Sulfonated xanthene derivatives

• US 6,130,101 A
• Filed: 09/23/1997
• Issued: 10/10/2000
• Est. Priority Date: 09/23/1997
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Leung, Wai-Yee, Mao, Fei, Haugland, Richard P.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US08/935,963
• Time in Patent Office: 1,113 Days
• US Class Current: 436/546
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/827 Lectins
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation

Abstract

The present invention describes xanthene dyes, including rhodamines, rhodols and fluoresceins that are substituted one or more times by a sulfonic acid or a salt of a sulfonic acid. The dyes of the invention, including chemically reactive dyes and dye-conjugates are useful as fluorescent probes, particularly in biological samples.

Claims

1. A compound having the formula ##STR51## or the formula ##STR52## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁ -C₁₈ alkyl, or C₁ -C₁₈ alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or --SO₃ X where X is H or a counterion

R¹ and R⁶ are H;

or R¹ taken in combination with R², or R⁵ taken in combination with R⁶, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO₃ X;

A is NR⁸ R⁹,

R⁸ and R⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R⁸ in combination with R⁹ forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R⁸ in combination with R², or R⁹ in combination with R³, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

C is OR¹⁷ or NR¹⁸ R¹⁹ ;

where R¹⁷ is H, or C₁ -C₁₈ alkyl;

where R¹⁸ and R¹⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R¹⁸ in combination with R¹⁹ forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R¹⁸ in combination with R⁴, or R¹⁹ in combination with R⁵, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

B is O or N⁺ R¹⁸ R¹⁹ ;

R¹⁰ is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or R¹⁰ is a saturated or unsaturated C₁ -C₁₈ alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;

or R¹⁰ has the formula ##STR53## where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;

or C₁ -C₁₈ alkyl, C₁ -C₁₈ alkoxy, C₁ -C₁₈ alkylthio, C₁ -C₁₈ alkanoylamino, C₁ -C₁₈ alkylaminocarbonyl, C₂ -C₃₆ dialkylaminocarbonyl, C₁ -C₁₈ alkyloxycarbonyl, or C₆ -C₁₈ arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R¹⁶, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid; and

R¹¹ is H, hydroxy, CN or a C₁ -C₆ alkoxy;

or R¹⁰ in combination with R¹¹ forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;

or R¹¹ in combination with R¹² forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH₃ ;

or R¹⁰ when taken in combination with R¹¹ is a carbonyl oxygen;

provided that either at least one of R², R³, R⁴, and R⁵ is --SO₃ X;

or R⁸ in combination with R², or R⁹ in combination with R³, or R¹⁸ in combination with R⁴, or R¹⁹ in combination with R⁵, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH₂ SO₃ X moiety.

2. A compound, as claimed in claim 1, wherein R³ and R⁴ are each --SO₃ X.

3. A compound, as claimed in claim 1, wherein R¹⁰ has the formula ##STR54## wherein R¹² is a carboxylic acid, a salt of carboxylic acid, or --SO₃ X;

and R¹¹ is not present.

4. A compound, as claimed in claim 3, wherein at least three of R¹³, R¹⁴, R¹⁵, and R¹⁶ are F or Cl.

5. A compound, as claimed in claim 3, wherein one of R¹⁴ and R¹⁵ is a carboxylic acid or salt of a carboxylic acid or is --S-(CH₂)_n COOH, wherein n is 1-15, and wherein the other of R¹⁴ or R¹⁵ is H, F or Cl.

6. A compound, as claimed in claim 1, wherein A is NR⁸ R⁹, B is O and C is OR¹⁷.

7. A compound, as claimed in claim 1, wherein A is NR⁸ R⁹, B is N⁺ R¹⁸ R¹⁹, and C is NR¹⁸ R¹⁹.

8. A compound, as claimed in claim 7, wherein R³ and R⁴ are each --SO₃ X.

9. A compound, as claimed in claim 7, wherein R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

and R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties.

10. A compound, as claimed in claim 9, wherein R⁹ and R¹⁸ are independently H, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl.

11. A compound, as claimed in claim 9, wherein R⁸ in combination with R², and R¹⁹ in combination with R⁵, each form a 5- or 6-membered ring that is saturated;

and R³ and R⁴ are each --SO₃ X.

12. A compound, as claimed in claim 9, wherein R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted by --CH₂ SO₃ X;

and R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by --CH₂ SO₃ X.

13. A compound, as claimed in claim 8, wherein

R¹, R², R⁵, and R⁶ are H;

R⁸, R⁹, R¹⁸, and R¹⁹ are H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl.

14. A compound, as claimed in claim 1, having the formula ##STR55## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, C₁ -C₁₈ alkyl, C₁ -C₁₈ alkoxy, or --SO₃ X;

R⁸ and R⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl;

or R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and that is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

R¹⁸ and R¹⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl;

or R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and that is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, Cl, F, amino, nitro, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, or --S-(CH₂)_n COOH where n=1-15;

provided that R³ and R⁴ are each --SO₃ X;

or

R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted --CH₂ SO₃ X; and

R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by --CH₂ SO₃ X.

15. A compound having the formula ##STR56## or the formula ##STR57## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁ -C₁₈ alkyl, or C₁ -C₁₈ alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or --L-R_x ;

or --SO₃ X where X is H or a counterion

R¹ and R⁶ are H;

or R¹ taken in combination with R², or R⁵ taken in combination with R⁶, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO₃ X;

A is NR⁸ R⁹,

R⁸ and R⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R⁸ in combination with R⁹ forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or --L-R_x ;

or R⁸ in combination with R², or R⁹ in combination with R³, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

C is OR¹⁷ or NR¹⁸ R¹⁹ ;

where R¹⁷ is H, or C₁ -C₁₈ alkyl;

or --L-R_x ;

where R¹⁸ and R¹⁹ are independently H, C₁ -C₆ alkyl, C¹ -C⁶ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R¹⁸ in combination with R¹⁹ forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or --L-R_x ;

or R¹⁸ in combination with R⁴, or R¹⁹ in combination with R⁵, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

B is O or N⁺ R¹⁸ R¹⁹ ;

R¹⁰ is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or R¹⁰ is a saturated or unsaturated C₁ -C₁₈ alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;

or --L-R_x ;

or R¹⁰ has the formula ##STR58## where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;

or --L-R_x ;

or C₁ -C₁₈ alkyl, C₁ -C₁₈ alkoxy, C₁ -C₁₈ alkylthio, C₁ -C₁₈ alkanoylamino, C₁ -C₁₈ alkylaminocarbonyl, C₂ -C₃₆ dialkylaminocarbonyl, C₁ -C₁₈ alkyloxycarbonyl, or C₆ -C₁₈ arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R¹⁶, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid; and

R¹¹ is H, hydroxy, CN or a C₁ -C₆ alkoxy;

or R¹⁰ in combination with R¹¹ forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;

or R¹¹ in combination with R¹² forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH₃ ;

or R¹⁰ when taken in combination with R¹¹ is a carbonyl oxygen;

for all --L-R_x, each L is a covalent linkage; and

R_x is a reactive group;

provided that either at least one of R², R³, R⁴, and R⁵ is --SO₃ X;

or R⁸ in combination with R², or R⁹ in combination with R³, or R¹⁸ in combination with R⁴, or R¹⁹ in combination with R⁵, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH₂ SO₃ X moiety; and

that at least one of R², R³, R⁴, R⁵, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, and R¹⁹ is --L-R_x.

16. A compound, as claimed in claim 15, wherein each L is independently a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorus-nitrogen bonds.

17. A compound, as claimed in claim 15, wherein R_x is an acrylamide, an activated ester of a carboxylic acid, hydroxy, an aldehyde, an alkyl halide, a sulfonate, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, 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, a maleimide, a phosphoramidite, a sulfonyl halide, or a thiol group.

18. A compound, as claimed in claim 15, wherein L is a single covalent bond and R_x is a carboxylic acid, an activated ester of a carboxylic acid, an amine, an azide, a hydrazine, a haloacetamide, an alkyl halide, an isothiocyanate, or a maleimide group.

19. A compound, as claimed in claim 15, wherein R² and R⁵ are F or Cl.

20. A compound, as claimed in claim 15, wherein one of R¹³, R¹⁴, R¹⁵, and R¹⁶ is --L-R_x.

21. A compound, as claimed in claim 15, wherein one of R⁸, R⁹, R¹⁷, R¹⁸, and R¹⁹ is --L-R_x.

22. A compound, as claimed in claim 15, wherein A is NR⁸ R⁹, B is N⁺ R¹⁸ R¹⁹, and C is NR¹⁸ R¹⁹.

23. A compound, as claimed in claim 15, wherein R¹⁰ has the formula ##STR59## wherein R¹² is a carboxylic acid, a salt of carboxylic acid, or --SO₃ X;

and R¹¹ is not present.

24. A compound, as claimed in claim 23, wherein at least three of R¹³, R¹⁴, R¹⁵, and R¹⁶ are F or Cl.

25. A compound, as claimed in claim 23, wherein one of R¹⁴ and R¹⁵ is a carboxylic acid or salt of a carboxylic acid or is --S-(CH₂)_n COOH, wherein n is 1-15, and wherein the other of R¹⁴ or R¹⁵ is H, F or Cl.

26. A compound, as claimed in claim 15, wherein R³ and R⁴ are each --SO₃ X.

27. A compound, as claimed in claim 22, wherein R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

and R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties.

28. A compound, as claimed in claim 27, wherein R⁹ and R¹⁸ are independently H, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl.

29. A compound, as claimed in claim 27, wherein R⁸ in combination with R², and R¹⁹ in combination with R⁵, each form a 5- or 6-membered ring that is saturated;

and R³ and R⁴ are each --SO₃ X.

30. A compound, as claimed in claim 27, wherein R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, that is substituted by --CH₂ SO₃ X;

and R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by --CH₂ SO₃ X.

31. A compound, as claimed in claim 26, wherein

R¹, R², R⁵, and R⁶ are H;

R⁸, R⁹, R¹⁸, and R¹⁹ are H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl.

32. A compound, as claimed in claim 15, having the formula ##STR60## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I;

or C₁ -C₁₈ alkyl

R⁸ and R⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl;

or R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and that is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

R¹⁸ and R¹⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl;

or R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and that is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, Cl, F, amino, nitro, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, --S-(CH₂)_n COOH where n=1-15, or --L-R_x.

33. A compound having the formula ##STR61## or the formula ##STR62## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁ -C₁₈ alkyl, or C₁ -C₁₈ alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or --L-S_c ;

or --SO₃ X where X is H or a counterion

R¹ and R⁶ are H;

or R¹ taken in combination with R², or R⁵ taken in combination with R⁶, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO₃ X;

A is OR⁷ or NR⁸ R⁹,

R⁷ is H, C₁ -C₁₈ alkyl;

or --L-S_c ;

R⁸ and R⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R⁹ in combination with R⁹ forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or --L-S_c ;

or R⁸ in combination with R², or R⁹ in combination with R³, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

C is OR¹⁷ or NR¹⁸ R¹⁹ ;

where R¹⁷ is H, or C₁ -C₁₈ alkyl;

or --L-S_c ;

where R¹⁸ and R¹⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R¹⁸ in combination with R¹⁹ forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or --L-S_c ;

or R¹⁸ in combination with R⁴, or R¹⁹ in combination with R⁵, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

B is O or N⁺ R¹⁸ R¹⁹ ;

R¹⁰ is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or R¹⁰ is a saturated or unsaturated C₁ -C₁₈ alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;

or --L-S_c ;

or R¹⁰ has the formula ##STR63## where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;

or --L-S_c ;

or C₁ -C₁₈ alkyl, C₁ -C₁₈ alkoxy, C₁ -C₁₈ alkylthio, C₁ -C₁₈ alkanoylamino, C₁ -C₁₈ alkylaminocarbonyl, C₂ -C₃₆ dialkylaminocarbonyl, C₁ -C₁₈ alkyloxycarbonyl, or C₆ -C₁₈ arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R¹⁶, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid; and

R¹¹ is H, hydroxy, CN or a C₁ -C₆ alkoxy;

or R¹⁰ in combination with R¹¹ forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;

or R¹¹ in combination with R¹² forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH₃ ;

or R¹⁰ when taken in combination with R¹¹ is a carbonyl oxygen;

for all --L-S_c, each L is a covalent linkage; and

S_c is a conjugated substance;

provided that either at least one of R², R³, R⁴, and R⁵ is --SO₃ X;

or R⁸ in combination with R², or R⁹ in combination with R³, or R¹⁸ in combination with R⁴, or R¹⁹ in combination with R⁵, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH₂ SO₃ X moiety; and

that at least one of R², R³, R⁴, R⁵, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, and R¹⁹ is --L-S_c.

34. A compound, as claimed in claim 33, wherein each L is independently a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorus-nitrogen bonds.

35. A compound, as claimed in claim 33, wherein S_c is an amino acid, peptide, protein, polysaccharide, ion-complexing moiety, nucleotide, nucleic acid polymer, hapten, drug, lipid, lipid assembly, non-biological organic polymer, polymeric microparticle, animal cell, plant cell, bacterium, yeast, virus or protist.

36. A compound, as claimed in claim 35, wherein S_c is further substituted by one or more additional fluorescent or non-fluorescent dyes.

37. A compound, as claimed in claim 33, wherein S_c is a peptide, protein, polysaccharide, nucleotide, or nucleic acid polymer.

38. A compound, as claimed in claim 33, wherein S_c is an antibody, an avidin, a streptavidin, a lectin, a growth factor, an actin, or a toxin.

39. A compound, as claimed in claim 38, wherein S_c is an antibody to a fluorophore.

40. A compound, as claimed in claim 33, wherein one of R¹³, R¹⁴, R¹⁵, and R¹⁶ is --L-Sc.

41. A compound, as claimed in claim 33, wherein one of R⁷, R⁸, R⁹, R¹⁷, R¹⁸, and R¹⁹ is --L-Sc.

42. A compound, as claimed in claim 33, wherein A is NR⁸ R⁹, B is N⁺ R¹⁸ R¹⁹, and C is NR¹⁸ R¹⁹.

43. A compound, as claimed in claim 33, wherein R¹⁰ has the formula ##STR64## wherein R¹² is a carboxylic acid, a salt of carboxylic acid, or --SO₃ X;

and R¹¹ is not present.

44. A compound, as claimed in claim 43, wherein at least three of R¹³, R¹⁴, R¹⁵, and R¹⁶ are F or Cl.

45. A compound, as claimed in claim 43, wherein one of R¹⁴ and R¹⁵ is a carboxylic acid or salt of a carboxylic acid or is --S-(CH₂)_n COOH, wherein n is 1-15, and wherein the other of R¹⁴ or R¹⁵ is H, F or Cl.

46. A compound, as claimed in claim 33, wherein R³ and R⁴ are each --SO₃ X.

47. A compound, as claimed in claim 42, wherein R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

and R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties.

48. A compound, as claimed in claim 47, wherein R⁹ and R¹⁸ are independently H, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl.

49. A compound, as claimed in claim 47, wherein R⁸ in combination with R², and R¹⁹ in combination with R⁵, each form a 5- or 6-membered ring that is saturated;

and R³ and R⁴ are each --SO₃ X.

50. A compound, as claimed in claim 47, wherein R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, that is substituted by --CH₂ SO₃ X;

and R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is at a carbon atom substituted by --CH₂ SO₃ X.

51. A compound, as claimed in claim 46, wherein

R¹, R², R⁵, and R⁶ are H;

R⁸, R⁹, R¹⁸, and R¹⁹ are H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl.

52. A compound, as claimed in claim 33, having the formula ##STR65## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, C₁ -C₈ alkyl, C₁ -C₁₈ alkoxy, or --SO₃ X;

R⁸ and R⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl;

or R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and that is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

R¹⁸ and R¹⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl;

or R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and that is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, Cl, F, amino, nitro, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, or --S-(CH₂)_n COOH where n=1-15;

provided that R³ and R⁴ are each --SO₃ X;

or

R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted by --CH₂ SO₃ X; and

R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by --CH₂ SO₃ X.

72. A compound, as claimed in claim 33, wherein the conjugated substance is a nucleic acid base, nucleoside, nucleotide, or a nucleic acid polymer.

73. A compound, as claimed in claim 72, wherein the conjugated substance is a nucleoside triphosphate, a deoxynucleoside triphosphate, or a dideoxynucleoside triphosphate.

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

combining a dye solution comprising a compound of the formula ##STR66## or the formula ##STR67## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁ -C₁₈ alkyl, or C₁ -C₁₈ alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or --L-R_x ;

--L-S_c ;

or --SO₃ X where X is H or a counterion;

R¹ and R⁶ are H;

or R¹ taken in combination with R², or R⁵ taken in combination with R⁶, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO₃ X;

A is OR⁷ or NR⁸ R⁹,

R⁷ is H, C₁ -C₁₈ alkyl;

or --L-R_x ;

or --L-S_c ;

R⁸ and R⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R⁸ in combination with R⁹ forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or --L-R_x ;

or --L-S_c ;

or R⁸ in combination with R², or R⁹ in combination with R³, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

C is OR¹⁷ or NR¹⁸ R¹⁹ ;

where R¹⁷ is H, or C₁ -C₁₈ alkyl;

or --L-R_x ;

or --L-S_c ;

where R¹⁸ and R¹⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, a salt of C₁ -C₆ carboxyalkyl, or a salt of C₁ -C₆ sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or R¹⁸ in combination with R¹⁹ forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine, each of which is optionally substituted by methyl, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alkyl;

or --L-R_x ;

or --L-S_c ;

or R¹⁸ in combination with R⁴, or R¹⁹ in combination with R⁵, or both, form a 5- or 6-membered ring that is saturated or unsaturated, and is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

B is O or N⁺ R¹⁸ R¹⁹ ;

R¹⁰ is H, F, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or R¹⁰ is a saturated or unsaturated C₁ -C₁₈ alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;

or --L-R_x ;

or --L-S_c ;

or R¹⁰ has the formula ##STR68## where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;

or --L-R_x ;

or --L-S_c ;

or C₁ -C₁₈ alkyl, C₁ -C₁₈ alkoxy, C₁ -C₁₈ alkylthio, C₁ -C₁₈ alkanoylamino, C₁ -C₁₈ alkylaminocarbonyl, C₂ -C₃₆ dialkylaminocarbonyl, C₁ -C₁₈ alkyloxycarbonyl, or C₆ -C₁₈ arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R¹⁶, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid; and

R¹¹ is H, hydroxy, CN or a C₁ -C₆ alkoxy;

or R¹⁰ in combination with R¹¹ forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;

or R¹¹ in combination with R¹² forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH₃ ;

or R¹⁰ when taken in combination with R¹¹ is a carbonyl oxygen;

each L is a covalent linkage;

each R_x is a reactive group; and

each S_c is a conjugated substance;

provided that either at least one of R², R³, R⁴, and R⁵ is --SO₃ X;

or R⁸ in combination with R², R⁹ in combination with R³, or R¹⁸ in combination with R⁴, or R¹⁹ in combination with R⁵, form a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by at least one --CH₂ SO₃ X moiety;

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

54. A method, as claimed in claim 53, further comprising combining the sample with an additional detection reagent that has spectral properties that are detectably different from said optical response.

55. A method, as claimed in claim 53, further comprising the step of determining a characteristic of the sample by comparing the optical response with a standard response parameter.

56. A method, as claimed in claim 55, wherein the sample comprises cells, and the characteristic determined is the viability of the cells.

57. A method, as claimed in claim 53, wherein the sample comprises cells, and the step of combining comprises electroporation, shock treatment, high extracellular ATP, pressure microinjection, scrape loading, patch clamp loading, or phagocytosis.

58. A method, as claimed in claim 53, further comprising tracing the temporal or spatial location of the optical response within the sample.

59. A method, as claimed in claim 53, wherein each L is independently a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorus-nitrogen bonds.

60. A method, as claimed in claim 53, wherein for said compound

at least one of R¹³, R¹⁴, R¹⁵ and R¹⁶ is --L-R_x or --L-S_c ;

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

S_c is a peptide, a protein, a polysaccharide, 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.

61. A method, as claimed in claim 53, wherein for said compound

at least one of R¹³, R¹⁴, R¹⁵, and R¹⁶ is --L-S_c ;

and S_c is an antibody to a fluorophore.

62. A method, as claimed in claim 53, wherein for said compound A is NR⁸ R⁹, B is N⁺ R¹⁸ R¹⁹, and C is NR¹⁸ R¹⁹.

63. A method, as claimed in claim 62, wherein said compound has the formula ##STR69## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, C₁ -C₁₈ alkyl, C₁ -C₁₈ alkoxy, or --SO₃ X;

R⁸ and R⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl;

or R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and that is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

R¹⁸ and R¹⁹ are independently H, C₁ -C₆ alkyl, C₁ -C₆ carboxyalkyl, a salt of C₁ -C₆ carboxyalkyl, C₁ -C₆ sulfoalkyl, or a salt of C₁ -C₆ sulfoalkyl;

or R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and that is optionally substituted by one or more C₁ -C₆ alkyls or --CH₂ SO₃ X moieties;

R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, Cl, F, amino, nitro, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, or --S-(CH₂)_n COOH where n=1-15;

provided that R³ and R⁴ are each --SO₃ X;

or

R⁸ in combination with R² forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted by --CH₂ SO₃ X; and

R¹⁹ in combination with R⁵ forms a 5- or 6-membered ring that is saturated or unsaturated, and is substituted at a carbon atom by --CH₂ SO₃ X.

64. A method, as claimed in claim 53, wherein for said compound

at least one of R¹³, R¹⁴, R¹⁵, and R¹⁶ is --L-S_c ;

and S_c is an antibody to an antibody that is present in said sample.

65. A compound having the formula ##STR70## or the formula ##STR71## wherein R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁ -C₁₈ alkyl, or C₁ -C₁₈ alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁ -C₆ alcohol;

or --L-R_x ;

or --SO₃ X where X is H or a counterion

R¹ and R⁶ are H;

or R¹ taken in combination with R², or R⁵ taken in combination with R⁶, or both, form a fused aromatic six membered ring that is optionally substituted one or more times by --SO₃ X;

A is OR⁷,

R⁷ is H, C₁ -C₁₈ alkyl;

or --L-R_x ;

C is OR¹⁷ ;

where R¹⁷ is H, or C₁ -C₁₈ alkyl;

or --L-R_x ;

B is O;

R¹⁰ has the formula ##STR72## where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, --SO₃ X, a carboxylic acid, a salt of carboxylic acid, CN, nitro, hydroxy, azido, amino, hydrazino;

or --L-R_x ;

or C₁ -C₁₈ alkyl, C₁ -C₁₈ alkoxy, C₁ -C₁₈ alkylthio, C₁ -C₁₈ is alkanoylamino, C₁ -C₁₈ alkylaminocarbonyl, C₂ -C₃₆ dialkylaminocarbonyl, C₁ -C₁₈ alkyloxycarbonyl, or C₆ -C₁₈ arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C₁ -C₆ alcohol, --SO₃ X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R⁶, when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid; and

R¹¹ is H, hydroxy, CN or a C₁ -C₆ alkoxy;

or R¹⁰ in combination with R¹¹ forms a 5-membered spirolactone ring or a 5-membered spirosultone ring;

or R¹¹ in combination with R¹² forms a 5- or 6-membered spirolactone ring or a 5- or 6-membered spirosultone ring that is optionally and independently substituted by H, F or CH₃ ;

or R¹⁰ when taken in combination with R¹¹ is a carbonyl oxygen;

for all --L-R_x, each L is a covalent linkage; and

R_x is a reactive group;

provided that at least two of R², R³, R⁴, and R⁵ is --SO₃ X; and

that at least one of R², R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷ is --L-R_x.

66. A compound, as claimed in claim 65, wherein one of R¹³, R¹⁴, R¹⁵, and R¹⁶ is --L-R_x.

67. A compound, as claimed in claim 65, wherein R³ and R⁴ are each --SO₃ X.

68. A compound, as claimed in claim 65, wherein R¹² is a carboxylic acid, a salt of carboxylic acid, or --SO₃ X;

and R¹¹ is not present.

69. A compound, as claimed in claim 65, wherein each L is independently a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorus-nitrogen bonds.

70. A compound, as claimed in claim 65, wherein R_x is an acrylamide, an activated ester of a carboxylic acid, hydroxy, an aldehyde, an alkyl halide, a sulfonate, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, 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, a maleimide, a phosphoramidite, a sulfonyl halide, or a thiol group.

71. A compound, as claimed in claim 65, wherein L is a single covalent bond and R_x is a carboxylic acid, an activated ester of a carboxylic acid, an amine, an azide, a hydrazine, a haloacetamide, an alkyl halide, an isothiocyanate, or a maleimide group.