Nucleic acids labelled with extended rhodamine dyes

• US 6,781,001 B2
• Filed: 02/09/2001
• Issued: 08/24/2004
• Est. Priority Date: 06/03/1999
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y. L., Menchen, Steven M., Benson, Scott C.
• Primary Examiner(s): Celsa, Bennett
• Assistant Examiner(s): Epperson, Jon D.
• Application Number:
  US09/780,600
• Publication Number:
  US 20010007905A1
• Time in Patent Office: 1,292 Days
• Field of Search: 546/28, 546/30, 546/44, 546/71, 546/73, 546/75, 546/79, 546/108, 546/41, 546/101, 546/104, 546/111, 544/59, 544/63, 544/180, 544/336, 544/359, 548/131, 548/206, 548/240, 548/250, 548/311.4, 548/311.7, 549/382, 549/384, 536/22.1, 536/26.1, 536/26.2
• US Class Current: 549/382
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 221/08 Aza-anthracenes
• CPC Class Codes: C07D 221/10 Aza-phenanthrenes
• CPC Class Codes: C07D 471/06 Peri-condensed systems
• CPC Class Codes: C07D 487/06 Peri-condensed systems
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Extended rhodamine compounds exhibiting favorable fluorescence characteristics having the structure

are disclosed. In addition, novel intermediates for synthesis of these dyes are disclosed, such intermediates having the structure

In addition, methods of making and using the dyes as fluorescent labels are disclosed.

Claims

1. A labelled nucleic acid compound having the formula:

2. The labelled nucleic acid compound of claim 1 wherein

Y₁ is taken together with R₁ or R₁₁ and is C₂ or C₃ alkyleno or alkyleno independently substituted with one or more Z₁;

or

Y₂ is taken together with R₁ or R₁₁ and is C₂ or C₃ alkyleno or alkyleno independently substituted with one or more Z₁;

or

Y₃ is taken together with R₄ or R₅ or R₆ or R₁₃ and is C₂ or C₃ alkyleno or alkyleno independently substituted with one or more Z₁;

or

Y₄ is taken together with R₄ or R₅ or R₆ or R₁₃ and is C₂ or C₃ alkyleno or alkyleno independently substituted with one or more Z₁.

3. The labelled nucleic acid compound of claim 2 wherein the C₂ or C₃ substituted alkyleno is gem disubstituted with C₁-C₃ alkyl.

4. The labelled nucleic acid compound of claim 2 wherein the C₂ or C₃ substituted alkyleno is gem disubstituted with methyl.

5. The labelled nucleic acid compound of claim 1 wherein R₈ is alkyl independently substituted with one or more substituents selected from halogen, —

C(O)R, and —

S(O)₂R wherein R is independently selected from —

OH, O-alkyl, —

NH₂, N-alkyl and a linking group.

6. The labelled nucleic acid compound of claim 1 wherein R₈ is —

CF₃.

7. The labelled nucleic acid compound of claim 1 wherein R₈ is aryl or aryl independently substituted with one or more Z₁.

8. The labelled nucleic acid compound of claim 1 wherein R₈ is selected from the structures:

wherein LG is a linking group.

9. The labelled nucleic acid compound of claim 1 wherein NUC comprises a nucleobase selected from uracil, cytosine, deazaadenine, and deazaguanosine.

10. The labelled nucleic acid compound of claim 1 having the structure:

wherein B is a nucleobase;

W₁ and W₂ taken separately are selected from —

H, —

OH, and —

F; and

W₃ is selected from —

OH, monophosphate, diphosphate, triphosphate and phosphate analog.

11. The labelled nucleic acid compound of claim 1 having the structure:

wherein B is a nucleobase.

12. The labelled nucleic acid compound of claim 1 having the structure:

wherein B is a nucleobase.

13. The labelled nucleic acid compound of claim 1 having the structure:

wherein B is a nucleobase.

14. The labelled nucleic acid compound of claim 1 wherein L is attached to a nucleobase of NUC and to D to form the structure:

15. The labelled nucleic acid compound of claim 1 wherein L is attached to a nucleobase of NUC and to D to form the structure:

 

NUC—

C≡

C—

CH₂OCH₂CH₂NR₃X—

D

wherein R₃ is selected from —

H and (C₁-C₆) alkyl; and

X is selected from the structures;

where n ranges from 1 to 5;

φ

is aryldiyl; and

R₁ is selected from —

H, (C₁-C₆) alkyl and protecting group.

16. The labelled nucleic acid compound of claim 1 wherein L is attached at R₈ of D.

17. The labelled nucleic acid compound of claim 1 wherein NUC is a polynucleotide and L is attached to the polynucleotide at a position selected from the 5′

terminus, the phosphodiester backbone, a nucleobase, and the 3′

terminus.

18. The labelled nucleic acid compound of claim 17 wherein L comprises an aminohexyl linking group, NUC is a polynucleotide and L is attached to the polynucleotide at the 5′

terminus.