Sulfonated diarylrhodamine dyes

• US 7,018,431 B2
• Filed: 06/24/2003
• Issued: 03/28/2006
• Est. Priority Date: 11/25/1997
• Status: Expired due to Term

• Current Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y. L., Menchen, Steven M., Benson, Scott C., Taing, Meng, Khan, Shaheer H., Rosenblum, Barnett B., Sun, Daqing, Zhen, Weiguo
• Primary Examiner(s): NORTHINGTON DAVIS, ZINNA
• Application Number:
  US10/602,974
• Publication Number:
  US 20040096397A1
• Time in Patent Office: 1,008 Days
• Field of Search: 857/4, 857/9, 548/417, 549/381
• US Class Current: 8/574
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 221/10 Aza-phenanthrenes
• CPC Class Codes: C07D 311/78 Ring systems having three or more relevant rings
• CPC Class Codes: C07D 311/94 condensed with rings other than six-membered or with ring systems containing such rings
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 11/02 derived from diarylmethanes
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes

Abstract

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.

Claims

1. A sulfonated diarylrhodamine dye comprising a compound of the structure:

wherein R², R^2′

, R¹² and R^12′

when taken alone are each independently selected from hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,

R² when taken together with R¹ forms a ring structure having from 4 to 7 ring members optionally substituted by one or more of C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ 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 R¹ forms a ring structure having from 4 to 7 ring members optionally substituted by one or more of C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,

R¹² when taken together with R¹³ forms a ring structure having from 4 to 7 ring members optionally substituted by one or more of C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ 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 R¹³ forms a ring structure having from 4 to 7 ring members optionally substituted by one or more of C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,

R² when taken together with R³ forms a ring structure having from 5 to 7 ring members optionally substituted by one or more of C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,

R² when taken together with R³ forms a ring structure having from 5 to 7 ring members optionally substituted by one or more of C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety,

R¹² when taken together with R¹¹ forms a ring structure having from 5 to 7 ring members optionally substituted by one or more of C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ 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 RR¹¹ forms a ring structure having from 5 to 7 ring members optionally substituted by one or more of C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyl, C₁-C₁₂ alkyldiyl, C₁-C₁₂ substituted alkyldiyl, phenyl, substituted phenyl, benzyl, substituted benzyl, biphenyl, substituted biphenyl, naphthyl, substituted naphthyl, heterocycle, substituted heterocycle, water-solubilizing group or linking moiety, and

R¹, R³, R⁴, R⁵, R⁶, R⁸, R¹¹, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ are each independently selected from hydrogen, fluorine, chlorine, C₁-C₈ alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—

CH₂NH₂), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—

CH₂OH), methoxy (—

OCH₃), hydroxyalkyl (—

ROH), thiomethyl (—

CH₂SH), thioalkyl (—

RSH), alkylsulfone (—

SO₂R), arylthio (—

SAr), arylsulfone (—

SO₂Ar), sulfonamide (—

SO₂NR₂), alkylsulfoxide (—

SOR), arylsulfoxide (—

SOAr), amino (—

NH₂), ammonium (—

NH₃⁺), amido (—

CONR₂), nitrile (—

CN), C₁-C₈ 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 R¹, R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ is sulfonate.

2. The dye of claim 1 wherein at least one of R², R^2′

, R¹² and R^12′

is C₁-C₆ alkylsulfonate or C4-C₁₀ arylsulfonate.

3. The dye of claim 1 wherein the C₁-C₁₂ substituted alkyl, C₁-C₁₂ 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 C₁-C₁₂ substituted alkyl, C₁-C₁₂ 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 R¹, R³, R⁶, R⁸, R¹¹, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ are hydrogen.

9. The dye of claim 1 comprising at least one of

a first bridging group wherein R¹² when taken together with R¹³ forms a first ring structure having from 4 to 7 ring members, and

a second bridging group wherein R² when taken together with R¹ 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 (C₁-C₈) 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 R¹² when taken together with R¹¹ form a third ring structure having from 5 to 7 ring members;

and

a fourth bridging group wherein R² when taken together with R³ 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 (C₁-C₈) alkyl.

19. The dye of claim 18 wherein the gem substituents are methyl.

20. The dye of claim 1 wherein, when taken together, R³ and R⁴ 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, C₁-C₈ alkyl, carboxylate, sulfate, sulfonate, alkylsulfonate, aminomethyl (—

CH₂NH₂), aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (—

CH₂OH), methoxy (—

OCH₃), hydroxyalkyl (—

ROH), thiomethyl (—

CH₂SH), thioalkyl (—

RSH), alkylsulfone (—

SO₂R), arylthio (—

SAr), arylsulfone (—

SO₂Ar), sulfonamide (—

SO₂NR₂), alkylsulfoxide (—

SOR), arylsulfoxide (—

SOAr), amino (—

NH₂), ammonium (—

NH₃⁺), amido (—

CONR₂), nitrile (—

CN), C₁-C₈ 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: