METHOD OF DETECTING FLUORESCENCE

• US 20110060150A1
• Filed: 08/10/2010
• Published: 03/10/2011
• Est. Priority Date: 09/20/2002
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (Danaher Corporation)
• Inventors: Laikhter, Andrei, Yong, Yawfui, Behlke, Mark Aaron, Huang, Lingyan, Rose, Scott
• Granted Patent: US 8,114,979 B2
• Time in Patent Office: Days
• US Class Current: 552/209
• CPC Class Codes: C07C 225/36 the carbon skeleton being further substituted by singly-bound oxygen atoms
• CPC Class Codes: C07F 9/091 with hydroxyalkyl compounds with further substituents on alkyl
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• 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 1/325 Dyes with no other substituents than the amino groups
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer

Abstract

The invention provides novel anthraquinone compositions that are useful as broad-spectrum quenchers of fluorescence and provides methods for making and using them. The anthraquinone quenchers can be conjugated to a variety of biologically relevant compounds, including lipids, nucleic acids, polypeptides, and more specifically antigens, steroids, vitamins, drugs, haptens, metabolites, toxins, environmental pollutants, amino acids, peptides, proteins, nucleotides, oligonucleotides, polynucleotides, carbohydrates, and their analogs. The invention also provides kits comprising, in one or more containers, at least one anthraquinone quencher dye composition of the present invention, and instructions for using that composition.

Claims

1. A compound comprising a di-alpha amino anthraquinone comprising a first amino group and a second amino group attached at alpha-positions of the anthraquinone, wherein a first substituent attached to the first amino group is a phosphate ester or phosphite ester and wherein a second substituent attached to the second amino group is an alcohol or a protected alcohol.

2. The compound of claim 1, wherein said phosphite ester is a phosphoramidite.

3. The compound of claim 2, wherein said phosphoramidite is of formula (3):

4. The compound of claim 1, wherein said alcohol is protected with a trityl group.

5. The compound of claim 4, wherein the trityl group is a dimethoxytrityl group.

6. The compound of claim 5, wherein the protected alcohol substituent has the following structure:

7. The compound of claim 1, wherein a third and fourth substituent attached to the first and second amino groups respectively are selected independently from the groups hydrogen, alkyl, alkynyl, alkenyl, aryl, heteroaryl, cycloalkyl, heteroalkyl, alkoxy, alkoxycarbonyl, carbonyl, carbamoyl, alkylaryl, and heteroalkyl.

8. The compound of claim 1, wherein the di-alpha amino anthraquinone has the formula:

9. The compound of claim 1, further comprising an oligonucleotide labeled with the di-alpha amino anthraquinone.

10. The compound of claim 9, wherein said phosphite ester is a phosphoramidite.

11. The compound of claim 9, wherein said phosphoramidite is of formula (3):

12. The compound of claim 9, wherein said alcohol is protected with a trityl group.

13. The compound of claim 12, wherein the trityl group is a dimethoxytrityl group.

14. The compound of claim 12, wherein the protected alcohol substituent has the following structure:

15. The compound of claim 9, wherein a third and fourth substituent attached to the first and second amino groups respectively are selected independently from the groups hydrogen, alkyl, alkynyl, alkenyl, aryl, heteroaryl, cycloalkyl, heteroalkyl, alkoxy, alkoxycarbonyl, carbonyl, carbamoyl, alkylaryl, and heteroalkyl.

16. The compound of claim 9, wherein the oligonucleotide is further labeled with a fluorophore.

17. The compound of claim 16, wherein the fluorophore is selected from the group consisting of pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazle, cyanine, carbocyanine, slaicylate, anthranilate, coumarin, fluorescein and rhodamine.

18. The compound of claim 16, wherein the di-alpha amino anthraquinone is adapted to quench the fluorescence of the fluorophore.

19. The compound of claim 9, wherein the oligonucleotide is adapted to hybridize to a selected nucleic acid sequence.

20. The compound of claim 9, wherein the di-alpha amino anthraquinone has the formula: