Water-soluble rhodamine dye conjugates

• US 7,795,042 B2
• Filed: 03/12/2009
• Issued: 09/14/2010
• Est. Priority Date: 11/03/1999
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Linda G., Graham, Ronald J., Lu, Lily, Swartzman, Elana, Werner, William E.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/402,634
• Publication Number:
  US 20090305433A1
• Time in Patent Office: 551 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 536/4.1, 536/23.1, 536/24.3, 536/25.3, 536/26.6, 530/300, 530/350, 436/800
• US Class Current: 435/7.92
• CPC Class Codes: C07D 311/80 Dibenzopyrans; Hydrogenated dibenzopyrans
• CPC Class Codes: C07D 491/04 Ortho-condensed systems
• CPC Class Codes: C07D 491/06 Peri-condensed systems
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• 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 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: G01N 2458/00 Labels used in chemical analysis of biological material
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

The present invention provides novel, water-soluble, red-emitting fluorescent rhodamine dyes and red-emitting fluorescent energy-transfer dye pairs, as well as labeled conjugates comprising the same and methods for their use. The dyes, energy-transfer dye pairs and labeled conjugates are useful in a variety of aqueous-based applications, particularly in assays involving staining of cells, protein binding, and/or analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.

Claims

1. A rhodamine dye or a salt thereof, comprising a rhodamine-type parent xanthene ring having attached to the xanthene C9 carbon a phenyl group that is further substituted with an ortho carboxy or ortho sulfonate group or a salt thereof, one to three substituted or unsubstituted aminopyridinium groups and a substituted or unsubstituted alkylthio, arylthio, or heteroarylthio group, said rhodamine dye optionally including one or more linking moieties.

2. The rhodamine dye of claim 1 which comprises the structure:

3. A labeled rhodamine dye-polypeptide conjugate comprising the rhodamine dye of claim 1 and a polypeptide, wherein the polypeptide is selected from the group consisting of a peptide, a protein, and an antibody.

4. A method of detecting a rhodamine dye-antibody conjugate, in which said conjugate is a rhodamine dye-antibody conjugate according to claim 3, comprising the steps of:

(a) binding the conjugates to a peptide or protein, and

(b) detecting the rhodamine dye-antibody conjugate bound to the peptide or protein.

5. The method of claim 4 in which the conjugate is bound to the peptide or protein in the presence of a second antibody specific for binding said peptide or protein.

6. The method of claim 5 in which the second antibody is bound to a solid bead or particle.