monoester (PSO 2 − ER 13 ) a thiophosphate diester (PSOER 13 ER 14 ), a thiophosphonate (PSO 2 ═ ), a thiophosphonate monoester (PSO − ER 13 ) a thiophosphonate diester (PSER 13 ER 14 ), a phosphonamide (PONR 13 R 14 NR 19 R 20 ), a thioanalogue of a phosphonamide (PSNR 13 R 14 NR 19 R 20 ), a phosphoramide (PONR 13 R 14 NR 15 NR 19 R 20 ), a thioanalogue of a phosphoramide (PSNR 13 R 14 NR 15 NR 19 R 20 ), a phosphoramidite (PO 2 R 19 NR 13 R 14 ) or a thioanalogue of a phosphoramidite (POSR 19 NR 13 R 14 ), and E is O or S;
wherein Z is attached directly or indirectly through a saturated or unsaturated, linear or branched, substituted or unsubstituted carbon linker arm, which may further comprise one or more heteroatoms selected from sulfur, oxygen and nitrogen;
and wherein any of R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 , R 21 or R 22 may further comprise a heteroatom containing side chain wherein said side chain is joined to the R group by a linkage selected from an ether linkage (—OR 25 ), a thioether linkage (—SR 25 ), and an amine linkage (—NR 25 R 26 or —N + R 25 R 26 R 27 ), and
wherein R 25 , R 26 and R 27 are each independently selected from hydrogen, Z, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkoxy group, or when taken together, R 25 and R 26 , or R 26 and R 27 form a saturated or unsaturated five-membered or six-membered alkyl or heteroalkyl ring, and wherein any of R 25 , R 26 or R 27 may further comprise a heteroatom containing side chain.
2. The compound of claim 1 wherein at least one of R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 , R 21 or R 22 comprises a reactive group, said reactive group comprising a nucleophilic reactive group, an electrophilic reactive group, a terminal alkene, a terminal alkyne, a coordinate group or an alkylating agent.
3. The dye of claim 1 , having the structure:
wherein R 31 and R 32 are each independently selected from hydrogen, Z, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkoxy group, or when taken together, R 7 and R 22 , R 22 and R 32 , R 32 and R 31 , or R 31 and R 9 form a five-membered or six-membered saturated or unsaturated alkyl or heteroalkyl ring,
wherein any of R 31 and R 32 may further comprise a heteroatom containing side chain wherein said side chain is joined to the R group by a linkage selected from an ether linkage (—OR 25 ), a thioether linkage (—SR 25 ), and an amine linkage (—NR 25 R 26 or —N + R 25 R 26 R 27 ), and
wherein R 25 , R 26 and R 27 are each independently selected from hydrogen, Z, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkoxy group, or when taken together, R 25 and R 26 , or R 26 and R 27 form a saturated or unsaturated five-membered or six-membered alkyl or heteroalkyl ring, and wherein any of R 25 , R 26 or R 27 may further comprise a heteroatom containing side chain.
4. The dye of claim 3 , having the structure:
5. The dye of claim 1 having the structure:
wherein R 31 , R 32 , R 33 , R R 34 , R 35 and R 36 are independently selected from hydrogen, Q, Z, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, and a saturated or unsaturated, linear or branched, substituted or unsubstituted alkoxy group,
any of R 31 , R 32 , R 33 , R 34 , R 35 and R 36 may further comprise a heteroatom containing side chain wherein said side chain is joined to the R group by a linkage selected from an ether linkage (—OR 25 ), a thioether linkage (—SR 25 ), and an amine linkage (—NR 25 R 26 or —N + R 25 R 26 R 27 ), and
wherein R 25 , R 26 and R 27 are independently selected from hydrogen, Z, a saturated or unsaturated, linear or branched, substituted or unsubstituted alkyl group, and a saturated or unsaturated, linear or branched, substituted or unsubstituted alkoxy group, or when taken together, R 25 and R 26 , or R 26 and R 27 form a saturated or unsaturated five-membered or six-membered alkyl or heteroalkyl ring, and wherein any of R 25 , R 26 or R 27 may further comprise a heteroatom containing side chain.
6. The dye of claim 5 , having the structure:
7. The dye of claim 5 , having the structure:
8. The dye of claim 1 , having the structure:
9. The dye of claim 2 , wherein said nucleophilic reactive group is a thiol, amine or hydroxyl group.
10. The dye of claim 2 , wherein said electrophilic reactive group is an isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal or aldehyde group.
11. The dye of claim 2 , wherein said alkyl or alkoxy groups independently comprise from 1-18 carbons.
12. The dye of claim 11 , wherein said alkyl or alkoxy groups independently comprise from 1-6 carbons.
13. The dye of claim 11 , wherein said dye further comprises one or more charged groups or polar groups.
14. The dye of claim 13 , wherein at least one of said one or more charged groups or polar groups is monomeric.
15. The dye of claim 13 , wherein at least one of said one or more charged groups or polar groups is polymeric.
16. The dye of claim 13 , wherein at least one of said one or more charged groups or polar groups is anionic.
17. The dye of claim 13 , wherein at least one of said one or more charged groups or polar groups is cationic.
18. The dye of claim 2 , wherein said dye further comprises one or more anionic charged groups or polar groups and one or more cationic charged groups or polar groups.
19. The dye of claim 2 , wherein said dye is linked to a target molecule through said reactive group.
20. The dye of claim 19 , wherein said target molecule comprises a nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, lipid, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, glass particle, glass surface, plastic particle, plastic surface, silicaceous particle, silicaceous surface, organic molecule, dye or a derivative thereof.
21. The dye of claim 20 , wherein said nucleoside, nucleotide, oligonucleotide, or polynucleotide comprises one or more ribonucleoside moieties, ribonucleotide moieties, deoxyribonucleoside moieties, deoxyribonucleotide moieties, modified ribonucleosides, modified ribonucleotides, modified deoxyribonucleosides, modified deoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotide analogues or any combination thereof.
22. The dye of claim 19 , wherein said dye is linked to said target molecule through a linker arm.
23. The dye of claim 22 , wherein said linker arm is attached to said target molecule through a bond which comprises a covalent bond, a non-covalent bond, a polar bond or a coordinate bond.
24. The oligonucleotide or polynucleotide of claim 20 , wherein said oligonucleotide or polynucleotide comprises two or more dye molecules attached to separate nucleotides of said oligonucleotide or polynucleotide.
25. The oligonucleotide or polynucleotide of claim 24 , wherein said two or more dye molecules comprise the same dye molecules.
26. The oligonucleotide or polynucleotide of claim 24 , wherein said two or more dye molecules comprise different dye molecules.
27. A composite dye comprising the dye of claim 20 , wherein said target molecule comprises a dye.
28. The composite dye of claim 27 further comprising a nucleophilic reactive group, an electrophilic reactive group, a terminal alkene, a terminal alkyne, a coordinate group or an alkylating agent.
29. The dye of claim 2 , wherein said dye has been linked to a target specific moiety through said reactive group.
30. The dye of claim 29 , wherein said target specific moiety comprises a protein or a nucleic acid.
31. The dye of claim 30 , wherein said protein comprises an antibody or a fragment thereof.
32. The dye of claim 30 , wherein said nucleic acid comprises unmodified nucleotides, modified nucleotides, nucleotide analogues or any combination thereof.