4, 7-Dichlororhodamine dyes

• US 5,847,162 A
• Filed: 06/27/1996
• Issued: 12/08/1998
• Est. Priority Date: 06/27/1996
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Benson, Scott C., Rosenblum, Barnett B., Spurgeon, Sandra L., Lee, Linda
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/672,196
• Time in Patent Office: 894 Days
• Field of Search: 435/6, 536/22.1, 549/356, 549/381, 549/385, 549/227
• US Class Current: 549/227
• 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

Abstract

A class of 4,7-dichlororhodamine compounds useful as fluorescent dyes are disclosed having the structure ##STR1## wherein R₁ -R₆ are hydrogen, fluorine, chlorine, lower alkyl lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, lining group, or combinations thereof or, when taken together, R₁ and R₆ is benzo, or, when taken together, R₄ and R₅ is benzo; Y₁ -Y₄ are hydrogen or lower alkyl or, when taken together, Y₁ and R₂ is propano and Y₂ and R₁ is propano, or, when taken together, Y₃ and R₃ is propano and Y₄ and R₄ is propano; and X₁ -X₃ taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl carboxylate, sulfonic acid, --CH₂ OH, and linking group. In another aspect, the invention includes reagents labeled with the 4,7-dichlororhodamine dye compounds, including deoxynucleotides, dideoxynucleotides, and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.

Claims

1. A compound having the formula:

##STR32## wherein;

R₁ -R₆ taken separately are selected from the group consisting of hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group, and combinations thereof, or, when taken together, R₁ and R₆ is benzo, or, when taken together, R₄ and R₅ is benzo;

Y₁ -Y₄ taken separately are selected from the group consisting of hydrogen and lower alkyl or, when taken together, Y₁ and R₂ is propano and Y₂ and R₁ is propano, or, when taken together, Y₃ and R₃ is propano and Y₄ and R₄ is propano; and

X₁ -X₃ taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonic acid, --CH₂ OH, and linking group.

2. The compound of claim 1 wherein X₁ is carboxylate.

3. The compound of claim 1 wherein one of X₂ and X₃ is linking group.

4. The compound of claim 1 wherein R₂ and R₃ taken separately are hydrogen.

5. The compound of claim 1 wherein:

R₁ -R₆ taken separately are selected from the group consisting of hydrogen, methyl, and ethyl, or, when taken together, R₁ and R₆ is benzo, or when taken together, R₄ and R₅ is benzo;

Y₁ -Y₄ taken separately are selected from the group consisting of hydrogen, methyl, and ethyl, or, when taken together, Y₁ and R₂ is propano and Y₂ and R₁ is propano, or, when taken together, Y₃ and R₃ is propano and Y₄ and R₄ is propano;

X₁ is carboxylate; and

X₂ and X₃ taken separately are selected from the group consisting of hydrogen and linking group.

6. The compound of claim 1 wherein:

R₁ -R₆ taken separately are hydrogen;

Y₁ -Y₄ taken separately are hydrogen;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

7. The compound of claim 1 wherein:

R₁ and R₄ taken separately are methyl;

R₂, R₃, R₅, and R₆ are hydrogen;

one of Y₁ and Y₂ is ethyl, the other being hydrogen;

one of Y₃ and Y₄ is ethyl, the other being hydrogen;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

8. The compound of claim 1 wherein:

R₁ -R₆ taken separately are hydrogen;

Y₁ -Y₄ taken separately are methyl;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

9. The compound of claim 1 wherein:

R₁ and Y₂ taken together are propano;

R₂ and Y₁ taken together are propano;

R₃ and Y₃ taken together are propano;

R₄ and Y₄ taken together are propano;

R₅ and R₆ are hydrogen;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

10. The compound of claim 1 wherein:

R₁ is methyl;

R₂ -R₆ taken separately are hydrogen;

one of Y₁ and Y₂ is ethyl, the other being hydrogen;

Y₃ and Y₄ taken separately are hydrogen;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

11. The compound of claim 1 wherein:

R₁ is methyl;

R₂ -R₆ taken separately are hydrogen;

one of Y₁ and Y₂ is ethyl, the other being hydrogen;

Y₃ and Y₄ taken separately are methyl;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

12. The compound of claim 1 wherein:

R₁, R₂, R₅, and R₆ taken separately are hydrogen;

Y₁ and Y₂ taken separately are methyl;

R₃ and Y₃ taken together are propano;

R₄ and Y₄ taken together are propano;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

13. The compound of claim 1 wherein:

R₁, R₂, R₅, and R₆ taken separately are hydrogen;

Y₁ and Y₂ taken separately are hydrogen;

R₃ and Y₃ taken together are propano;

R₄ and Y₄, taken together are propano;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

14. The compound of claim 1 wherein:

R₁ is methyl;

R₂, R₅ and R₆ taken separately are hydrogen;

one of Y₁ and Y₂ is ethyl, the other being hydrogen;

R₃ and Y₃ taken together are propano;

R₄ and Y₄ taken together are propano propano;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.

15. The compound of claim 1 wherein:

R_1-R6 taken separately are hydrogen;

Y₁ and Y₂ taken separately are hydrogen;

Y₃ and Y₄ taken separately are methyl;

X₁ is carboxylate; and

one of X₂ and X₃ is linking group, the other being hydrogen.