Reactive derivatives of sulforhodamine 101 with enhanced hydrolytic stability

• US 6,562,632 B1
• Filed: 08/04/1998
• Issued: 05/13/2003
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Szalecki, Wojciech, Haugland, Richard P.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US09/129,015
• Time in Patent Office: 1,743 Days
• Field of Search: 546/37, 546/546, 546/800
• US Class Current: 436/546
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C07K 14/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 16/4283 against an allotypic or isotypic determinant on Ig
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C08B 37/0021 Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
• 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: C09B 62/002 with the linkage of the reactive group being alternatively specified
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

The invention describes reactive dyes having an alkyl spacer attached via a sulfonamide bond to a sulforhodamine 101 fluorophore, and a variety of useful conjugates prepared therefrom. The increased length of the covalent linkage due to the alkyl spacer results in dye-conjugates having a number of surprisingly advantageous properties relative to previous sulforhodamine 101-labeled conjugates, including enhanced solubility and increased fluorescence. The reactive dyes of the present invention are more stable than the known compound sulforhodamine 101 sulfonyl chloride. Novel reactive dyes are described for selective modification of groups other than amines, including thiols and photoreactive derivatives.

Claims

1. A reactive dye having the formula

or the formula

wherein

R² is H, C₁-C₆ alkyl or C₁-C₆ acyl;

and

n=2 to 8; and

R is a halomethylbenzamidyl, maleimidyl, (3,5-dichloro-2,4,6-triazin-1-yl)amino, isocyanato or an isothiocyanato;

or

n=1 to 7; and

R is a carboxylic acid, an alkali or alkaline earth metal salt of a carboxylic acid, or R is —

(C═

O)—

R_x, where R_x is a chloride, fluoride or azide;

or R_x is selected so as to make R a hydroxybenzotriazolyl ester, a succinimidyl ester, a sulfosuccinimidyl ester, an alkali or alkaline earth metal salt of a sulfosuccinimidyl ester, a symmetric anhydride that links two sulforhodamine 101 fluorophores, a mixed anhydride of a sulforhodamine 101 fluorophore and a chloroformate having 2-8 carbons, a mixed anhydride of a sulforhodamine 101 fluorophore and a carboxylic acid or perfluorinated carboxylic acid having 2-8 carbons, a mixed anhydride of a sulforhodamine 101 fluorophore and a sulfonic or fluorinated sulfonic acid having 1-8 carbons, or an ester of a phenol or a naphthol that is further substituted one or more times by nitro, sulfo, carboxy, alkali or alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro, or trifluoromethyl;

or R_x is selected so as to make R a carboxylic acid activated by a carbodiimide having 2-14 carbons.

2. A reactive dye, as claimed in claim 1, wherein

n=2-8, and R is a halomethylbenzamido, maleimido, maleimidyl benzamido, maleimidyl alkylamido, azidobenzamido, azidoperfluorobenzamido, (3,5-dichloro-2,4,6-triazin-1-yl)amino, isocyanato or an isothiocyanato.

3. A reactive dye, as claimed in claim 2, wherein n=5 or 6.

4. A reactive dye, as claimed in claim 2, wherein R is maleimido or a halomethylbenzamido.

5. A reactive dye, as claimed in claim 2, wherein R is maleimido.

6. A reactive dye, as claimed in claim 5, having the formula

7. A reactive dye, as claimed in claim 1, wherein n=1-7 and R is a carboxylic acid, an alkali or alkaline earth metal salt of a carboxylic acid, or R is —

(C═

O)—

R_x, where R_x is a chloride, fluoride or azide;

or R_x is selected so as to make R a hydroxybenzotriazolyl ester, a succinimidyl ester, a sulfosuccinimidyl ester, an alkali or alkaline earth metal salt of a sulfosuccinimidyl ester, a symmetric anhydride that links two sulforhodamine 101 fluorophores, a mixed anhydride of a sulforhodamine 101 fluorophore and a chloroformate having 2-8 carbons, a mixed anhydride of a sulforhodamine 101 fluorophore and a carboxylic acid or perfluorinated carboxylic acid having 2-8 carbons, a mixed anhydride of a sulforhodamine 101 fluorophore and a sulfonic or fluorinated sulfonic acid having 1-8 carbons, or an ester of a phenol or a naphthol that is further substituted one or more times by nitro, sulfo, carboxy, alkali or alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro, or trifluoromethyl;

or R_x is selected so as to make R a carboxylic acid activated by a carbodiimide having 2-14 carbons.

8. A reactive dye, as claimed in claim 7, wherein R_x is selected so as to make R an ester of a phenol or a naphthol that is further substituted one or more times by nitro, sulfo, carboxy, alkali or alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro, or trifluoromethyl.

9. A reactive dye, as claimed in claim 7, wherein R has the formula —

(C═

O)—

R_x, wherein R_x is chloride, fluoride, or azide;

or R_x is selected so as to make R a succinimidyl ester, sulfosuccinimidyl ester, or an alkali or alkaline earth metal salt of sulfosuccinimidyl ester.

10. A reactive dye, as claimed in claim 7, wherein R_x is selected so as to make R a succinimidyl ester, sulfosuccinimidyl ester, or an alkali or alkaline earth metal salt of sulfosuccinimidyl ester.

11. A reactive dye, as claimed in claim 10, wherein n=4 or 5.

12. A reactive dye, as claimed in claim 10, wherein R_x is selected so as to make R a succinimidyl ester.

13. A reactive dye, as claimed in claim 12, having the formula

14. A kit for the fluorescent labeling of an organic substance, where said substance has at least one functional group that is an amine, a thiol, an alcohol, a phenol, an aldehyde, a ketone, a phosphate, an imidazole, a hydrazine, a hydroxylamine, a disubstituted amine, a trisubstituted amine, a halide, an epoxide, a sulfonate ester, or a carboxylic acid;

comprising;

a) a reactive dye of the formula

 

or the formula

 

wherein

R2 is H, C1-C6 alkyl or C1-C6 acyl; and

n=2 to 8; and

R is a halomethylbenzamidyl, maleimidyl, (3,5-dichloro-2,4,6-triazin-1-yl)amino, isocyanato or an isothiocyanato;

or

n=1 to 7; and

R is a carboxylic acid, an alkali or alkaline earth metal salt of a carboxylic acid, or R is —

(C═

O)—

R_x, where R_x is a chloride, fluoride or azide;

or R_x is selected so as to make R a hydroxybenzotriazolyl ester, a succinimidyl ester, a sulfosuccinimidyl ester, an alkali or alkaline earth metal salt of a sulfosuccinimidyl ester, a symmetric anhydride that links two sulforhodamine 101 fluorophores, a mixed anhydride of a sulforhodamine 101 fluorophore and a chloroformate having 2-8 carbons, a mixed anhydride of a sulforhodamine 101 fluorophore and a carboxylic acid or perfluorinated carboxylic acid having 2-8 carbons, a mixed anhydride of a sulforhodamine 101 fluorophore and a sulfonic or fluorinated sulfonic acid having 1-8 carbons, or an ester of a phenol or a naphthol that is further substituted one or more times by nitro, sulfo, carboxy, alkali or alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro, or trifluoromethyl;

or R_x is selected so as to make R a carboxylic acid activated by a carbodiimide having 2-14 carbons;

such that R will spontaneously react with said functional group to yield a labeled substance; and

b) instructions for combining the reactive dye with the substance and recovering the labeled substance.

15. A kit, as claimed in claim 14, wherein said substance is an amino acid, an amino acid polymer, a nucleotide, an oligonucleotide, an antisense oligonucleotide, a nucleic acid polymer, or a carbohydrate.

16. A kit, as claimed in claim 14, wherein said substance is an amino acid polymer.

17. A kit, as claimed in claim 14, wherein said substance is an oligonucleotide or nucleic acid polymer.

18. A kit, as claimed in claim 14, wherein said substance is a carbohydrate.

19. A kit, as claimed in claim 14, wherein said functional group is an amine or a thiol.