Sulfonated diarylrhodamine dyes
View Patent ↗Sulfonated diarylrhodamine compounds are useful as fluorescent labels of nucleosides, nucleotides, polynucleotides, and polypeptides. The compounds find particular application in the area of fluorescent nucleic acid analysis, e.g., automated DNA sequencing and fragment analysis, detection of probe hybridization in hybridization arrays, detection of nucleic acid amplification products, and the like.
1. A sulfonated diarylrhodamine dye comprising a compound of the structure:
wherein R 2 , R 2′ , R 12 and R 12′ when taken alone are each independently selected from hydrogen, C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,
R 2 when taken together with R 1 forms a ring structure having from 4 to 7 ring members optionally substituted by one or more of C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,
R 2′ when taken together with R 1 forms a ring structure having from 4 to 7 ring members optionally substituted by one or more of C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,
R 12 when taken together with R 13 forms a ring structure having from 4 to 7 ring members optionally substituted by one or more of C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,
R 12′ when taken together with R 13 forms a ring structure having from 4 to 7 ring members optionally substituted by one or more of C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,
R 2 when taken together with R 3 forms a ring structure having from 5 to 7 ring members optionally substituted by one or more of C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,
R 2 when taken together with R 3 forms a ring structure having from 5 to 7 ring members optionally substituted by one or more of C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,
R 12 when taken together with R 11 forms a ring structure having from 5 to 7 ring members optionally substituted by one or more of C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,
R 12′ when taken together with RR 11 forms a ring structure having from 5 to 7 ring members optionally substituted by one or more of C 1 -C 12 alkyl, C 1 -C 12 substituted alkyl, C 1 -C 12 alkyldiyl, C 1 -C 12 substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety, and
R 1 , R 3 , R 4 , R 5 , R 6 , R 8 , R 11 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21 are each independently selected from hydrogen, fluorine, chlorine, C 1 -C 8 alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—CH 2 NH 2 ), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—CH 2 OH), methoxy (—OCH 3 ), hydroxyalkyl (—ROH), thiomethyl (—CH 2 SH), thioalkyl (—RSH), alkylsulfone (—SO 2 R), arylthio (—SAr), arylsulfone (—SO 2 Ar), sulfonamide (—SO 2 NR 2 ), alkylsulfoxide (—SOR), arylsulfoxide (—SOAr), amino (—NH 2 ), ammonium (—NH 3 + ), amido (—CONR 2 ), nitrile (—CN), C 1 -C 8 alkoxy (—OR), phenoxy, phenolic, tolyl, phenyl, aryl, benzyl, heterocycle, phosphonate, phosphate, quaternary amine, sulfate, polyethyleneoxy, water-solubilizing group, or linking moiety;
wherein at least one of R 1 , R 3 , R 4 , R 5 , R 6 , R 8 , R 9 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21 is sulfonate.
2. The dye of claim 1 wherein at least one of R 2 , R 2′ , R 12 and R 12′ is C 1 -C 6 alkylsulfonate or C4-C 10 arylsulfonate.
3. The dye of claim 1 wherein the C 1 -C 12 substituted alkyl, C 1 -C 12 substituted alkyldiyl, substituted phenyl, substituted benzyl, substituted biphenyl, substituted naphthyl, substituted heterocycle are substituted with at least one sulfonate substituent.
4. The dye of claim 1 wherein the C 1 -C 12 substituted alkyl, C 1 -C 12 substituted alkyldiyl, substituted phenyl, substituted benzyl, substituted biphenyl, substituted naphthyl, substituted heterocycle are substituted with at least one carboxyl substituent.
5. The dye of claim 1 which comprises a linking moiety selected from azido, monosubstituted primary amine, disubstituted secondary amine, thiol, hydroxyl, halide, epoxide, N-hydroxysuccinimidyl ester, carboxyl, isothiocyanate, sulfonyl chloride, sulfonate ester, silyl halide, chlorotriazinyl, succinimidyl ester, pentafluorophenyl ester, maleimide, haloacetyl, epoxide, alkylhalide, allyl halide, aldehyde, ketone, acylazide, anhydride, iodoacetamide or an activated ester.
6. The dye of claim 1 wherein the water-solubilizing group is selected from carboxylate, sulfonate, phosphonate, phosphate, quaternary amine, sulfate, polyhydroxyl, or water-soluble polymer.
7. The dye of claim 1 which comprises heterocycle selected from pyrrole, indole, furan, benzofuran, thiophene, benzothiophene, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2-imidazole, 4-imidazole, 3-pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl, 5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O,3-N)-oxazole, 5-(1-O,3-N)-oxazole, 4-(1-S,3-N)-thiazole, 5-(1-S,3-N)-thiazole, 2-benzoxazole, 2-benzothiazole, 4-(1,2,3-N)-benzotriazole, or benzimidazole.
8. The dye of claim 1 wherein R 1 , R 3 , R 6 , R 8 , R 11 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21 are hydrogen.
9. The dye of claim 1 comprising at least one of
a first bridging group wherein R 12 when taken together with R 13 forms a first ring structure having from 4 to 7 ring members, and
a second bridging group wherein R 2 when taken together with R 1 forms a second ring structure having from 4 to 7 ring members.
10. The dye of claim 9 wherein at least one of the first and second ring structures is a five membered ring structure.
11. The dye of claim 10 wherein the five membered ring structure comprises at least one gem disubstituted carbon.
12. The dye of claim 11 wherein the gem substituents are (C 1 -C 8 ) alkyl.
13. The dye of claim 11 wherein the gem substituents are methyl.
14. The dye of claim 10 wherein the five membered ring structure comprises at least one of a linking moiety or a water-solubilizing group.
15. The dye of claim 1 comprising at least one of a third bridging group wherein R 12 when taken together with R 11 form a third ring structure having from 5 to 7 ring members; and
a fourth bridging group wherein R 2 when taken together with R 3 forms a fourth ring structure having from 5 to 7 ring members.
16. The dye of claim 15 wherein at least one of the third and fourth ring structures is a six membered ring structure.
17. The dye of claim 16 wherein the six membered ring structure comprises one gem disubstituted carbon.
18. The dye of claim 17 wherein the gem substituents are (C 1 -C 8 ) alkyl.
19. The dye of claim 18 wherein the gem substituents are methyl.
20. The dye of claim 1 wherein, when taken together, R 3 and R 4 form a fused aromatic ring.
21. The dye of claim 20 wherein the fused aromatic ring comprises at least one substituent selected from fluorine, chlorine, C 1 -C 8 alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—CH 2 NH 2 ), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—CH 2 OH), methoxy (—OCH 3 ), hydroxyalkyl (—ROH), thiomethyl (—CH 2 SH), thioalkyl (—RSH), alkylsulfone (—SO 2 R), arylthio (—SAr), arylsulfone (—SO 2 Ar), sulfonamide (—SO 2 NR 2 ), alkylsulfoxide (—SOR), arylsulfoxide (—SOAr), amino (—NH 2 ), ammonium (—NH 3 + ), amido (—CONR 2 ), nitrile (—CN), C 1 -C 8 alkoxy (—OR), phenoxy, phenolic, tolyl, phenyl, aryl, benzyl, heterocycle, phosphonate, phosphate, quaternary amine, sulfate, polyethyleneoxy, water-solubilizing group and linking moiety.
22. The dye of claim 1 comprising the structure:
23. The dye of claim 1 comprising the structure:
24. The dye of claim 1 comprising the structure:
25. The dye of claim 1 comprising the structure:
26. The dye of claim 1 comprising the structure:
27. The dye of claim 1 comprising the structure:
28. The dye of claim 1 comprising the structure:
29. The dye of claim 1 comprising the structure:
30. The dye of claim 1 comprising the structure:
31. The dye of claim 1 comprising the structure:
32. The dye of claim 1 comprising the structure: