Reactive derivatives of sulforhodamine 101 with enhanced hydrolytic stability
AssigneeMolecular Probes, Inc.
InventorsRichard P. Haugland, Wojciech Szalecki
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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.
Claims34
A method of detecting a complementary member of a specific binding pair in a sample, said specific binding pair having a first member and a complementary member, comprising:
- a) adding to said sample a fluorescent conjugate of the first member of the specific binding pair;
- wherein said fluorescent conjugate is prepared by the reaction of at least one functional group on said first member with a reactive dye;
- where said functional group 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, a purine, a pyrimidine, or a carboxylic acid;
- and said reactive dye has the formula ##STR24## or the formula ##STR25## wherein R.sup.2 is H, C.sub.1 -C.sub.6 alkyl or C.sub.1 -C.sub.6 acyl;
- n=1-8;
- and
- R is a halomethylbenzamidyl, maleimidyl, (3,5-dichloro-2,4,6-triazin-1-yl)amino, isocyanato or an isothiocyanato;
- or R is a carboxylic acid, an alkali or alkaline earth metal salt of a carboxylic acid, or R is an activated ester of a carboxylic acid;
- b) allowing sufficient time for said fluorescent conjugate to form a complex with said complementary member;
- and
- c) detecting said complex, where the presence of said complex indicates the location of said complementary member.
A fluorescent conjugate of a specific binding pair member, prepared by the reaction of a member of a specific binding pair selected from the group consisting of amino acids, amino acid polymers, nucleotides, nucleic acid polymers and carbohydrates, wherein said specific binding pair member possesses at least one reactive functional group;
- with a reactive dye having the formula ##STR26## or the formula ##STR27## wherein R.sup.2 is H, C.sub.1 -C.sub.6 alkyl or C.sub.1 -C.sub.6 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 an acid chloride, an acid fluoride, or an acyl azide;
- or R is a hydroxybenzotriazolyl ester, a succinimidyl ester, a sulfosuccinimidyl ester, an alkali or alkaline earth metal salt of a sulfosuccinimidyl ester, a symmetric anhydride, a mixed anhydride of a chloroformate having 2-8 carbons, a mixed anhydride of a carboxylic acid or perfluorinated carboxylic acid having 2-8 carbons, a mixed anhydride of 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 is a carboxylic acid activated by a carbodiimide having 2-14 carbons.
Dependencies are inferred from the claim text.