Chemically reactive unsymmetrical cyanine dyes and their conjugates

• US 5,863,753 A
• Filed: 08/19/1997
• Issued: 01/26/1999
• Est. Priority Date: 10/27/1994
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Millard, Paul J., Yue, Stephen T., Haugland, Richard P., Singer, Victoria L.
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US08/914,439
• Time in Patent Office: 525 Days
• Field of Search: 435/34, 435/4, 435/29, 435/6, 436/94, 436/800, 536/1.11, 536/26.73, 536/25.6
• US Class Current: 435/34
• CPC Class Codes: C07D 215/18 Halogen atoms or nitro radicals
• 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: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1402 Data analysis by thresholding or gating operations performed on the acquired signals or stored data
• 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 comprises cyanine dyes, in particular chemically reactive dyes, conjugates of reactive cyanine dyes, the non-covalent complexes of nucleic acids with the dyes and dye-conjugates of the invention, and a method of forming a nucleic acid complex with the dyes and dye-conjugates of the present invention. The dyes of the invention are useful for the preparation of dye-conjugates. The presence of a reactive group on the unsymmetrical cyanine dyes of the invention facilitates their covalent conjugation to a variety of substances, both biological and synthetic.

Claims

1. A compound of the formula ##STR41## wherein each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

an alkoxy group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or -L-R_x ;

R² is an alkyl group having 1-6 carbons;

or -L-R_x ;

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

n=0, 1 or 2;

ψ

^- is a compatible counterion;

Y is --CR³ ═

CR⁴ --;

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

R⁵ is an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl;

or R⁵ is a TAIL;

or R⁵ is -L-R_x ;

R⁴ is H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or a halogen;

or an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons;

or --OR⁸, --SR⁸, --(NR⁸ R⁹);

or a TAIL;

or -L-R_x ;

where R⁸ and R⁹, which can be the same or different, are independently alkyl groups having 1-6 carbons;

or 1-2 alicyclic or aromatic rings;

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

R³, R⁶ and R⁷ are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or -L-R_x ;

or R⁶ and R⁷ form a fused aromatic ring such that said compound has the formula ##STR42## wherein R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or --OR⁸, --SR⁸, or --(NR⁸ R⁹);

or a TAIL;

or -L-R_x ;

L is a single covalent bond, or L is a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

R_x is a reactive group;

TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP;

wherein

LINK is a single covalent bond, --O--, --S--, or --NR²⁰ --;

where R²⁰ is H, a linear or branched alkyl having 1-8 carbons, or R²⁰ is -SPACER'"'"'-CAP'"'"';

SPACER and SPACER'"'"', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

CAP and CAP'"'"', which may be the same or different, are --O--R²¹, --S--R²¹, --NR²¹ R²², or --N⁺ R²¹ R²² R²³ ψ

^- ;

wherein

R²¹, R²², and R²³ are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons;

or, one or more of R²¹, R²² and R²³, taken in combination with SPACER or SPACER'"'"' or R²⁰ forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S;

where ψ

^- is a compatible counterion;

or

CAP and CAP'"'"' are independently ##STR43## where R²¹, R²², R²³, and ψ

^- are as defined previously;

such that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-R_x ; and

where more than one of R¹ R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-R_x, each -L-R_x is optionally the same or different;

provided that when R⁵ is -L-R_x and m=1, R⁴ is not hydrogen.

2. A compound, as claimed in claim 1, wherein R_x is a reactive group that is a carboxylic acid, an activated ester of a carboxylic acid, an acyl azide, an acyl halide, an acyl nitrile, an aldehyde, an anhydride, an acrylamide, an alkyl halide, a sulfonate, an aniline, an aryl halide, a thiol, an azide, an aziridine, a boronate, a carbodiimide, a diazoalkane, an epoxide, a glycol, a haloacetamide, a halotriazine, a hydrazine, a hydroxylamine, an imido ester, an isocyanate, an isothiocyanate, a ketone, an aldehyde, a maleimide, a phosphoramidite, a silyl halide, or a sulfonyl halide.

3. A compound, as claimed in claim 1, wherein R_x is an activated ester of a carboxylic acid, an aldehyde, an alkyl halide, a haloacetamide, a halotriazine, a hydrazine, an isothiocyanate, a maleimide, a thiol, or an azide.

4. A compound, as claimed in claim 1, wherein R⁴ is not hydrogen.

5. A compound, as claimed in claim 1, wherein m=1.

6. A compound, as claimed in claim 1 wherein R⁶ and R⁷ form a fused aromatic ring;

and at least three of R¹¹, R¹², R¹³ and R¹⁴ are H.

7. A compound, as claimed in claim 1, wherein R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons.

8. A compound, as claimed in claim 1, wherein R⁵ is -L-R_x .

9. A compound, as claimed in claim 8, wherein R⁴ is -L-R_x, and m=1.

10. A compound, as claimed in claim 9, wherein R¹ and R³ are H;

R² is methyl or ethyl;

X is O or S;

R⁵ is an alkyl having 1-6 carbons, or an aryl or heteroaryl;

or R⁵ is a TAIL;

R⁶ and R⁷ form a fused aromatic ring;

at least three of R¹¹, R¹², R¹³, and R¹⁴ are H; and

n=0 or 1.

11. A compound, as claimed in claim 8, wherein R⁴ is an alkyl that is saturated or unsaturated, linear or branched having 1-6 carbons;

or an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons;

or --OR⁸, --SR⁸, --(NR⁸ R⁹);

or R⁴ is a TAIL.

12. A compound, as claimed in claim 1, wherein R¹ and R³ are H;

R² is methyl or ethyl;

X is O or S;

R⁵ is an alkyl having 1-6 carbons, or an aryl or heteroaryl;

or R⁵ is a TAIL;

or R⁵ is -L-R_x ;

R⁶ and R⁷ form a fused aromatic ring;

at least three of R¹¹, R¹², R¹³, and R¹⁴ are H; and

n=0 or 1.

13. A compound, as claimed in claim 12, wherein R⁵ is aryl or heteroaryl.

14. A compound, as claimed in claim 12, wherein one of R⁴ and R⁵ is -L-R_x.

15. A compound of the formula ##STR44## wherein each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

an alkoxy group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or -L-S_c ;

R² is an alkyl group having 1-6 carbons;

or -L-S_c ;

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;

n=0, 1 or 2;

ψ

^- is a compatible counterion;

Y is --CR³ ═

CR⁴ --;

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

R⁵ is an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl;

or R⁵ is a TAIL;

or R⁵ is -L-S_c ;

R⁴ is H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or a halogen;

or an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons;

or --OR⁸, --SR⁸, --(NR⁸ R⁹);

or a TAIL;

or -L-S_c ;

where R⁸ and R⁹, which can be the same or different, are independently alkyl groups having 1-6 carbons;

or 1-2 alicyclic or aromatic rings;

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

R³, R⁶ and R⁷ are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or -L-S_c ;

or R⁶ and R⁷ form a fused aromatic ring such that said compound has the formula ##STR45## wherein R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or --OR⁸, --SR⁸, or --(NR⁸ R⁹);

or a TAIL;

or -L-S_c ;

L is a single covalent bond, or L is a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

S_c is a conjugated substance;

TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP;

wherein

LINK is a single covalent bond, --O--, --S--, or --NR²⁰ --;

where R²⁰ is H, a linear or branched alkyl having 1-8 carbons, or R²⁰ is -SPACER'"'"'-CAP'"'"';

SPACER and SPACER'"'"', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

CAP and CAP'"'"', which may be the same or different, are --O--R²¹, --S--R²¹, --NR²¹ R²², or --N⁺ R²¹ R²² R²³ ψ

^- ;

wherein

R²¹, R²², and R²³ are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons;

or, one or more of R²¹, R²² and R²³, taken in combination with SPACER or SPACER'"'"' or R²⁰ forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S;

where ψ

^- is a compatible counterion;

or

CAP and CAP'"'"' are independently ##STR46## where R²¹, R²², R²³, and ψ

^- are as defined previously;

such that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-S_c ; and

where more than one of R¹ R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-S_c, each -L-S_c is optionally the same or different;

provided that when R⁵ is -L-S_c and m=1, R⁴ is not hydrogen.

16. A compound, as claimed in claim 15, wherein S_c is a member of a specific binding pair.

17. A compound, as claimed in claim 16, wherein S_c is a drug or a hapten.

18. A compound, as in claim 15, wherein S_c is an amino acid, peptide, protein, polysaccharide, a nucleic acid base, nucleoside, nucleotide or a nucleic acid polymer, lipid, lipophilic polymer, non-biological organic polymer, polymeric microparticle, animal cell, plant cell, bacterium, yeast, or virus.

19. A compound, as in claim 18, wherein S_c is an amino acid, peptide, protein, nucleotide, a nucleic acid polymer, a polysaccharide, or a non-biological organic polymer or polymeric microparticle.

20. A compound, as in claim 19, wherein S_c is a protein, or a nucleic acid polymer.

21. A compound, as claimed in claim 15, wherein S_c is an enzyme, or an enzyme substrate.

22. A compound, as claimed in claim 15, wherein neither R¹ nor R² is -L-S_c.

23. A compound, as claimed in claim 15, wherein R⁴ is not hydrogen.

24. A compound, as claimed in claim 15, wherein m=1.

25. A compound, as claimed in claim 15 wherein R⁶ and R⁷ form a fused aromatic ring;

and at least three of R¹¹, R¹², R¹³ and R¹⁴ are H.

26. A compound, as claimed in claim 15 wherein R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons.

27. A compound, as claimed in claim 15, wherein R⁵ is -L-S_c.

28. A compound, as claimed in claim 15, wherein R⁴ is -L-S_c and m=1.

29. A compound, as claimed in claim 15, wherein R¹ and R³ are H;

R² is methyl or ethyl;

X is O or S;

R⁵ is an alkyl having 1-6 carbons, or an aryl or heteroaryl;

or R⁵ is a TAIL;

R⁶ and R⁷ form a fused aromatic ring;

at least three of R¹¹, R¹², R¹³, and R¹⁴ are H; and

n=0 or 1.

30. A compound, as claimed in claim 15, wherein R⁴ is an alkyl that is saturated or unsaturated, linear or branched having 1-6 carbons;

or an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons;

or --OR⁸, --SR⁸, --(NR⁸ R⁹);

or R⁴ is a TAIL.

31. A compound, as claimed in claim 15 wherein R¹ and R³ are H;

R² is methyl or ethyl;

X is O or S;

R⁵ is an alkyl having 1-6 carbons, or an aryl or heteroaryl;

or R⁵ is a TAIL;

R⁶ and R⁷ form a fused aromatic ring;

at least three of R¹¹, R¹², R¹³, and R¹⁴ are H; and

n=0 or 1.

32. A compound, as claimed in claim 31, wherein R⁵ is aryl or heteroaryl.

33. A complex comprising a nucleic acid polymer non-covalently bound to a dye of the formula ##STR47## wherein each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

an alkoxy group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or -L-R_x ;

or -L-S_c ;

R² is an alkyl group having 1-6 carbons;

or -L-R_x ;

or -L-S_c ;

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;

n=0, 1 or 2;

ψ

^- is a compatible counterion;

Y is --CR³ ═

CR⁴ --;

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

R⁵ is an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl;

or R⁵ is a TAIL;

or R⁵ is -L-R_x ;

or R⁵ is -L-S_c ;

R⁴ is H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or a halogen;

or an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons;

or --OR⁸, --SR⁸, --(NR⁸ R⁹);

or a TAIL;

or -L-R_x ;

or -L-S_c ;

where R⁸ and R⁹, which can be the same or different, are independently alkyl groups having 1-6 carbons;

or 1-2 alicyclic or aromatic rings;

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

R³, R⁶ and R⁷ are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or -L-R_x ;

or -L-S_c ;

or R⁶ and R⁷ form a fused aromatic ring such that said compound has the formula ##STR48## wherein R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or --OR⁸, --SR⁸, or --(NR⁸ R⁹);

or a TAIL;

or -L-R_x ;

or -L-S_c ;

each L is a single covalent bond, or L is a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

R_x is a reactive group;

S_c is a conjugated substance;

TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP;

wherein

LINK is a single covalent bond, --O--, --S--, or --NR²⁰ --;

where R²⁰ is H, a linear or branched alkyl having 1-8 carbons, or R²⁰ is -SPACER'"'"'-CAP'"'"';

SPACER and SPACER'"'"', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

CAP and CAP'"'"', which may be the same or different, are --O--R²¹, --S--R²¹, --NR²¹ R²², or --N⁺ R²¹ R²² R²³ ψ

^- ;

wherein

R²¹, R²², and R²³ are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons;

or, one or more of R²¹, R²² and R²³, taken in combination with SPACER or SPACER'"'"' or R²⁰ forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S;

where ψ

^- is a compatible counterion;

or

CAP and CAP'"'"' are independently ##STR49## ;

where R²¹, R²², R²³ , and ψ

^- are as defined previously;

such that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-R_x or -L-S_c ; and

where more than one of R¹ R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-R_x or -L-S_c, each -L-R_x and -L-S_c is optionally the same or different;

provided that when R⁵ is -L-R_x or -L-S_c and m=1, R⁴ is not hydrogen.

34. A complex, as claimed in claim 33, wherein one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-S_c, and S_c is a member of a specific binding pair.

35. A complex, as claimed in claim 34, wherein the specific binding pair member is a drug, a hapten, an enzyme, an enzyme substrate, an oligonucleotide, or a peptide.

36. A complex, as claimed in claim 33, wherein S_c is an organic polymer.

37. A complex, as claimed in claim 36, wherein S_c is a polymeric microparticle, or a polymeric membrane.

38. A complex, as claimed in claim 36, wherein S_c is poly(ethylene glycol) or a polysaccharide.

39. A complex, as claimed in claim 33, wherein R⁴ is not hydrogen, m=1, R⁶ and R⁷ form a fused aromatic ring;

and at least three of R¹¹, R¹², R¹³ and R¹⁴ are H.

40. A compound, as claimed in claim 39, wherein R⁴ is an alkyl that is saturated or unsaturated, linear or branched having 1-6 carbons;

or an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons;

or --OR⁸, --SR⁸, --(NR⁸ R⁹);

or R⁴ is a TAIL.

41. A complex, as claimed in claim 33, wherein R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons and R⁴ is -L-R_x or -L-S_c.

42. A complex, as claimed in claim 33, wherein R⁵ is -L-R_x or -L-S_c.

43. A complex, as claimed in claim 33, wherein R⁴ is -L-R_x or -L-S_c.

44. A method of forming a nucleic acid complex, comprising

a) combining a sample that contains or is thought to contain a nucleic acid, with a mixture containing a compound of the formula ##STR50## wherein each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

an alkoxy group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or -L-R_x ;

or -L-S_c ;

R² is an alkyl group having 1-6 carbons;

or -L-R_x ;

or -L-S_c ;

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;

n=0, 1 or 2;

ψ

^- is a compatible counterion;

Y is --CR³ ═

CR⁴ --;

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

R⁵ is an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl;

or R⁵ is a TAIL;

or R⁵ is -L-R_x ;

or R⁵ is -L-S_c ;

R⁴ is H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or a halogen;

or an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons;

or --OR⁸, --SR⁸, --(NR⁸ R⁹);

or a TAIL;

or -L-R_x ;

or -L-S_c ;

where R⁸ and R⁹, which can be the same or different, are independently alkyl groups having 1-6 carbons;

or 1-2 alicyclic or aromatic rings;

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

R³, R⁶ and R⁷ are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or -L-R_x ;

or -L-S_c ;

or R⁶ and R⁷ form a fused aromatic ring such that said compound has the formula ##STR51## wherein R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or --OR⁸, --SR⁸, or --(NR⁸ R⁹);

or a TAIL;

or -L-R_x ;

or -L-S_c ;

each L is a single covalent bond, or L is a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

R_x is a reactive group;

S_c is a conjugated substance;

TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP;

wherein

LINK is a single covalent bond, --O--, --S--, or --NR²⁰ --;

where R²⁰ is H, a linear or branched alkyl having 1-8 carbons, or R²⁰ is -SPACER'"'"'-CAP'"'"';

SPACER and SPACER'"'"', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

CAP and CAP'"'"', which may be the same or different, are --O--R²¹, --S--R²¹, --NR²¹ R²², or --N⁺ R²¹ R²² R²³ ψ

^- ;

wherein

R²¹, R²², and R²³ are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons;

or, one or more of R²¹, R²² and R²³, taken in combination with SPACER or SPACER'"'"' or R²⁰ forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S;

where ψ

^- is a compatible counterion;

or

CAP and CAP'"'"' are independently ##STR52## ;

where R²¹, R²², R²³, and ψ

^- are as defined previously;

such that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-R_x or -L-S_c ; and

where more than one of R¹ R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-R_x or -L-S_c, each -L-R_x and -L-S_c is optionally the same or different;

provided that when R⁵ is -L-R_x or -L-S_c and m=1, R⁴ is not hydrogen; and

b) incubating the sample and mixture for a time sufficient for the compound to associate non-covalently with the nucleic acid in the sample to form one or more nucleic acid complexes.

45. A method, as claimed in claim 44, wherein said nucleic acid complex exhibits a detectable optical response.

46. A method, as claimed in claim 45, wherein the detectable optical response is a fluorescence response.

47. A method, as claimed in claim 45, wherein one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is -L-S_c.

48. A method, as claimed in claim 47, further comprising separating the nucleic acid complex from the sample.

49. A method, as claimed in claim 47, wherein S_c is an organic polymer.

50. A method, as claimed in claim 49, wherein S_c is poly(ethylene glycol) or a polysaccharide.

51. A method, as claimed in claim 47, wherein S_c is a polymeric microparticle, or a polymeric membrane.

52. A method, as claimed in claim 51, wherein the polymeric microparticle is a magnetic microparticle and, further comprising separating the nucleic acid complex from the sample by removing the magnetic microparticle from the sample.

53. A method, as claimed in claim 47, wherein S_c is a first member of a specific binding pair having a first and second member.

54. A method, as claimed in claim 53, wherein the formation of said complex indicates the location of the second member of the specific binding pair in the sample.

55. A method, as claimed in claim 53, wherein the first member is a drug, a hapten, an enzyme substrate, a nucleic acid polymer, a peptide, or a protein.

56. A method, as claimed in claim 53, wherein the first member is a biotin, a digoxigenin, an avidin, a streptavidin, or an antibody.

57. A method, as claimed in claim 54, wherein the sample comprises one or more cells, bacteria, virus, yeast cells, or a polymeric membrane or polymeric microparticles.

58. A method, as claimed in claim 44, wherein R⁴ is not hydrogen, m=1, R⁶ and R⁷ form a fused aromatic ring;

and at least three of R¹¹, R¹², R¹³ and R¹⁴ are H.

59. A method, as claimed in claim 44, wherein R⁴ is an alkyl that is saturated or unsaturated, linear or branched having 1-6 carbons;

or an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons;

or --OR⁸, --SR⁸, --(NR⁸ R⁹);

or R⁴ is a TAIL.

60. A method, as claimed in claim 44, wherein R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl having 3-10 carbons and R⁴ is -L-R_x or -L-S_c.

61. A method, as claimed in claim 44, wherein R⁵ is -L-R_x or -L-S_c.

62. A method, as claimed in claim 44, wherein R⁴ is -L-R_x or -L-S_c.

63. A compound of the formula ##STR53## wherein X is O or S;

R² is an alkyl group having 1-6 carbons;

n=0, 1 or2;

ψ

^- is a compatible counterion;

R⁴ is an aryl or heteroaryl that is optionally substituted by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, alkoxy or carboxyalkyl, each alkyl group having 1-6 carbons;

or is substituted by TAIL;

or is substituted by -L-R_x ;

R⁵ is an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

an aryl or heteroaryl;

or R⁵ is a TAIL;

or R⁵ is -L-R_x ;

R³, R⁶ and R⁷ are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or R⁶ and R⁷ form a fused aromatic ring such that said compound has the formula ##STR54## wherein R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or an alkoxy having 1-6 carbons;

or --OR⁸, --SR⁸, or --(NR⁸ R⁹) where R⁸ and R⁹, which can be the same or different, are independently alkyl groups having 1-6 carbons;

or 1-2 alicyclic or aromatic rings;

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

or a TAIL;

L is a single covalent bond, or L is a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

R_x is a reactive group;

TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP;

wherein

LINK is a single covalent bond, --O--, --S--, or --NR²⁰ --;

where R²⁰ is H, a linear or branched alkyl having 1-8 carbons, or R²⁰ is -SPACER'"'"'-CAP'"'"';

SPACER and SPACER'"'"', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

CAP and CAP'"'"', which may be the same or different, are --O--R²¹, --S--R²¹, --NR²¹ R²², or --N⁺ R²¹ R²² R²³ ψ

^- ;

wherein

R²¹, R²², and R²³ are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons;

or, one or more of R²¹, R²² and R²³, taken in combination with SPACER or SPACER'"'"' or R²⁰ forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N, S;

where ψ

^- is a compatible counterion;

or

CAP and CAP'"'"' are independently ##STR55## where R²¹, R²², R²³, and ψ

^- are as defined previously.

64. A compound, as claimed in claim 63, wherein R⁴ is a naphthyl, a phenyl, a thienyl, a benzothiazolyl, a furanyl, an oxazolyl, a benzoxazolyl, or a pyridinyl that is optionally substituted by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, alkoxy or carboxyalkyl, each alkyl group having 1-6 carbons;

or by TAIL;

or -L-R_x.

65. A compound, as claimed in claim 64, wherein R⁴ is substituted by TAIL or -L-R_x.

66. A compound, as claimed in claim 65, wherein R⁴ is substituted by TAIL having the formula LINK-SPACER-CAP;

wherein LINK is a single covalent bond, --O--, --S--, or --NR²⁰ --;

where R²⁰ is H, a linear or branched alkyl having 1-8 carbons, or R²⁰ is -SPACER'"'"'-CAP'"'"';

SPACER and SPACER'"'"' independently have the formula --(CH₂)_k --, where k=1-8,

CAP and CAP'"'"', which may be the same or different, are --NR²¹ R²² or --N⁺ R²¹ R²² R²³ ψ

^- ;

where R²¹, R²², and R²³ are independently methyl or ethyl.

67. A compound, as claimed in claim 64, wherein R⁵ is an alkyl having 1-6 carbons.

68. A compound of the formula ##STR56## or of the formula ##STR57## wherein each R¹ is independently H;

or an alkyl group having from 1-6 carbons;

an alkoxy group having from 1-6 carbons;

or a trifluoromethyl;

or a halogen;

or -L-R_x ;

R² is an alkyl group having 1-6 carbons;

or -L-R_x ;

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;

n=0, 1 or 2;

R⁵ is an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or R⁵ is a cyclic substituent that is an aryl or heteroaryl;

or a cycloalkyl;

or R⁵ is a TAIL;

or R⁵ is -L-R_x ;

R⁴ is NR¹⁹ ;

where R¹⁹ is an alkyl group having 1-6 carbons;

or an alicyclic or aromatic ring;

or R¹⁹ is -SPACER-CAP;

or R¹⁹ is -L-R_x ;

R³, R⁶ and R⁷ are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or -L-R_x ;

or R⁶ and R⁷ form a fused aromatic ring such that said compound has the formula ##STR58## or the formula ##STR59## wherein R¹¹, R¹², R¹³, and R¹⁴, which may be the same or different, are independently H;

or an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons;

or --OR⁸, --SR⁸, or --(NR⁸ R⁹), where R⁸ and R⁹, which can be the same or different, are independently alkyl groups having 1-6 carbons;

or 1-2 alicyclic or aromatic rings;

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

or a TAIL;

or -L-R_x ;

L is a single covalent bond, or L is a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

R_x is a reactive group;

TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP;

wherein

LINK is a single covalent bond, --O--, --S--, or --NR²⁰ --;

where R²⁰ is H, a linear or branched alkyl having 1-8 carbons, or R²⁰ is -SPACER'"'"'-CAP'"'"';

SPACER and SPACER'"'"', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds;

or single, double, triple or aromatic carbon--carbon bonds;

or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen--nitrogen or nitrogen-oxygen bonds;

or aromatic or heteroaromatic bonds;

CAP and CAP'"'"', which may be the same or different, are --O--R²¹, --S--R²¹, --NR²¹ R²², or --N⁺ R²¹ R²² R²³ ψ

^- ;

wherein

R²¹, R²², and R²³ are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons;

or, one or more of R²¹, R²² and R²³, taken in combination with SPACER or SPACER'"'"' or R²⁰ forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S;

where ψ

^- is a compatible counterion;

or

CAP and CAP'"'"' are independently ##STR60## where R²¹, R²², R²³, and ψ

^- are as defined previously.

69. A compound, as claimed in claim 68, wherein R⁵ is aryl or heteroaryl;

X is O or S;

n is 0 or 1; and

R⁶ and R⁷ are H;

or taken in combination, form a fused aromatic ring.

70. A compound, as claimed in claim 69, wherein R⁵ is phenyl;

and R¹⁹ is -SPACER-CAP;

wherein SPACER and has the formula --(CH₂)_k --, where k=1-8;

CAP is --NR²¹ R²² or --N⁺ R²¹ R²² R²³ ψ

^- ;

where R²¹, R²², and R²³ are independently methyl or ethyl, or CAP is ##STR61##