Dimers of unsymmetrical cyanine dyes containing pyridinium moieties

• US 5,410,030 A
• Filed: 04/05/1993
• Issued: 04/25/1995
• Est. Priority Date: 04/05/1993
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Yue, Stephen T., Haugland, Richard P.
• Primary Examiner(s): Ivy, C. Warren
• Assistant Examiner(s): NORTHINGTON DAVIS, ZINNA
• Application Number:
  US08/043,665
• Time in Patent Office: 750 Days
• Field of Search: 546/165, 546/256, 546/152, 546/176, 536/23.1, 536/24.3, 536/25.32
• US Class Current: 536/23.1
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups

Abstract

The invention relates to nucleic acid stains that are homo- or heterodimers of unsymmetrical cyanine dyes, where at least one dye unit of the dimer is a substituted or unsubstituted benzazolium- or naphthazolium-pyridinium derivative, which dye unit is linked to another unsymmetrical cyanine dye unit, that is the same or different by an aliphatic chain containing one or more heteroatoms; such that the fluorescence of the dye molecules, when bound to nucleic acids, is enhanced. These dimers of unsymmetrical cyanine dyes have spectral properties and binding affinities superior to related monomers. In addition, the dyes can be tailored to absorb and emit in a wide range of wavelengths, which can be further varied by energy transfer mixtures, making the dyes useful for applications that involve simultaneous use of multiple dyes.

Claims

1. A compound of the formula:

space="preserve" listing-type="equation">α

--(CH═

CH).sub.n --β

--BRIDGE--γ

--(CH═

CH).sub.n'"'"' --δ

where each of α and

δ

is independently a benzazolium ring moiety that is a benzoxazolium, benzothiazolium, benzimidazolium, 3,3-dialkylindolium heterocycle or a naphthazolium ring moiety that is a naphthoxazolium, naphthothiazolium, napthimidazolium or benzo-3,3-dialkylindolium heterocycle, each of the nitrogen atoms of which is substituted by alkyl with 1 to 6 carbons, which benzazolium ring moiety is linked to --(CH═

CH)_n -- or --(CH═

CH)_n'"'"' -- respectively through a 2-position of the ring moiety, wherein the remaining substituents of the ring moiety are independently hydrogen, halogen, trifluoromethyl, alkyl, alkoxy, amino, mono- or dialkylamino, the alkyl portions of which contain 1 to 6 carbons;

n and n'"'"' are independently 0, 1 or 2;

β

is a first 2-methine-substituted pyridinium;

##STR17## or a first 4-methine-substituted pyridinium;

##STR18## that is linked to --(CH═

CH)_n -- through an exocyclic methine residue of β

, which pyridinium is optionally substituted by hydrogen, halogen, trifluoromethyl, alkyl, alkoxy, amino, mono- or dialkylamino, the alkyl portions of which contain 1 to 6 carbons;

BRIDGE is an aliphatic chain containing a backbone of 4 to 19 methylene groups (--CH₂ --), which is optionally interspersed at one or more intervals with a heteroatom, each of which is independently N, O or S, wherein each N heteroatom is additionally substituted by H, alkyl or two alkyl groups with 1 to 6 carbons, which alkyl substituents may be the same or different, provided that any heteroatom is separated from another heteroatom by at least 2 methylene groups, wherein one methylene terminus of BRIDGE is attached to a nitrogen atom of β and

another methylene terminus of BRIDGE is attached to a nitrogen atom of γ

;

γ

is a second 2-methine-substituted pyridinium or a second 4-methine-substituted pyridinium or a 2-methine-substituted quinolinium;

##STR19## or a 4-methine-substituted quinolinium;

##STR20## that is linked to --(CH═

CH)_n'"'"' -- through an exocyclic methine residue of γ

, which pyridinium or quinolinium is optionally substituted by hydrogen, halogen, trifluoromethyl, alkyl, alkoxy, amino, mono- or dialkylamino, the alkyl portions of which contain 1 to 6 carbons;

and Ψ

is a counterion or counterions that neutralize positive charges on the compound.

2. A compound, as claimed in claim 1, where α

and δ

are each benzothiazolium or benzoxazolium moieties.

3. A compound, as claimed in claim 2, where β

and γ

are each pyridinium moieties.

4. A compound, as claimed in claim 2, in which n and n'"'"' are both 0.

5. A compound, as claimed in claim 2, in which n and n'"'"' are both 1.

6. A compound, as claimed in claim 1, where β

and γ

are each pyridinium moieties.

7. A compound, as claimed in claim 6, where α

and δ

are each benzoxazolium moieties.

8. A compound, as claimed in claim 6, where α

and δ

are each benzothiazolium moieties.

9. A compound, as claimed in claim 1, where BRIDGE has the formula:

space="preserve" listing-type="equation">--(CH.sub.2).sub.p --J--(CH.sub.2).sub.q --K--(CH.sub.2).sub.r --L--(CH.sub.2).sub.s --

where s is 2-12 and p, q and r are independently 0 or 2-6, provided that if any of p, q or r is 0 then the adjacent moiety J, K or L is absent;

and wherein J, K and L are independently O, S, imino (--NH--), immonium (--NH₂⁺ --), alkylimino (--NR¹ --), alkylimmonium (--NHR₁⁺ --) or dialkylimmonium (--NR¹ R²⁺ --), wherein R¹ and R² independently have 1 to 6 carbons.

10. A compound, as claimed in claim 9, where α

and δ

are each benzothiazolium or benzoxazolium moieties.

11. A compound, as claimed in claim 10, where β

and γ

are each pyridinium moieties.

12. A compound, as claimed in claim 11, in which n and n'"'"' are both 0.

13. A compound, as claimed in claim 11, in which n and n'"'"' are both 1.

14. A compound, as claimed in claim 9, where β

and γ

are each pyridinium moieties.

15. A compound, as claimed in claim 14, where α

and δ

are each benzoxazolium moieties.

16. A compound, as claimed in claim 14, where α

and δ

are each benzothiazolium moieties.

17. A compound, as claimed in claim 9, where at least one of J, K and L is imino (--NH--), immonium (--NH₂⁺ --), alkylimino (--NR¹ --), alkylimmonium (--NHR₁⁺ --) or dialkylimmonium (--NR¹ R²⁺ --).

18. A compound, as claimed in claim 9, where at least two of J, K and L are dimethylammonium.

19. A compound of the formula:

##STR21## where X is S or O;

R³ is C₁ to C₆ alkyl;

Ψ

is a counterion or counterions that neutralize positive charges on the compound;

and wherein n and n'"'"' are independently 0, 1 or 2.

20. A compound, as claimed in claim 19, wherein X is S or O, R³ is methyl and n and n'"'"' are both 0.

21. A compound, as claimed in claim 19, wherein X is S or O, R³ is methyl and n and n'"'"' are both 1.

22. A compound comprising a nucleic acid polymer bound to one or more dye molecules of the formula:

space="preserve" listing-type="equation">α

--(CH═

CH).sub.n --β

--BRIDGE--γ

--(CH═

CH).sub.n'"'"' --δ

where each of α and

δ

is independently a benzazolium ring moiety that is a benzoxazolium, benzothiazolium, benzimidazolium, 3,3-dialkylindolium heterocycle or a naphthazolium ring moiety that is a naphthoxazolium, naphthothiazolium, napthimidazolium or benzo-3,3-dialkylindolium heterocycle, each of the nitrogen atoms of which is substituted by alkyl with 1 to 6 carbons, which benzazolium ring moiety is linked to --(CH═

CH)_n -- or --(CH═

CH)_n'"'"' -- respectively through a 2-position of the ring moiety, wherein the remaining substituents of the ring moiety are independently hydrogen, halogen, trifluoromethyl;

alkyl, alkoxy, amino, mono- or dialkylamino, the alkyl portions of which contain 1 to 6 carbons;

n and n'"'"' are independently 0, 1 or 2;

β

is a first 2-methine-substituted pyridinium;

##STR22## or a first 4-methine-substituted pyridinium;

##STR23## that is linked to --(CH═

CH)_n -- through an exocyclic methine residue of β

, which pyridinium is optionally substituted by hydrogen, halogen, trifluoromethyl, alkyl, alkoxy, amino, mono- or dialkylamino, the alkyl portions of which contain 1 to 6 carbons;

BRIDGE is an aliphatic chain containing a backbone of 4 to 19 methylene groups (--CH₂ --), which is optionally interspersed at one or more intervals with a heteroatom, each of which is independently N, O or S, wherein each N heteroatom is additionally substituted by H, alkyl or two alkyl groups with 1 to 6 carbons, which alkyl substituents may be the same or different, provided that any heteroatom is separated from another heteroatom by at least 2 methylene groups, wherein one methylene terminus of BRIDGE is attached to a nitrogen atom of β and

another methylene terminus of BRIDGE is attached to a nitrogen atom of γ

;

γ

is a second 2-methine-substituted pyridinium or a second 4-methine-substituted pyridinium or a 2-methine-substituted quinolinium;

##STR24## or a 4-methine-substituted quinolinium;

##STR25## that is linked to --(CH═

CH)_n'"'"' -- through an exocyclic methine residue of γ

, which pyridinium or quinolinium is optionally substituted by hydrogen, halogen, trifluoromethyl, alkyl, alkoxy, amino, mono- or dialkylamino, the alkyl portions of which contain 1 to 6 carbons;

and Ψ

is a counterion or counterions that neutralize positive charges on the compound such that the fluorescence of said dye molecules when bound to the nucleic acid polymer is enhanced.

23. A compound, as claimed in claim 22, wherein BRIDGE has the formula:

space="preserve" listing-type="equation">--(CH.sub.2).sub.p --J--(CH.sub.2).sub.q --K--(CH.sub.2).sub.r --L--(CH.sub.2).sub.s --

where s is 2-12, and p, q and r are independently 0 or 2-6, provided that if any of p, q or r is 0 then the adjacent moiety J, K or L is absent;

and wherein J, K and L are independently O, S, imino (--NH--), immonium (--NH₂⁺ --), alkylimino (--NR¹ --), alkylimmonium (--NHR₁⁺ --) or dialkylimmonium (--NR¹ R²⁺ --), wherein R¹ and R² independently have 1 to 6 carbons.

24. A compound, as claimed in claim 22, wherein the dye has the formula:

##STR26## where X is S or O;

R³ is C₁ to C₆ alkyl;

and wherein n and n'"'"' are independently 0, 1 or 2.

25. A compound, as claimed in claim 22, where the nucleic acid polymer is DNA.

26. A compound, as claimed in claim 22, where the nucleic acid polymer is RNA.

27. A compound, as claimed in claim 22, where the nucleic acid polymer is intracellular.

28. A compound, as claimed in claim 22, where the fluorescence enhancement of the dye molecules when bound to the nucleic acid is greater than about 50-fold.

29. A compound, as claimed in claim 22, containing at least two different dye molecules having different spectral properties, where a first dye molecule has an emission maximum that substantially overlaps an excitation maximum of a second dye molecule.

30. A compound, as claimed in claim 29, where one of the different dye molecules are selected from the group consisting of:

##STR27## where X is S or O;

R³ is C₁ to C₆ alkyl;

and wherein n and n'"'"' are independently 0, 1 or 2.