Modified carbocyanine dyes and their conjugates

• US 20020064794A1
• Filed: 10/01/2001
• Published: 05/30/2002
• Est. Priority Date: 09/29/2000
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Leung, Wai-Yee, Cheung, Ching-Ying, Yue, Stephen
• Granted Patent: US 6,974,873 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 41/0057 Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
• CPC Class Codes: C07C 66/02 Anthraquinone carboxylic acids
• CPC Class Codes: C07D 209/08 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 209/16 Tryptamines
• CPC Class Codes: C07D 209/18 Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 209/24 with an alkyl or cycloalkyl radical attached to the ring nitrogen atom
• CPC Class Codes: C07D 209/54 Spiro-condensed
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 209/96 Spiro-condensed ring systems
• CPC Class Codes: C07D 401/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 403/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 403/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 403/14 containing three or more hetero rings
• 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: C07D 471/04 Ortho-condensed systems
• CPC Class Codes: C07D 498/04 Ortho-condensed systems
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C09B 23/02 : the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
• CPC Class Codes: C09B 23/06 : three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 : five >CH- groups
• CPC Class Codes: C09B 23/107 : four >CH- groups
• CPC Class Codes: C12Q 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C40B 70/00 : Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• 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 31/22 : using chemical indicators G01N31/02 takes precedence
• CPC Class Codes: G01N 33/533 : with fluorescent label
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/581 : with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 : with fluorescent label
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

Chemically reactive carbocyanine dyes incorporating an indolium ring moiety that is substituted at the 3-position by a reactive group or by a conjugated substance, and their uses, are described. Conjugation through this position results in spectral properties that are uniformly superior to those of conjugates of spectrally similar dyes wherein attachment is at a different position. The invention includes derivative compounds having one or more benzo nitrogens.

Claims

1. A compound of the formula:

2. A compound, as claimed in claim 1, wherein

the atoms of Y are selected from —

CH, —

C, and —

CR¹;

α

is 1;

the atoms of W are selected from —

CH, —

C, —

CR^1′

, and —

N(R¹²)_β

′

, where each β

′

is 0 or 1;

each R² and R¹² is independently —

L—

R_x;

or —

L—

S_c;

or a C₁-C₂₂ alkyl, which optionally incorporates up to six hetero atoms, and is optionally substituted;

δ

is 0 or 1, and δ

+all β

′

=1; and

δ

is 1 only if W contains no ring nitrogen atoms;

Z is O, S, or CR¹³R¹⁴.

3. A compound, as claimed in claim 2, wherein

the atoms of W are selected from —

CH, —

C, —

CR^1′

, and —

N(R¹²)_β

′

, where β

′

is 1, but no more than one of the atoms in W is —

N(R¹²)_β

′

;

δ

is 0; and

Z is CR¹³R¹⁴.

4. A compound, as claimed in claim 2, wherein

the atoms of W are selected from —

CH, —

C, and —

CR^1′

;

δ

is 1; and

Z is CR¹³R¹⁴.

5. A compound, as claimed in claim 2, wherein,

R³ is —

L—

R_x;

or —

L—

S_c; and

R⁴ is independently a C₁-C₆ alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amnino;

the atoms of Y are selected from —

CH, —

C, and —

CR¹, where each R¹ is independently —

L—

R_x;

or —

L—

S_c ;

or amino, sulfo, trifluoromethyl, or halogen;

or C₁-C₆ alkyl, which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

the atoms of W are selected from —

CH, —

C, —

CR^1′

, and —

N(R¹²)_β

′

, where each β

′

is 0 or 1, but no more than one of such atoms is —

N(R¹²)_β

′

, and each R^1′

is independently —

L—

R_x;

or —

L—

S_c;

or amino, sulfo, trifluoromethyl, or halogen;

or C₁-C₆ alkyl, which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

R² and R¹² are independently —

L—

R_x;

or —

L—

S_c;

or a C₁-C₆ alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino;

Z is CR¹³R¹⁴;

one of R¹³ and R¹⁴ is a C₁-C₆ alkyl and the other of R¹³ and R¹⁴ is —

L—

R_x;

or —

L—

S_c;

or a C₁-C₂₂ alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino;

each of R²¹, R²², and R²³ are H;

or any two adjacent substituents of R²¹, R²², R²³, when taken in combination, forms a 4-, 5-, or 6-membered saturated or unsaturated hydrocarbon ring that is optionally substituted one or more times by C₁-C₆ alkyl, halogen, or a carbonyl oxygen.

6. A compound, as claimed in claim 5, wherein

R³ is —

L—

R_x;

or —

L—

S_c; and

R⁴ is C₁-C₆ alkyl; and

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, and incorporating the formula —

(CH₂)_d(CONH(CH₂)_e)_z′

—

, where d is 0-5, e is 1-5 and z′

is 0 or 1, 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, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds;

or aromatic or heteroaromatic bonds;

R_x is a reactive group that is an activated ester of a carboxylic acid, an amine, a carboxylic acid, a halotriazine, a hydrazide, a maleimide, a reactive platinum complex;

S_c is a conjugated substance that is an antibody, an amino acid, an avidin or streptavidin, a biotin, a blood component protein, a blood vessel proliferation inhibition factor, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a non-biological microparticle, a nucleotide, an oligonucleotide, a peptide toxin, a phospholipid-binding protein, a small-molecule drug, a structural protein, an NTA, or a tyramide; and

the atoms of Y are selected from —

CH, —

C, and —

CR¹, where R¹ is sulfo;

the atoms of W are selected from —

CH, —

C, —

CR^1′

, and —

N(R¹²)_β

′

, where each R^1′

is sulfo or Br;

R² and R¹² are independently a C₁-C₆ alkyl or C₁-C₆ alkyl substituted by sulfo;

Z is CR¹³R¹⁴, where R¹³ and R¹⁴ are independently C₁-C₆ alkyl

R²¹, R²², and R²³ are H.

7. A compound, as claimed in claim 6, where n=1.

8. A compound, as claimed in claim 6, where n=2.

9. A compound, as claimed in claim 6, where n=3.

10. A compound, as claimed in claim 1, wherein

R³ is —

L—

R_x; and

R⁴ is independently a C₁-C₂₂ alkyl, which optionally incorporates up to six hetero atoms, and is optionally substituted;

or R³ and R⁴ taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by —

L—

R_x; and

each R_x is a reactive group that is independently an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine (including hydrazides), an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, or a photoactivatable group;

no more than one of the atoms within Y is —

N(R²)_β

, and each R¹ is independently —

L—

R_x;

or —

L—

S_c;

or amino, sulfo, trifluoromethyl, or halogen;

or C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy; and

each S_c is a conjugated substance that is an amino acid, peptide, polypeptide, protein, nucleotide, oligonucleotide, nucleic acid polymer, a sugar, a polysaccharide, an oligosaccharide, a fluorescent dye, or a microsphere;

α

is 1 only when Y contains no ring nitrogen atoms,

no more than one of the atoms within W is —

N(R¹²)_β

′

, and each R^1′

is independently —

L—

R_x;

or —

L—

S_c;

or amino, sulfo, trifluoromethyl, or halogen;

or C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

R² and R¹² are independently —

L—

R_x;

or —

L—

S_c;

or is a C₁-C₂₂ alkyl, which optionally incorporates up to six hetero atoms, and is optionally substituted;

δ

is 1 only when W contains no ring nitrogen atoms;

Z is CR¹³R¹⁴;

R¹³ and R¹⁴ are independently —

L—

R_x;

or —

L—

S_c;

or a C₁-C₂₂ alkyl, which optionally incorporates up to six hetero atoms, is optionally substituted;

or R¹³ and R¹⁴ taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted by —

L—

R_x;

or —

L—

S_c; and

R²¹, R²², and R²³ are independently H, F, Cl, C₁-C₆ alkyl, C₁-C₆ alkoxy, aryloxy, a nitrogen heterocycle, an iminium ion, —

L—

R_x, or —

L—

S_C.

11. A compound, as claimed in claim 10, where n=1.

12. A compound, as claimed in claim 10, where n=2.

13. A compound, as claimed in claim 10, where n=3.

14. A compound, as claimed in claim 1, wherein

R³ is —

L—

S_c; and

R⁴ is independently a C₁-C₂₂ alkyl, which optionally incorporates up to six hetero atoms, and is optionally substituted;

or R³ and R⁴ taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by —

L—

S_c; and

L is a single covalent bond, or a covalent linkage and

S_c is a conjugated substance that is an antibody, an amino acid, an avidin or streptavidin, a biotin, blood vessel proliferation inhibition factor, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a non-biological microparticle, a nucleotide, an oligonucleotide, a peptide toxin, a phospholipid-binding protein, a small-molecule drug, a structural protein, an NTA, or a tyramide;

no more than one of the atoms within Y is —

N(R²)_β

, and each R¹ is independently —

L—

R_x;

or —

L—

S_c;

or amino, sulfo, trifluoromethyl, or halogen;

or C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy; and

—

R_x is a reactive group that is an activated ester of a carboxylic acid, an amine, a carboxylic acid, a halotriazine, a hydrazide, a maleimide, a reactive platinum complex; and

α

is 1 only when Y contains no ring nitrogen atoms,

no more than one of the atoms within W is —

N(R¹²)_β

′

, and each R^1′

is independently —

L—

R_x;

or —

L—

S_c;

or amino, sulfo, trifluoromethyl, or halogen;

or C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

R² and R¹² are independently —

L—

R_x;

or —

L—

S_c;

or is a C₁-C₂₂ alkyl, which optionally incorporates up to six hetero atoms, and is optionally substituted;

δ

is 1 only when W contains no ring nitrogen atoms;

Z is CR¹³R¹⁴;

R¹³ and R¹⁴ are independently —

L—

R_x;

or —

L—

S_c;

or a C₁-C₂₂ alkyl, which optionally incorporates up to six hetero atoms, is optionally substituted;

or R¹³ and R¹⁴ taken in combination complete a five- or six-membered saturated or unsaturated ring that is optionally substituted by —

L—

R_x;

or —

L—

S_c; and

R²¹, R²², and R²³, are independently H, F, Cl, C₁-C₆ alkyl, C₁-C₆ alkoxy, aryloxy, a nitrogen heterocycle, an iminium ion, —

L—

R_x, or —

L—

S_C.

15. A compound, as claimed in claim 14, where n=1.

16. A compound, as claimed in claim 14, where n=2.

17. A compound, as claimed in claim 14, where n=3.

19. A compound, as claimed in claim 18, where n=2.

20. A compound, as claimed in claim 19, wherein

R³ is —

L—

R_x; and

R⁴ is a C₁-C₆ alkyl; and

L is a covalent linkage that incorporates the formula —

(CH₂)_d(CONH(CH₂)_e)_z′

—

, or —

(CH₂)_d(CONH(CH₂)_eNH₂)_z′

—

, —

(CH₂)_d(CONH(CH₂) _eNHCO)—

_z′

,

where d is 0-5, e is 1-5 and z′

is 0 or 1;

R_x is a reactive group that is an activated ester of a carboxylic acid, an amine, a carboxylic acid, a halotriazine, a hydrazide, a maleimide, a reactive platinum complex;

R⁷ is sulfo, and R⁶, R⁸, and R⁹ are H;

or R⁶ and R⁷ combine to form a fused benzo ring that is substituted one or more times by sulfo, and R⁸ and R⁹ are H;

R¹⁷ is H or Br, and R¹⁶ and R¹⁸ are H.

21. A compound, as claimed in claim 20, where —

L is —

(CH₂)_d(CONH(CH₂)_e)_z′

— and

—

R_x is a carboxylic acid, or a succinimidyl ester of a carboxylic acid, a hydrazide, or a maleimide.

22. A compound, as claimed in claim 19, wherein

R³ is —

L—

S_c; and

R⁴ is a C₁-C₆ alkyl; and

L is a covalent linkage that incorporates the formula —

(CH₂)_d(CONH(CH₂)_e)_z′

—

, or —

(CH₂)_d(CONH(CH₂)_eNH₂)_z′

—

, —

(CH₂)_d(CONH(CH₂)_eNHCO)—

_z′

,

where d is 0-5, e is 1-5 and z′

is 0or 1;

S_c is a conjugated substance that is an actin, an antibody or fragment thereof, an avidin or streptavidin, a biotin, a dextran, an enzyme, a fluorescent protein, a lectin, a lipopolysaccharide, a microorganism, a non-biological microparticle, a nucleotide, an oligonucleotide, a peptide toxin, a phosphotidylserine-binding protein, a protein A or G, a small-molecule drug, an NTA, or a tyramide;

R⁷ is sulfo, and R⁶, R⁸, and R⁹ are H;

or R⁶ and R⁷ combine to form a fused benzo ring that is substituted one or more times by sulfo, and R⁸ and R⁹ are H;

R¹⁷ is H, sulfo, or Br, and R¹⁶ and R¹⁸ are H.

23. A compound, as claimed in claim 22, where 40 t —

L is —

(CH₂)_d(CONH(CH₂)_e)_z and S_c is a conjugated substance that is an antibody or fragment thereof, a fluorescent protein, a lectin, an oligonucleotide, a small-molecule drug, an NTA, or a tyramide.

25. A compound, as claimed in claim 24, where n=1.

26. A compound, as claimed in claim 24, where n=2.

27. A compound, as claimed in claim 24, where n=3.

28. A compound, as claimed in claim 24, wherein

R³ is —

L—

R_x; and

R⁴ is a C₁-C₆ alkyl; and

L is a covalent linkage that incorporates the formula —

(CH₂)_d(CONH(CH₂)_e)_z′

—

, or —

(CH₂)_d(CONH(CH₂)_eNH₂)_z′

—

, —

(CH₂)_d(CONH(CH₂)_eNHCO)—

_z′

,

where d is 0-5, e is 1-5 and z′

is 0 or 1;

R_x is a reactive group that is an activated ester of a carboxylic acid, an amine, a carboxylic acid, a halotriazine, a hydrazide, a maleimide, a reactive platinum complex;

R⁷ and R¹⁷ are sulfo, and R⁶, R⁸, R⁹, R¹⁶ , R¹⁸, and R¹⁹ are H.

29. A compound, as claimed in claim 28, where n=1.

30. A compound, as claimed in claim 28, where n=2.

31. A compound, as claimed in claim 28, where n=3.

32. A compound, as claimed in claim 28, where —

L is —

(CH₂)_d(CONH(CH₂)_e)_z′

— and

—

R_x is a carboxylic acid, or a succinimidyl ester of a carboxylic acid, a hydrazide, or a maleimide.

33. A compound, as claimed in claim 24, wherein

R³ is —

L—

S_c; and

R⁴ is a C₁-C₆ alkyl; and

L is a covalent linkage that incorporates the formula —

(CH₂)_d(CONH(CH₂)_e)_z′

—

, or —

(CH₂)_d(CONH(CH₂)_eNH₂)_z′

—

, —

(CH₂)_d(CONH(CH₂)_eNHCO)—

_z′

,

where d is 0-5, e is 1-5 and z′

is 0 or 1;

S_c is a conjugated substance that is an actin, an antibody or fragment thereof, an avidin or streptavidin, a biotin, a dextran, an enzyme, a fluorescent protein, a lectin, a lipopolysaccharide, a microorganism, a non-biological microparticle, a nucleotide, an oligonucleotide, a peptide toxin, a phosphotidylserine-binding protein, a protein A or G, a small-molecule drug, an NTA, or a tyramide;

R⁷ and R¹⁷ are sulfo, and R⁶, R⁸, R⁹, R¹⁶, R¹⁸, and R¹⁹ are H.

34. A compound, as claimed in claim 33, where —

L is —

(CH₂)_d(CONH(CH₂)_e)_z′

— and

S_c is a conjugated substance that is antibody or fragment thereof, a fluorescent protein, a lectin, an oligonucleotide, a small-molecule drug, an NTA, or a tyramide.

18. A compound of the formula

24. A compound of the formula:

35. A compound of the formula:

36. A compound of the formula:

37. A compound of the formula:

38. A compound of the formula:

39. A compound of the formula:

40. A compound of the formula:

41. A compound of the formula:

42. A compound, as claimed in claim 41, wherein

R³ is C₅-C₆ carboxyalkyl;

and R⁴ is methyl;

R² is methyl or sulfopropyl;

R⁶ and R⁷ form a fused benzo ring that is optionally substituted one or more times by sulfo;

or R⁷ is independently H or sulfo, and R⁶ is H;

both R⁸ and R⁹ are H.

43. A method of staining a biological sample, comprising:

combining a dye solution comprising a compound of the formula

or its salts, wherein

R³ is —

L—

R_x;

or —

L—

S_c; and

R⁴ is independently —

L—

R_x;

or —

L—

S_c;

or a C₁-C₂₂ alkyl or C₇-C₂₂ arylalkyl, each alkyl portion of which optionally incorporates up to six hetero atoms, selected from N, O and S, and each alkyl portion of which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino, or C₃-C₁₈ trialkylammonium;

or R³ and R⁴ taken in combination complete a five- or six-membered saturated or unsaturated ring that is substituted by —

L—

R_x;

or —

L—

S_c ; and

L is a single covalent bond, or 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, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds;

or aromatic or heteroaromatic bonds;

R_x is a reactive group;

S_c is a conjugated substance;

R⁶ through R⁹ are independently H, —

L—

R_x;

or —

L—

S_c;

or amino, sulfo, trifluoromethyl, or halogen;

or C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

or any two adjacent substituents of R⁶ through R⁹ form a fused benzo ring that is optionally substituted one or more times by —

L—

R_x;

or by —

L—

S_c;

or by amino, sulfo, trifluoromethyl, or halogen;

or by C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂-C₁₂ dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

the atoms of W are selected from —

CH, —

C, —

CR^1′

, and —

N(R¹²)_β

′

, where β

′

is 1, but no more than one of such atoms is —

N(R¹²)_β

′

; and

each R^1′

is independently —

L—

R_x;

or —

L—

S_c;

or amino, sulfo, trifluoromethyl, or halogen;

or C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₂C₁₂ dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;

each R² and R¹² is independently —

L—

R_x;

or —

L—

S_c;

or a C₁-C₂₂ alkyl, which optionally incorporates up to six hetero atoms, and is optionally substituted one or more times by amino, carboxy, or sulfo;

δ

is 0 or 1, and δ

+all β

′

=1; and

δ

is 1 only when W contains no ring nitrogen atoms;

one of R¹³ and R¹⁴ is a C₁-C₆ alkyl and the other of R¹³ and R¹⁴ is —

L—

R_x;

or —

L—

S_c;

or a C₁-C₂₂ alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, C₁-C₆ alkoxy, C₁-C₆ alkylamino, or C₂-C₁₂ dialkylamino;

with a biological sample in a concentration sufficient to yield a detectable optical response under the desired conditions.

44. A method, as claimed in claim 43, wherein the sample comprises cells.

45. A method, as claimed in claim 43, wherein the sample comprises proteins or nucleic acid polymers in a microarray.

46. A method, as claimed in claim 43, wherein the sample comprises a ligand that is a member of a specific binding pair, for which S_c is a complementary member.

47. A method, as claimed in claim 43, wherein the sample is contained in or on a solid or semi-solid matrix that is a membrane, an electrophoretic gel, a silicon chip, a glass slide, a microwell plate, or a microfluidic chip.

48. A method, as claimed in claim 43, wherein for said compound

R_x is a carboxylic acid, an activated ester of a carboxylic acid, an amine, an azide, a hydrazide, a haloacetamide, an alkyl halide, an isothiocyanate, or a maleimide group;

and

S_c is an antibody, a peptide, a lectin, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid polymer, an ion-complexing moiety, a lipid, or a non-biological organic polymer or polymeric microparticle, that is optionally bound to one or more additional fluorophores that are the same or different.

49. A method, as claimed in claim 43, wherein for the compound, R³ is the only —

L—

S_c, and S_c is a conjugated substance that is an antibody or fragment thereof, a fluorescent protein, a lectin, a nucleotide, an oligonucleotide, a peptide, a small-molecule drug, an NTA, or a tyramide.

50. A kit for fluorescent labeling of a biological sample, comprising

a dye solution comprising a compound of the formula

or its salts, wherein

R³ is —

L—

R_x;

or —

L—

S_c; and

R⁴ is C₁-C₆ alkyl; and

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, and incorporating the formula —

(CH₂)_d(CONH(CH₂)_e)_z′

—

, where d is 0-5, e is 1-5 and z′

is 0 or 1, 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, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds;

or aromatic or heteroaromatic bonds;

R_x is a reactive group that is an activated ester of a carboxylic acid, an amine, a carboxylic acid, a halotriazine, a hydrazide, a maleimide, a reactive platinum complex;

S_c is a conjugated substance that is an antibody, an amino acid, an angiostatin or endostatin, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG-binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a metal-binding protein, a microorganism or portion thereof, a neuropeptide, a non-biological microparticle, a nucleotide, an oligonucleotide, a peptide toxin, a phospholipid-binding protein, a small-molecule drug, a structural protein, an NTA, or a tyramide; and

R⁷ is sulfo, and R⁶, R⁸, and R⁹ are H;

or R⁶ and R⁷ combine to form a fused benzo ring that is substituted one or more times by sulfo, and R⁸ and R⁹ are H;

the atoms of W are selected from —

CH, —

C, —

CR^1′

, and —

N(R¹²)_β

′

, where each β

′

is 1, but no more than one of such atoms is —

N(R¹²)_β

′

, where each R^1′

is sulfo or Br;

R² and R¹² are independently a C₁-C₆ alkyl or C₁-C₆ alkyl substituted by sulfo;

δ

is 0 or 1, and δ

+all β

′

=1; and

δ

is 1 only when W contains no ring nitrogen atoms;

R¹³ and R¹⁴ are independently C₁-C₆ alkyl;

each of R²¹, R²², and R²³ are H;

or any two adjacent substituents of R²¹, R²², R²³, when taken in combination, forms a 4-, 5-, or 6-membered saturated or unsaturated hydrocarbon ring that is optionally substituted one or more times by C₁-C₆ alkyl, halogen, or a carbonyl oxygen; and

a buffer.