Dimers of unsymmetrical cyanine dyes

• US 5,582,977 A
• Filed: 01/06/1994
• Issued: 12/10/1996
• Est. Priority Date: 09/16/1991
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Johnson, Iain D., Yue, Stephen T., Haugland, Richard P.
• Primary Examiner(s): Guzo, David
• Application Number:
  US08/180,763
• Time in Patent Office: 1,069 Days
• Field of Search: 435/968, 435/6, 435/7.1, 435/34, 435/40.5, 536/23.1, 546/152, 546/165, 546/176
• US Class Current: 435/6.12
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72

Abstract

The invention relates to dimers of unsymmetrical cyanine dyes, typically dimers of benzthiazole or benzoxazole derivatives, that exhibit enhanced fluorescence on binding with DNA or RNA, The dimers generally have the formula: ##STR1## where R¹ and R², which may be the same or different, are alkyl groups having 1-6 carbons;

X is O, S, or N--R³, where R³ is H or an alkyl group having 1-6 carbons;

Z is O, S, or N--R⁴, where R⁴ is H or an alkyl group having 1-6 carbons;

n and s, which may be the same or different, =0, 1, or 2;

Y is HC═CH; and

p, m, q, and r=0 or 1, such that p+m=1 and q+r=1; and

where -BRIDGE- has the general formula:

--(CH.sub.2).sub.α --[A.sup.1 --(CH.sub.2).sub.β --].sub.I

[A² --(CH₂).sub.γ --]_II A³ --(CH₂).sub.δ --

where α, β, γ, and δ, which may be the same or different, are integers greater than 1 and less than 5;

I and II, which may be the same or different, =0 or 1; and

A¹, A², and A³, which may be the same or different, are independently O; S; (CH₂).sub.μ where μ=0 or 1; --(NR⁵)-- where R⁵ is H or an alkyl group having 1-6 carbons; or--(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-6 carbons.

Claims

1. A compound of the formula:

##STR8## where R¹ and R², which may be the same or different, are alkyl groups having 1-6 carbons;

X is O, S, or N--R³, where R³ is H or an alkyl group having 1-6 carbons;

Z is O, S, or N--R⁴, where R⁴ is H or an alkyl group having 1-6 carbons;

n and s, which may be the same or different, =0, 1, or 2;

Y is HC═

CH; and

p, m, q, and r=0 or 1, such that p+m=1 and q+r=1; and

where -BRIDGE- has the general formula;

space="preserve" listing-type="equation">--(CH.sub.2).sub.α

--[A.sup.1 --(CH.sub.2).sub.β

--].sub.I [A.sup.2 --(CH.sub.2).sub.γ

--].sub.II A.sup.3 --(CH.sub.2).sub.δ

--

where α

, β

, γ

, and δ

, which may be the same or different, are integers greater than 1 and less than 5;

I and II, which may be the same or different, =0 or 1;

A¹ and A², which may be the same or different, are independently O;

S;

(CH₂).sub.μ

where μ

=0 or 1;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-6 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-6 carbons, and

A³ is S;

(CH₂).sub.μ

where μ

=0 or 1;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-6 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-6 carbons.

2. A compound, as claimed in claim 1, where A¹, A² and A³, which may be the same or different, are independently (CH₂).sub.μ

, --(NR⁵)--, or --N⁺ R⁶ R⁷)--.

3. A compound, as claimed in claim 1, where X and Z are the same;

R¹ and R² are the same;

n=s and m=r.

4. A compound, as claimed in claim 1, where m and r are each 1.

5. A compound, as claimed in claim 1, where X and Z, which may be the same or different, are O or S.

6. A compound, as claimed in claim 1, where n is not equal to s.

7. A compound, as claimed in claim 1, where A¹ and A³, which may be the same or different, are independently --(NR⁵)-- where R⁵ is H or an alkyl group having 1-2 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-2 carbons; and

II=0.

8. A compound, as claimed in claim 1, where R¹ and R², which may be the same or different, are alkyl groups having 1-2 carbons;

X and Z, which may be the same or different are O or S;

m and r=1;

in the BRIDGE formula

α

, β

, γ

, and δ

, which may be the same or different, are 2 or 3;

A¹, A², and A³, which may be the same or different, are (CH₂).sub.μ

where μ

=0;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-2 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-2 carbons.

9. A compound, as claimed in claim 3, where R¹ and R² are alkyl groups having 1-2 carbons;

X and Z are O or S;

m and r=1;

in the BRIDGE formula

α

, β

, γ

, and δ

, which may be the same or different, are 2 or 3;

A¹, A², and A³, which may be the same or different, are (CH₂).sub.μ

where μ

=0;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-2 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-2 carbons.

10. A fluorescent compound comprising a nucleic acid polymer bound to a dye of the general formula:

##STR9## where R¹ and R², which may be the same or different, are alkyl groups having 1-6 carbons;

X is O, S, or N--R³, where R³ is H or an alkyl group having 1-6 carbons;

Z is O, S, or N--R⁴, where R⁴ is H or an alkyl group having 1-6 carbons;

n and s, which may be the same or different, =0, 1, or 2;

Y is HC═

CH; and

p, m, q, and r=0 or 1, such that p+m=1 and q+r=1; and

where -BRIDGE- has the general formula;

space="preserve" listing-type="equation">--(CH.sub.2).sub.α

--[A.sup.1 --(CH.sub.2).sub.β

--].sub.I [A.sup.2 --(CH.sub.2).sub.γ

--].sub.II A.sup.3 --(CH.sub.2).sub.δ

--

where α

, β

, γ

, and δ

, which may be the same or different, are integers greater than 1 and less than 5;

I and II, which may be the same or different, =0 or 1; and

A¹, A², and A³, which may be the same or different, are independently O;

S;

(CH₂).sub.μ

where μ

=0 or 1;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-6 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-6 carbons.

11. A compound, as claimed in claim 10, where A¹, A² and A³, which may be the same or different, are independently (CH₂).sub.μ

, --(NR⁵)--, or --(N⁺ R⁶ R⁷)--.

12. A compound, as claimed in claim 10, where A¹ and A³, which may be the same or different, are independently --(NR⁵)-- where R⁵ is H or an alkyl group having 1-2 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-2 carbons; and

II=0.

13. A compound, as claimed in claim 10, where R¹ and R², which may be the same or different, are alkyl groups having 1-2 carbons;

X and Z, which may be the same or different are O or S;

m and r=1;

in the BRIDGE formula

α

, β

, γ

, and δ

, which may be the same or different, are 2 or 3;

A¹, A², and A³, which may be the same or different, are (CH₂).sub.μ

where μ

=0;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-2 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-2 carbons.

14. A compound, as claimed in claim 11, where X and Z are the same;

R¹ and R² are the same;

n=s and m=r.

15. A compound, as claimed in claim 11, where m and r are each 1;

and where X and Z, which may be the same or different, are O or S.

16. A compound, as claimed in claim 11, where n is not equal to s.

17. A compound as claimed in claim 11, where the nucleic acid polymer is DNA.

18. A compound as claimed in claim 11, where the nucleic acid polymer is RNA.

19. A compound, as claimed in claim 14, where R¹ and R² are alkyl groups having 1-2 carbons;

X and Z are O or S;

m and r=1;

in the BRIDGE formula

α

, β

, γ

, and δ

, which may be the same or different, are 2 or 3;

A¹, A², and A³, which may be the same or different, are (CH₂).sub.μ

where μ

=0;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-2 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-2 carbons.

20. A method of forming a fluorescent nucleic acid complex, comprising:

a) combining a sample thought to contain nucleic acid polymers with a dye of the general formula;

##STR10## where R¹ and R², which may be the same or different, are alkyl groups having 1-6 carbons;

X is O, S, or N--R³, where R³ is H or an alkyl group having 1-6 carbons;

Z is O, S, or N--R⁴, where R⁴ is H or an alkyl group having 1-6 carbons;

n and s, which may be the same or different, =0, 1, or 2;

Y is HC═

CH; and

p, m, q, and r=0 or 1, such that p+m=1 and q+r=1; and

where -BRIDGE- has the general formula;

space="preserve" listing-type="equation">--(CH.sub.2).sub.α

--[A.sup.1 --(CH.sub.2).sub.β

--].sub.I [A.sup.2 --(CH.sub.2).sub.γ

--].sub.II A.sup.3 --(CH.sub.2).sub.δ

--

where α

, β

, γ

, and δ

, which may be the same or different, are integers greater than 1 and less than 5;

I and II, which may be the same or different, =0 or 1; and

A¹, A², and A³, which may be the same or different, are independently O;

S;

(CH₂).sub.μ

where μ

=0 or 1;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-6 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-6 carbons;

b) after the dye has been allowed to combine with any nucleic acid polymers in said sample, detecting fluorescence resulting from the dye combining with nucleic acid polymers.

21. A method, as in claim 20, wherein the nucleic acid polymers in the sample are contained in cells.

22. A method, as in claim 20, wherein the sample contains nucleic acid polymers in solution.

23. A method, as in claim 20, where R¹ and R², which may be the same or different, are alkyl groups having 1-2 carbons;

X and Z, which may be the same or different are O or S;

m and r=1;

in the BRIDGE formula

α

, β

, γ

, and δ

, which may be the same or different, are 2 or 3;

A¹, A², and A³, which may be the same or different, are (CH₂).sub.μ

;

--(NR⁵)-- where R⁵ is H or an alkyl group having 1-2 carbons;

or --(N⁺ R⁶ R⁷)-- where R⁶ and R⁷, which may be the same or different, are independently hydrogen or an alkyl group having 1-2 carbons.

24. A method, as in claim 21, wherein said fluorescence indicates loss of cellular membrane integrity.