Sulfonated diarylrhodamine dyes

• US 6,583,168 B1
• Filed: 11/28/2000
• Issued: 06/24/2003
• Est. Priority Date: 11/25/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Sun, Daqing, Zhen, Weiguo, Lam, Joe Y. L., Menchen, Steven M., Benson, Scott C., Taing, Meng, Khan, Shaheer H., Rosenblum, Barnett B.
• Primary Examiner(s): NORTHINGTON DAVIS, ZINNA
• Application Number:
  US09/724,855
• Time in Patent Office: 938 Days
• Field of Search: 514/410, 514/453, 549/381, 549/382, 548/417
• US Class Current: 514/410
• 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. Sulfonated diarylrhodamine compounds having the structures:

wherein R², R^2′

, R¹² and R^12′

is hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂ 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, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ is 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⁺), anildo (—

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;

with the proviso that at least one of R¹, R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ is sulfonate.

2. The compound 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 compound of claim 1 wherein the alkyldiyl, substituted phenyl, substituted benzyl, substituted biphenyl, substituted heterocycle and substituted naphthyl is substituted with sulfonate.

4. The compound of claim 1 wherein the alkyldiyl, substituted phenyl, substituted benzyl, substituted biphenyl, substituted heterocycle and substituted naphthyl is substituted with carboxyl.

5. The compound of claim 1 wherein the linking moiety is 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 compound of claim 1 wherein the water-solubilizing group is carboxylate, sulfonate, phosphonate, phosphate, quaternary amine, sulfate, polyhydroxyl, or water-soluble polymer.

7. The compound of claim 1 wherein the heterocycle is 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 compound of claim 1 wherein R¹, R³, R⁶, R⁸, R¹¹, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²¹ are hydrogen.

9. The compound of claim 1 comprising a first bridging group which when taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members;

and/or

a second bridging group which when taken together with the C-2-bonded-nitrogen and the C-1 and C-2 carbons forms a second ring structure having from 4 to 7 members.

10. The compound of claim 9 wherein one or both of the first and second ring structures has five members.

11. The compound of claim 10 wherein the five membered ring structure includes one gem disubstituted carbon.

12. The compound of claim 11 wherein the gem substituents are (C₁-C₈) alkyl.

13. The compound of claim 11 wherein the gem substituents are methyl.

14. The compound of claim 10 wherein the five membered ring is substituted with linking moiety or water-solubilizing group.

15. The compound of claim 1 comprising a third bridging group which when taken together with the C-12-bonded nitrogen and the C-11 and C-12 carbons forms a third ring structure having from 5 to 7 members;

and/or

a fourth bridging group which when taken together with the C-2-bonded nitrogen and the C-2 and C-3 carbons forms a fourth ring structure having from 5 to 7 members.

16. The compound of claim 15 wherein one or both of the third and fourth ring structures has six members.

17. The compound of claim 16 wherein the six membered ring structure includes one gem disubstituted carbon.

18. The compound of claim 17 wherein the gem substituents are (C₁-C₈) alkyl.

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

20. The compound of claim 1 having the structures:

wherein L² and L¹² are linkers selected from the group consisting of alkyldiyl, substituted phenyl, substituted benzyl, substituted biphenyl, substituted heterocycle and substituted naphthyl.

21. The compound of claim 1 comprising a fused aromatic ring bonded across the C-3 and C-4 carbons, the C-4 and C-5 carbons, the C-9 and C-10 carbons, or the C-10 and C-11 carbons, including substituted forms thereof.

22. The compound of claim 1 comprising a fused aromatic ring bonded across the C-3 and C-4 carbons and the C-10 and C-11 carbons, including substituted forms thereof.

23. A kit for labelling a polynucleotide, comprising a sulfonated diarylrhodamine compound including a linking moiety according to claim 5 and a polynucleotide.