Aza-benzazolium containing cyanine dyes

• US 6,664,047 B1
• Filed: 04/24/2000
• Issued: 12/16/2003
• Est. Priority Date: 04/30/1999
• 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): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/557,275
• Time in Patent Office: 1,331 Days
• Field of Search: 435/34, 435/4, 435/29, 435/6, 435/968, 435/94, 436/800, 546/165, 546/176, 546/270, 546/271
• 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: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2001/302 Stain compositions
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• 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
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Unsymmetrical cyanine dyes that incorporate an aza-benzazolium ring moiety are described, including cyanine dyes substituted by a cationic side chain, monomeric and dimeric cyanine dyes, chemically reactive cyanine dyes, and conjugates of cyanine dyes. The subject dyes are virtually non-fluorescent when diluted in aqueous solution, but exhibit bright fluorescence when associated with nucleic acid polymers such as DNA or RNA, or when associated with detergent-complexed proteins. A variety of applications are described for detection and quantitation of nucleic acids and detergent-complexed proteins in a variety of samples, including solutions, electrophoretic gels, cells, and microorganisms.

Claims

1. A compound having formula

wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —

C—

, CH or N(R²)^β

, provided at least one of said ring atoms is N(R²)^β

, wherein aromatic carbon atoms are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —

L—

R_x and —

L—

S_c;

X is selected from the group consisting of O and S,

α

is 0 or 1 and β

is 0 or 1 provided that α

+all β

=1;

R² is selected from the group consisting of —

L—

R_x, —

L—

S_c, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, or amino;

n=0, 1 or 2;

Y is —

CR³═

CR⁴—

;

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

R³, R⁴, R⁸, and R⁷ are independently selected from the group consisting of hydrogen, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL, BRIDGE, —

L—

R_x and —

L—

S_c wherein R⁸ and R⁹ 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—

W—

(CH₂)₂—

where W is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 H carbons;

or R⁶ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, halogen, —

OR⁸, —

SR⁸, —

(NR⁸R⁹) a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R³⁰, R³¹, and R³² are independently selected from the group consisting of hydrogen, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl; and

,

when present, BRIDGE is attached to a DYE compound provided that no more than one of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ is BRIDGE;

wherein

L and BRIDGE are independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;

R_x is a reactive group;

S_c is a conjugated substance;

TAIL is a heteroatom-containing moiety,

DYE is a compound having formula

wherein A, X, R², α

, n, Y_m, Y_p, R⁵, R⁶, R⁷, R³⁰, R³¹ and R³² are as defined above provided that BRIDGE not be any of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹², R¹³, R¹⁴ and R¹⁵.

2. The compound according to claim 1, having the formula selected from the group consisting of

3. The compound according to claim 1, wherein said TAIL comprises formula LINK-SPACER-CAP;

wherein

LINK is a single covalent bond, —

O—

, —

S—

, or —

NR²⁰—

;

where RF²⁰ is hydrogen, a linear or branched alkyl having 1-8 carbons, or -SPACER′

-CAP′

;

SPACER and SPACER′

are individually covalent linkages that are 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;

CAP and CAP′

are individually —

O—

R²¹, —

S—

R²¹, —

NR²¹R²², or —

N⁺R⁺R²¹R²³Ψ

⁻

;

wherein

R²¹, R²¹, and R²³ are independently hydrogen or a linear or branched alkyl having 1-6 carbons or cycloalkyl having 3-8 carbons, wherein said alkyl is optionally further substituted by substituents selected from the group consisting of halogen, hydroxy, alkoxy having 1-8 carbons, amino, carboxy, sulfo and phenyl, wherein said phenyl is optionally further substituted by substituents selected from the group consisting of halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, sulfoalkyl and carboxyalkyl having 1-8 carbons;

or one or more of R²¹, R²² and R²³, taken in combination with SPACER and R²⁰ or SPACER alone 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

4. The compound according to claim 2, wherein each R² is independently ethyl or methyl, each n is independently 0 or 1, and R³⁰, R³¹, and R³² are each hydrogen.

5. The compound according to claim 2, wherein at least one R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ is a CYCLIC SUBSTITUENT selected from the group consisting of aryl, heteroaryl, and cycloalkyl having 3-10 carbons wherein said CYCLIC SUBSTITUENT is individually and optionally substituted by TAIL, halogen, amino, or an alkyl containing moiety comprising 1-6 carbons.

6. The compound according to claim 3, wherein said TAIL comprises LINK that is a single covalent bond or NR²⁰ wherein R²⁰ is hydrogen or an alkyl;

SAPCER and SPACER′

that are independently a linear alkyl having 1-8 carbons or a 6-membered carbon ring;

CAP and CAP′

that are individually —

NR²¹R²², or —

N⁺R²¹R²²R²³ Ψ

⁻

, wherein R²¹, R²², and R²³ are independently hydrogen, alkyl or cycloalkyl.

7. The compound according to claim 6, wherein R⁴ is a TAIL or BRIDGE.

8. The compound according to claim 6, wherein R⁵ is a TAIL, a CYCLIC SUBSTITUENT, or BRIDGE.

9. The compound according to claim 8, wherein R⁵ is a TAIL or a BRIDGE wherein TAIL and BRIDGE incorporate at least one quaternary nitrogen atom.

10. The compound according to claim 2, wherein each R³, R¹¹, R¹², R¹³ and R¹⁴ is hydrogen.

11. The compound according to claim 3, wherein

R⁵ is an alkyl and

R⁴ is selected from the group consisting of halogen, CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL, BRIDGE, —

L—

R_x, and —

L—

S_c.

12. The compound according to claim 1, wherein S_c is selected from the group consisting of an amino acid, a peptide, a protein, a polysaccharide, a nucleotide, an oligonucleotide, a nucleic acid, a lipid, a polymeric microparticle, a biological cell, a DNA-binding protein and a virus.

13. The compound according to claim 12, wherein S_c is an oligonucleotide, a nucleic acid, or a DNA-binding protein.

14. The compound according to claim 6, wherein

each X is O;

each n is independently=0, 1, or 2;

each m=1;

each R³⁰, R³¹, and R³² are H; and

R⁵ is selected from the group consisting of an alkyl, a TAIL, a CYCLIC SUBSTITUENT, and a BRIDGE.

15. A complex comprising:

a) a compound having formula

wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —

C—

, CH or N(R²)^β

, provided at least one of said ring atoms is N(R²)^β

wherein aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —

L—

R_x and —

L—

S_c;

X is selected from the group consisting of O and;

α

is 0 or 1 and β

is 0 or 1 provided that α

+all β

=1;

R² is selected from the group consisting of —

L—

R_x, —

L—

S_c, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, or amino;

n=0, 1 or 2;

Y is —

CR³═

CR⁴—

;

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

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL, BRIDGE, —

L—

R_x and —

L—

S_c wherein R⁸ and R⁹ 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—

W—

(CH₂)₂—

where W is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

or R⁶ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ling substituents R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, halogen, —

OR⁸, —

SR⁸, —

(NR⁸R⁹) a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c, and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R³⁰, R³¹, and R³² are independently selected from the group consisting of hydrogen alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl; and

,

when present, BRIDGE is attached to a DYE compound provided that no more than one of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ is BRIDGE;

wherein

L and BRIDGE are independently A single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;

R_x is a reactive group;

S_c is a conjugated substance;

TAIL is a heteroatom-containing moiety;

DYE is a compound having formula

wherein A, X, R², α

, n, Y_m, Y_p, R⁵, R⁶, R⁷, R³⁰, R³¹ and R³² are as defined above provided that BRIDGE not be any of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵; and

b) a nucleic acid polymer.

16. The complex according to claim 15, wherein said nucleic acid polymer is a chromosome or fragment thereof, or a natural or synthetic oligonucleotide.

17. The complex according to claim 16, wherein said nucleic acid is enclosed in a biological structure, free in solution, immobilized on a solid or semi-solid material or is extracted from a biological structure.

18. The complex according to claim 17, wherein said complex is enclosed in a biological structure present in a solution or on an inert matrix.

19. The complex according to claim 17, wherein said biological structure is a cell and said cell is undergoing apoptosis, necrosis, or is in a cycle of cell division.

20. The complex according to claim 18 or 19, wherein said complex is enclosed in a biological structure.

21. The complex according to claim 15, wherein at least one compound is substituted by —

L—

S_c wherein S_c is selected from the group consisting of hapten, nucleotide, oligonucleotide, nucleic acid polymer, protein, polysaccharide and DNA binding protein.

22. A complex comprising:

a) a compound having formula;

wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —

C—

, CH or N(R²)^β

, provided at least one of said ring atoms is N(R²)^β

wherein aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —

L—

R_x and —

L—

S_c;

X is selected from the group consisting of O and S;

α

is 0 or 1 and β

is 0 or 1 provided that α

+all β

=1;

R² is selected from the group consisting of —

L—

R_x, —

L—

S_c, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, or amino;

n=0, 1 or 2;

Y is —

CR³═

CR⁴—

;

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

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL, BRIDGE, —

L—

R_x and —

L—

S_c wherein R⁸ and R⁹ 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—

W—

(CH₂)₂—

where W is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

or R⁶ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, halogen, —

OR⁸, —

SR⁸, —

(NR⁸R⁹) a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R³⁰, R³¹, and R³² are independently selected from the group consisting of hydrogen, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl; and

,

when present, BRIDGE is attached to a DYE compound provided that no more than one of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ is BRIDGE;

wherein

L and BRIDGE are independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;

R_x is a reactive group;

S_c is a conjugated substance;

TAIL is a heteroatom-containing moiety;

DYE is a compound having formula

wherein A, X, R², α

, n, Y_m, Y_p, R⁵, R⁶, R⁷, R³⁰, R³¹ and R³² are as defined above provided that BRIDGE not be any of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵; and

,

b) a poly(amino acid).

23. The complex according to claim 22, wherein said complex further comprises a detergent.

24. The complex according to claim 23, where sad detergent is an alkyl sulfate or alkyl sulfonate salt.

25. The complex according to claim 24, wherein said poly(amino acids) are present on or in a solid or semi-solid matrix.

26. The complex according to claim 25, wherein said matrix is a membrane or an electrophoretic gel.

27. A kit comprising:

a) a stock solution comprising;

i) one or more compounds individually having formula;

wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —

C—

, CH or N(R²)^β

, provided at least one of said ring atoms is N(R²)^β

wherein aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —

L—

R_x and —

L—

S_c;

X is selected from the group consisting of O and S;

α

is 0 or 1 and β

is 0 or 1 provided that α

+all β

=1;

R² is selected from the group consisting of —

L—

R_x, —

L—

S_c, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, or amino;

n=0, 1 or 2;

Y is —

CR³═

CR⁴—

;

p and m=0 or 1, such that p+m1;

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL, BRIDGE, —

L—

R_x and —

L—

S_c wherein R⁸ and R⁹ 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—

W—

(CH₂)₂—

where W is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

or R⁶ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, halogen, —

OR⁸, —

SR⁸, —

(NR⁸R⁹) a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R³⁰, R³¹, and R³² are independently selected from the group consisting of H, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl; and

,

when present, BRIDGE is attached to a DYE compound provided that no more than one of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ is BRIDGE;

wherein

L and BRIDGE are independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;

R_x is a reactive group;

S_c is a conjugated substance;

TAIL is a heteroatom-containing moiety;

DYE is a compound having formula

wherein A, X, R², α

, n, Y_m, Y_p, R⁵, R⁶, R⁷, R³⁰, R³¹ and R³² are as defined above provided that BRIDGE not be any of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵;

ii) an organic solvent; and

,

b) a buffer suitable for dilution of said stock solution.

28. The kit according to claim 27, further comprising a standard, an additional detection reagent, a silicon chip, a glass slide, or any combination thereof.

29. The kit according to claim 28, wherein said additional detection reagent is selected from the group consisting of an organelle stain, a labeled immunoreagent, a drug, and an enzyme.

30. The kit according to claim 29, wherein said R⁵ is BRIDGE that is attached to said DYE.

31. The kit according to claim 29, wherein said kit comprises two to six individual said compounds.

32. The kit according to any one of claims claim 29, 30 or 31, wherein said organic solvent is DMSO.

33. A compound having formula:

wherein R² is an alkyl having 1-6 carbon atoms and wherein meta and ortho positions to R² are independently and optionally substituted with halogen or an alkyl having 1-6 carbons atoms;

X is selected from the group consisting of O and S;

n=0, 1 or 2;

α

is 0 or 1 and β

is 0 or 1 provided that α

+all β

=1;

Y is —

CR³═

CR⁴—

;

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

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, an alkyl having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NFR⁸R⁹), TAIL and BRIDGE wherein R⁸ and R⁹ 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—

W—

(CH₂)₂—

where W is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

or R⁶ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, halogen, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), CYCLIC SUBSTITUENT, TAIL, BRIDGE,

and an alkyl having 1-6 carbons;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL BRIDGE, a pair of electrons, sulfoalkyl and an alkyl having 1-6 carbons;

R³⁰, R³¹, and R³² are independently selected from the group consisting of H, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl; and

,

when present, BRIDGE is attached to a DYE compound provided that no more than one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³ and R¹⁴ is BRIDGE;

wherein;

BRIDGE is independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;

TAIL is a heteroatom-containing moiety;

DYE is a compound of the formula

wherein X, R², α

, β

, n, Y_m, Y_p, R⁵, R⁶, R⁷, R³⁰, R³¹, and R³² are as defined above provided that BRIDGE not be any of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³ and R¹⁴.

34. The compound according to claim 33, wherein

R² is methyl; said meta and ortho positions are optionally substituted by halogen;

n=0;

m=1;

R³ and R⁴ are independently selected from the group consisting of H, alkyl, CYCLIC SUBSTITUENT and TAIL;

R⁵ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently hydrogen, —

OR⁸, or an alkyl having 1-6 carbons wherein R⁸ is methyl;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, a pair of electrons, and methyl;

R³⁰ is hydrogen.

35. The compound according to claim 34, wherein X is S, said halogen is chlorine or bromine, R³ is hydrogen, R⁴ is hydrogen or an alkyl and R⁵ is selected from the group consisting of methyl, CYCLIC SUBSTITUENT, BRIDGE and TAIL wherein said CYCLIC SUBSTITUENT is an unsubstituted aryl and TAIL comprises formula —

(CH₂)₃N(CH₃)₃.

36. The compound according to claim 35 or 34, wherein said R⁵ is BRIDGE comprising formula —

(CH₂)₃N(CH₃)CH₂—

.

37. The compound according to any one of claims 36, 35 or 34, wherein said BRIDGE is attached to said DYE.

38. The compound according to claim 35, wherein said compound has the formula:

39. The compound according to claim 35, wherein said compound has the formula:

40. The compound according to claim 35, wherein said compound has the formula:

41. The compound according to claim 34, wherein X is O;

R³ is hydrogen;

R⁴ is selected from the group consisting of hydrogen, CYCLIC SUBSTITUENT, TAIL and an alkyl;

R⁵ is selected from the group consisting of methyl, CYCLIC SUBSTITUENT, BRIDGE and TAIL wherein said CYCLIC SUBSTITUENT is an unsubstituted aryl and said TAIL comprises LINK that is a single covalent bond, SPACER that is a phenyl ring and CAP comprising formula —

(CH)₂N⁺CH₃(CH₂CH₃)₂;

R¹¹, R¹², R¹³, and R¹⁴ are individually hydrogen or —

OCH₃.

42. The compound according to claim 41, wherein said compound has the formula:

43. The compound according to claim 41, wherein said compound has the formula:

44. The compound according to claim 41, wherein said compound has the formula:

45. The compound according to claim 41, wherein said compound has the formula:

46. The compound according to claim 41, wherein said compound has the formula:

47. A compound having formula:

wherein R² is an alkyl having 1-6 carbon atoms and fused 6-membered aromatic ring is optionally substituted at a ring carbon by methylthio;

X is selected from the group consisting of O and S;

n=0, 1 or 2;

α

is 0 or 1 and β

is 0 or 1 provided that α

+all β

=1;

Y is —

CR³═

CR⁴—

;

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

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, an alkyl having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL and BRIDGE wherein R⁸ and R⁹ 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—

W—

(CH₂)₂—

where W is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

or R⁶ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, halogen, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), CYCLIC SUBSTITUENT, TAIL, BRIDGE, and an alkyl having 1-6 carbons;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, a pair of electrons, sulfoalkyl and an alkyl having 1-6 carbons;

R³⁰, R³¹, and R³² are independently selected from the group consisting of H, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl; and

,

when present, BRIDGE is attached to a DYE compound provided that no more than one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³ and R¹⁴ is BRIDGE;

wherein;

BRIDGE is independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;

TAIL is a heteroatom-containing moiety;

DYE is a compound having formula

wherein X, R², α

, β

, n, Y_m, Y_p, R⁵, R⁶, R⁷, R³⁰, R³¹ and R³² are as defined above provided that BRIDGE not be any of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³ and R¹⁴.

48. The compound according to claim 47, wherein said 6-membered aromatic ring is substituted by methylthio;

X is S;

n is 0;

m is 1;

R₃, R⁴, R¹¹, R¹²;

R¹³ and R¹⁴ are hydrogen and R⁵ is methyl.

49. The compound according to claim 48, wherein said compound has the formula:

50. A solution for staining nucleic acid polymers or poly(amino acids) wherein said solution comprises:

a) one or more compounds having formula

wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —

C—

, CH or N(R²)^β

, provided at least one of said ring atoms is N(R²)^β

wherein aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —

L—

R_x and —

L—

S_c;

X is selected from the group consisting of O and S;

α

is 0 or 1 and β

is 0 or 1 provided that α

+all β

=1;

R² is selected from the group consisting of —

L—

R_x, —

L—

S_c, TAIL BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, or amino;

n=0,1 or 2;

Y is —

CR³═

CR⁴—

;

p and m=0 or 1, such that p+m1;

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —

OR⁵, —

SR⁸, —

(NR⁸R⁹), TAIL, BRIDGE, —

L—

R_x and —

L—

S_c wherein R⁸ and R⁹ 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—

W—

(CH₂)₂—

where W is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

or R⁶ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, halogen, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —

L—

R_x, —

L—

S_c, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;

R³⁰, R³¹, and R³² are independently selected from the group consisting of hydrogen, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, awl, and heteroaryl; and

,

when present BRIDGE is attached to a DYE compound provided that no more than one of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ is BRIDGE;

wherein

L and BRIDGE are independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;

R_x is a reactive group;

S_c is a conjugated substance;

TAIL is a heteroatom-containing moiety;

DYE is a compound having formula

wherein A, X, R⁷, α

, n, Y_m, Y_p, R⁵, R⁶, R⁷, R³⁰, R³¹ and R³² are as defined above provided that BRIDGE not be any of R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵; and

;

b) an organic solvent.

51. The solution according to claim 50, wherein said compound has the formula

or

52. The solution according to claim 51, wherein R² is methyl;

said 6-membered aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, halogen, methylthio;

n=0;

m=1;

R³ and R⁴ are independently selected from the group consisting of hydrogen, alkyl, CYCLIC SUBSTITUENT and TAIL;

R⁶ and R⁷ taken in combination form a fused 6-membered aromatic ring wherein ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently hydrogen, —

OR⁸, or an alkyl having 1-6 carbons wherein R⁸ is methyl;

R⁵ is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, a pair of electrons, and methyl;

R³⁰ is hydrogen.

53. The solution according to claim 52, wherein R³ is hydrogen, said CYCLIC SUBSTITUENT is an unsubstituted aryl and TAIL comprises LINK that is a single covalent bond, SPACER that is a phenyl ring or a linear alkyl and CAP comprising formula —

(CH)₂N⁺CH₃(CH₂CH₃)₂ or formula —

N(CH₃)₃.

54. The solution according to claim 53, wherein R⁵ is BRIDGE comprising formula —

(CH₂)₃N(CH₃)CH₂—

wherein DYE is attached to said BRIDGE.

55. The solution according to claim 53 or 54 wherein said organic solvent is DMSO.