Cyclic-substituted unsymmetrical cyanine dyes

• US 5,436,134 A
• Filed: 07/12/1993
• Issued: 07/25/1995
• Est. Priority Date: 04/13/1993
• 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., Millard, Paul J., Roth, Bruce L.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US08/090,890
• Time in Patent Office: 743 Days
• Field of Search: 435/34, 435/6, 435/29, 435/32, 435/33, 435/4, 435/39, 536/27, 536/28, 536/26.73, 536/1.11, 536/25.6, 558/161, 436/94, 436/800
• US Class Current: 435/34
• CPC Class Codes: C07D 213/64 attached in position 2 or 6
• CPC Class Codes: C07D 215/18 Halogen atoms or nitro radicals
• CPC Class Codes: C07D 215/227 only one oxygen atom which is attached in position 2
• CPC Class Codes: C07D 409/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 413/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 417/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 417/14 containing three or more hetero rings
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention describes the preparation and use of fluorescent stains for nucleic acids derived from unsymmetrical cyanine dyes. In particular, the invention describes unsymmetrical cyanine dyes having a saturated or unsaturated cyclic substituent. The dyes of the invention possess superior fluorescent characteristics when complexed with nucleic acids, and have utility in any application which requires detection of nucleic acids. The presence of the cyclic substituent results in improved permeability in a wide range of living cells, resulting in improved detection of intracellular nucleic acids.

Claims

1. A compound of the formula ##STR34## wherein:

each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

or a trifiuoromethyl;

or a halogen;

or --OR⁸,-- SR⁸ or --(NR⁸ R⁹) where R⁸ and R⁹, which can be the same or different, are independently H;

or alkyl groups having 1-6 carbons;

or 1-2 allcyclic, heteroalicyclic, aromatic or heterouromatic nngs, containing 1-4 heteroatoms, wherein the hetero atoms are O, N or S;

or R⁸ and R⁹ taken in combination are --(CH₂)₂ --L--(CH₂)₂ -- where L=a single bond, --O--, --CH₂ --, or --NR₁₀ --,where R¹⁰ is H or an alkyl group having 1-6 carbons; and

t=1-4;

R² is an alkyl group having 1-6 carbons;

X is O, S, Se or --NR¹⁵, where R¹⁵ is H or an alkyl group having 1-6 carbons;

or X is CR¹⁶ R¹⁷ where R¹⁶ and R¹⁷, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R¹⁶ and R¹⁷ taken in combination complete a five or six membered saturated ring;

n=0, 1 or 2;

Z^- is a biologically compatible counterion;

Q has the formula Q1 or Q2 ##STR35## wherein Y is --CR³ =CR⁴ --;

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

R⁵ is an alkyl, alkenyl, polyalkenyl, alkynyl or polyailkynyl group having 1-6 carbons;

or R⁵ is aa OMEGA;

R³, R⁴, R⁶ and R⁷, which may be the same or dillSrent, are independently H;

or an alkyl, alkenyl, polyalkenyl, alkynyl or polyatlcynyl group having 1-6 carbons;

or a halogen;

or --OH, --OR⁸, --SR⁸, --(NR⁸ R⁹);

or --OSO₂ R¹⁹ where R¹⁹ is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl;

or an OMEGA;

or R⁶ and R⁷, taken in combination are --(CH₂)_v -- where v=3 or 4, or R⁶ and R⁷ form a fused aromatic ring according to formula Q2;

R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H, or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons;

or a halogen;

or an OMEGA;

or --OH, --OR⁸, --SR⁸, or --(NR⁸ R⁹);

OMEGA is a cyclohexyl, cyclohexenyl, morpholino, piperidinyl, naphthyl, phenyl, thienyl, benzothiazolyl, furanyl, oxazolyl, benzoxazolyl or pyridinyl that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, areinc, alkylamino, dialkylamino, alkoxy or carboxyalkyl, having 1-6 carbons, and that is attached as R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, or R¹⁴ by a single bond;

such that at least one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴, is an OMEGA, and, where more than one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is an OMEGA, each OMEGA is optionally the same or different; and

such that when O has the formula Q1, n=O.

2. A compound according to claim 1 wherein X is O or S.

3. A compound according to claim 1 wherein each R¹ is H, and R² is ethyl or methyl.

4. A compound according to claim 1 wherein R⁵ is an OMEGA.

5. A compound according to claim 1 wherein m=1.

6. A compound according to claim 1 wherein R⁵ is an OMEGA, and R⁶ is H;

or R⁶ is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons;

or a halogen;

or R⁶ is --OH, --OR⁸, --SR⁸, (NR⁸ R⁹);

or R⁶ is --OSO₂ R¹⁹ ;

or R⁶ is an OMEGA.

7. A compound according to claim 1, wherein each R¹ is H, R³ and R⁴ are H, R² is ethyl or methyl, R⁵ is an OMEGA, and R⁶ is optionally H;

or R⁶ is an alkyl group having 1-6 carbons;

or R⁶ is a halogen;

or R⁶ iS --OH, --OR⁸, --SR⁸, --(NR⁸ R⁹);

or R⁶ is --OSO₂ R^19;

or R⁶ is an OMEGA.

8. A compound according to claim 7, wherein X=O or S.

9. A compound according to claim 7, wherein n=O or 1.

10. A compound according to claim 7, wherein m=1.

11. A compound according to claim 7, wherein R⁵ is a substituted or unsubstituted cyclohexyl, cyclohexenyl, morpholino, piperidinyl, naphthyl, phenyl, thienyl, benzothiazolyl, furanyl, oxazolyl, benzoxazolyl or pyridinyl.

12. A compound according to claim 7, wherein R⁵ is naphthyl or phenyl.

13. A compound according to claim 7, wherein R⁵ is phenyl.

14. A compound according to claim 1 of the formula ##STR36##

15. A compound according to claim 14, wherein R⁵ is an OMEGA.

16. A compound according to claim 14, wherein each R¹ is H, R² is ethyl or methyl, X=O or S, and n=O or 1.

17. A compound according to claim 14, wherein m=1, of the structure ##STR37## wherein each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or --OR⁸, --SR⁸ or --(NR⁸ R⁹) where R⁸ and R⁹, which can be the same or different, are independently H;

or alkyl groups having 1-6 carbons; and

t=1-4;

R³, R⁴, R⁵, R¹¹, R¹², R¹³, and R¹⁴ are independently H, halogen, an alkyl containing 1-6 carbons, --OH, --OR⁸, --SR⁸, --(NR⁸ R⁹), where R⁸ and R⁹, which may be the same or different, are independently H;

or alkyl groups having 1-6 carbons;

or 1-2 substituted or unsubstituted alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 14 heteroatoms, wherein the heteroatoms are O, N, or S;

or R⁸ and R⁹ taken in combination are --(CH₂)₂ --L--(CH₂)₂ '"'"' where L=O--, --NR₁₀ --, --CH₂ -- or a single bond where R¹⁰ is H or an alkyl group having 1-6 carbons;

or --OSO₂ R¹⁹ where R¹⁹ is alkyl having 1-6 carbons or perfluoroalkyl having 1-6 carbons or aryl;

or an OMEGA;

X is O or S; and

at least one of R⁴ and R⁵ is an OMEGA.

18. A compound according to claim 17, wherein each R¹ is H, and R² is ethyl or methyl.

19. A compound according to claim 17, wherein R⁵ is an OMEGA.

20. A compound according to claim 17 wherein R⁴ is halogen or --OSO₂ R¹⁹.

21. A compound according to claim 17 wherein R⁴ is --OR⁸, --SR⁸, or --(NR⁸ R⁹).

22. A compound according to claim 17 wherein R⁴ is alkyl having 1-6 carbons, or is an OMEGA which is a cycloalkyl having 3-16 carbons.

23. A compound according to claim 17 wherein R⁵ is a substituted or unsubstituted cyclohexyl, cyclohexenyl, morpholino, piperidinyl, naphthyl, phenyl, thienyl, benzothiazolyl, furanyl, oxazolyl, benzoxazolyl or pyridinyl.

24. A compound according to claim 17 wherein R⁵ is naphthyl or phenyl.

25. A compound according to claim 17 wherein R⁵ is phenyl.

26. A compound according to claim 17 wherein R³, R¹¹, R¹², R¹³ and R¹⁴ are H.

27. A fluorescent complex comprising a nucleic acid and one or more dye molecules of the formula ##STR38## wherein:

each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or --OR⁸, --SR⁸ or --(NR⁸ R⁹) where R⁸ and R⁹, which can be the same or different, are independently H;

or alkyl groups having 1-6 carbons;

or 1-2 aft cyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-4 heteroatoms, wherein the hetero atoms are O, N or S;

or R⁸ and R⁹ taken in combination are --(CH₂)₂ --L--(CH₂)₂ -- where L=a single bond, --O--, --CH₂ --, or --NR₁₀ -- where R¹⁰ is H or an alkyl group having 1-6 carbons; and

t=1-4;

R² is an alkyl group having 1-6 carbons;

X is O, S, Se or NR¹⁵, where R¹⁵ is H or an alkyl group having 1-6 carbons;

or X is CR¹⁶ R¹⁷ where R¹⁶ and R¹⁷, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R¹⁶ and R¹⁷ taken in combination complete a five or six membered saturated ring;

n=0, 1 or 2;

Z^- is a biologically compatible counter ion;

Q has the formula Q1 or Q2 ##STR39## wherein Y is --CR³ ═

CR⁴ --;

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

R⁵ is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons;

or R⁵ is an OMEGA;

R³, R⁴, R⁶ and R⁷, which may be the same or different, are independently H;

or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons;

or a halogen;

or --OH, --OR⁸, --SR⁸, --(NR⁸ R⁹);

or --OSO₂ R¹⁹ where R¹⁹ is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl;

or an OMEGA;

or R⁶ and R⁷, taken in combination are --(CH₂)_v -- where v=3 or 4, or R⁶ and R⁷ form a fused aromatic ring according to formula Q2;

R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H;

or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons;

or a halogen;

or an OMEGA;

or --OH, --OR⁸, --SR⁸, or --(NR⁸ R⁹);

OMEGA is a saturated or unsaturated, substituted or unsubstituted, cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the hetero atoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, alkoxy or carboxyalkyl, having 1-6 carbons, and that is attached as R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, or R¹⁴ by a single bond;

such that at least one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is an OMEGA, and, where more than one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is an OMEGA, each OMEGA is optionally the same or different.

28. A complex according to claim 27, wherein the nucleic acid is DNA.

29. A complex according to claim 27, wherein the nucleic acid is DNA.

30. A complex according to claim 27, wherein the nucleic acid is synthetic.

31. A complex according to claim 27, wherein the nucleic acid is biological.

32. A complex according to claim 27, wherein no more than 2 of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is an OMEGA, and each OMEGA is optionally the same or different.

33. A complex according to claim 27, wherein OMEGA is a substituted or unsubstituted cyclohexyl, cyclohexenyl, morpholino, piperidinyl, naphthyl, phenyl, thienyl, benzothiazolyl, furanyl, oxazolyl, benzoxazolyl or pyridinyl.

34. A complex according to claim 27, wherein X is O or S.

35. A complex according to claim 27 wherein each R¹ is H, and R² is ethyl or methyl.

36. A complex according to claim 27 wherein R⁵ is an OMEGA.

37. A complex according to claim 27, wherein each R¹ is H;

R³ and R⁴ are H, R² is ethyl or methyl;

R⁶ is optionally H;

or an alkyl group having 1-6 carbons;

or a halogen;

or --OH, --OR⁸, --SR⁸, --(NR⁸ R⁹);

or or an OMEGA;

X=O or S;

n=0or 1;

m=1; and

R⁵ is an OMEGA which is a substituted or unsubstituted cyclohexyl, cyclohexenyl, morpholino, piperidinyl, naphthyl, phenyl, thienyl, benzothiazolyl, furanyl, oxazolyl, benzoxazolyl or pyridinyl.

38. A complex according to claim 37, wherein R⁵ is phenyl.

39. A complex according to claim 27 of the formula ##STR40##

40. A complex according to claim 39, wherein each R¹ is H, R² is ethyl or methyl, X=O or S, n=0 or 1 and R⁵ is an OMEGA.

41. A complex according to claim 39, wherein m=1, of the structure ##STR41## wherein each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or --OR⁸, --SR⁸ or --(NR⁸ R⁹) where R⁸ and R⁹, which can be the same or different, are independently H;

or alkyl having 1-6 carbons; and

t=1-4;

R³, R⁴, R⁵, R¹¹, R¹², R¹³, and R¹⁴ are independently H, halogen, an alkyl containing 1-6 carbons, --OH, --OR⁸, --SR⁸, --(NR⁸ R⁹), where R⁸ and R⁹, which may be the same or different, are independently H;

or alkyl group having 1-6 carbons;

or 1-2 substituted or unsubstituted alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms, wherein the heteroatoms are O, N, or S;

or R⁸ and R⁹ taken in combination are --(CH₂)₂ --L--(CH₂)₂ --where L=--O--, --NR₁₀ --, --CH₂ -- or a single bond where R¹⁰ is H or an alkyl group having 1-6 carbons;

or --OSO₂ R¹⁹ where R¹⁹ is alkyl having 1-6 carbons or perfluoroalkyl having 1-6 carbons or aryl;

X is O or S; and

at least one of R⁴ and R⁵ is an OMEGA.

42. A complex according to claim 41, wherein each R¹ is H, and R² is ethyl or methyl.

43. A complex according to claim 41 wherein R⁴ is halogen or --OSO₂ R¹⁹.

44. A complex according to claim 41 wherein R⁴ is --OR⁸, --SR⁸, or --(NR⁸ R⁹).

45. A complex according to claim 41 wherein R⁴ is alkyl having 1-6 carbons, or is an OMEGA which is a cycloalkyl having 3-16 carbons.

46. A complex according to claim 41 wherein OMEGA is a substituted or unsubstituted cyclohexyl, cyclohexenyl, morpholino, piperidinyl, naphthyl, phenyl, thienyl, benzothiazolyl, furanyl, oxazolyl, benzoxazolyl or pyridinyl.

47. A complex according to claim 46 wherein R⁵ is phenyl.

48. A complex according to claim 41 wherein R³, R¹¹, R¹², R¹³ and R¹⁴ are H.

49. A method of staining nucleic acids in a sample, comprising

a) combining a sample that contains nucleic acids with a dye compound of the formula:

##STR42## wherein;

each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or --OR⁸, --SR⁸ or --(NR⁸ R⁹) where R⁸ and R⁹, which can be the same or different, are independently H;

alkyl groups having 1-6 carbons;

or 1-2 alicyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-4 heteroatoms, wherein the hetero atoms are O, N or S;

or R⁸ and R⁹ taken in combination are --(CH₂)₂ --L--(CH₂)₂ -- where L=--O--, --NR₁₀ '"'"', --CH₂ -- or a single bond where R¹⁰ is H or an alkyl group having 1-6 carbons; and

t=1-4;

R² is an alkyl group having 1-6 carbons;

X is O, S, Se or NR¹⁵, where R¹⁵ is H or an alkyl group having 1-6 carbons;

or X is CR¹⁶ R¹⁷ where R¹⁶ and R¹⁷, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R¹⁶ and R¹⁷ taken in combination complete a five or six membered saturated ring;

n=0, 1 or2;

Z^- is a biologically compatible counter ion;

Q has the ;

formula Q1 or Q2 ##STR43## wherein Y is --CR³ ═

CR⁴ --;

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

R⁵ is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons;

or R⁵ is an OMEGA;

R³, R⁴, R⁶ and R⁷, which may be the same or different, are independently H;

or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons;

or a halogen;

or --OH, --OR⁸, --SR⁸, --(NR⁸ R⁹);

or --OSO₂ R¹⁹ where R¹⁹ is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons or aryl;

or an OMEGA;

or R⁶ and R⁷, taken in combination are --(CH₂)_v -- where v=3 or 4, or R⁶ and R⁷ form a fused aromatic ring according to formula Q2;

R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H;

or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons;

or a halogen;

or an OMEGA;

or --OH, --OR⁸, --SR⁸, or --(NR⁸ R⁹);

OMEGA is a saturated or unsaturated substituted or unsubstituted, cyclic substituent that has a total of 3-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the hetero atoms are O, N or S, that is unsubstituted or optionally substituted by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, alkoxy or carboxyalkyl, having 1-16 carbons, and that is attached as R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, or R¹⁴ by a single bond;

such that at least one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is an OMEGA, and, where more than one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is an OMEGA, each OMEGA is optionally the same or different.

b) incubating the sample for a time sufficient for the dye compound to combine with the nucleic acid in the sample to form a nucleic acid-dye complex that gives a detectable fluorescent signal;

c) observing the detectable fluorescent signal of the nucleic acid-dye complex.

50. A method according to claim 49, of staining nucleic acids in a sample, wherein the nucleic acids are enclosed in a biological structure, comprising

a) combining a sample that contains nucleic acids, wherein the nucleic acids are enclosed in a biological structure, with a dye compound of the formula:

##STR44## b) incubating the sample for a time sufficient for the dye compound to combine with the nucleic acid in the sample to form a nucleic acid-dye complex that gives a detectable fluorescent signal;

c) observing the detectable fluorescent signal of the nucleic acid-dye complex.

51. A method according to claim 49, of staining nucleic acids in a sample, wherein the nucleic acids are not enclosed in a biological structure, comprising

a) combining a sample that contains nucleic acids, wherein the nucleic acids are not enclosed in a biological structure, with a dye compound of the formula:

##STR45## b) incubating the sample for a time sufficient for the dye compound to combine with the nucleic acid in the sample to form a nucleic acid-dye complex that gives a detectable fluorescent signal;

c) observing the detectable fluorescent signal of the nucleic acid-dye complex.

52. A method according to claim 49, wherein the sample is an electrophoretic gel.

53. A pyridinium or quinolinium salt of the formula:

##STR46## or of the formula;

##STR47## wherein R⁵ is an OMEGA;

B is methyl;

R⁶ and R⁷ are H;

R³, R¹¹, R¹², R¹³, and R¹⁴ are independently H or alkyl having 1-6 carbons

R⁴ is F, Cl, Br, I, or --OSO₂ R¹⁹ where R¹⁹ is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl.

54. A cyclic-substituted unsymmetrical cyanine dye, comprising a first heterocyclic ring system that is a substituted benzothiazolium, benzoxazolium, benzoselenazolium, benzimidazolium, or dialkylindolinium;

That is linked by a monomethine, trimethine, or pentamethine bridging moiety attached at the 2-position of said first ring system to the 2- or 4- position of a second heterocyclic ring system that is a substituted quinolinium or that is linked by a monomethine bridging moiety attached to the 2-position of a second heterocyclic ring system that is a substituted pyridinium;

wherein one or more substituents of said second ring system is an OMEGA, where OMEGA is a substituted or unsubstituted cyclohexyl, cyclohexenyl, morpholino, piperidinyl, naphthyl, phenyl, thienyl, benzothizolyl, furanyl, oxazolyl, benzoxazolyl or pyridinyl.