Sulfonated derivatives of 7-aminocoumarin
First Claim
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1. A compound of the formula ##STR44## wherein R5 is H;
- R6 is H, methyl, halogen, or SO3 X;
R7 is NR1 R2 ;
whereinR1 and R2 are independently H, C1 -C18 alkyl, C6 -C18 aryl, C1 -C18 alkanoyl, C7 -C18 arylalkanoyl;
or R1 in combination with R2 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine;
or R2 is H and R1 is a radical formed by a removal of the hydroxyl group from the carboxylic acid of an amino acid or from the carboxylic acid of the carboxy-terminal amino acid of a linear peptide consisting of 2-6 amino acids;
or R1 is a 2-nitrobenzyloxycarbonyl of the formula ##STR45## wherein R9 and R10 are H, C1 -C6 alkoxy, or R9 and R10 taken in combination are --O--CH2 --O--; and
R11 is H, CH3, a carboxylic acid or a biologically compatible salt of a carboxylic acid;
or R7 is a nitrogen atom that is linked by a single or double covalent bond to the anomeric carbon of a mono- or polysaccharide and formally replaces the anomeric oxygen atom of the mono- or polysaccharide;
R8 is H, halogen or SO3 X;
X is H, or a biologically compatible cation;
R3 is H, SO3 X, halogen, CN, NO2, or --L--RX or --L--SC ;
R4 is H, CN, C1 -C18 alkyl, C1 -C18 perfluoroalkyl, CH2 SO3 X;
or --L--RX or --L--SC ;
whereineach L is a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O 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 and carbon-sulfur bonds;
RX is a reactive group; and
SC is a conjugated substance;
provided that at least one of R3, R6 or R8 is SO3 X.
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Abstract
The present invention describes 7-aminocoumarin dyes that are substituted one or more times at the 3-, 6- and/or 8-positions by a sulfonic acid or a salt of a sulfonic acid, said dyes being useful as fluorescent probes or in the preparation of enzyme substrates, caged probes, or adducts with reducing sugars. The dyes of the invention optionally possess a reactive group useful for preparing fluorescent conjugates, which conjugates and methods for their preparation are described herein.
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Citations
27 Claims
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1. A compound of the formula ##STR44## wherein R5 is H;
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R6 is H, methyl, halogen, or SO3 X; R7 is NR1 R2 ;
whereinR1 and R2 are independently H, C1 -C18 alkyl, C6 -C18 aryl, C1 -C18 alkanoyl, C7 -C18 arylalkanoyl; or R1 in combination with R2 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine; or R2 is H and R1 is a radical formed by a removal of the hydroxyl group from the carboxylic acid of an amino acid or from the carboxylic acid of the carboxy-terminal amino acid of a linear peptide consisting of 2-6 amino acids; or R1 is a 2-nitrobenzyloxycarbonyl of the formula ##STR45## wherein R9 and R10 are H, C1 -C6 alkoxy, or R9 and R10 taken in combination are --O--CH2 --O--; and R11 is H, CH3, a carboxylic acid or a biologically compatible salt of a carboxylic acid; or R7 is a nitrogen atom that is linked by a single or double covalent bond to the anomeric carbon of a mono- or polysaccharide and formally replaces the anomeric oxygen atom of the mono- or polysaccharide; R8 is H, halogen or SO3 X; X is H, or a biologically compatible cation; R3 is H, SO3 X, halogen, CN, NO2, or --L--RX or --L--SC ; R4 is H, CN, C1 -C18 alkyl, C1 -C18 perfluoroalkyl, CH2 SO3 X;
or --L--RX or --L--SC ;
whereineach L is a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O 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 and carbon-sulfur bonds; RX is a reactive group; and SC is a conjugated substance; provided that at least one of R3, R6 or R8 is SO3 X. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method of detecting a complementary member of a specific binding pair in a sample, comprising:
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a) adding to said sample a dye-conjugate of a first member of a specific binding pair for which there is a complementary member, where said first member of a specific binding pair is attached to one or more dye molecules, which may be the same or different, having the formula ##STR47## wherein R5 is H; R6 is H, methyl, halogen, or SO3 X; R7 is NR1 R2 ;
whereinR1 and R2 are independently H, C1 -C18 alkyl;
or R1 in combination with R2 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine;or R1 is a 2-nitrobenzyloxycarbonyl of the formula ##STR48## wherein R9 and R10 are H, C1 -C6 alkoxy, or R9 and R10 taken in combination are --O--CH2 --O--; and R11 is H, CH3, a carboxylic acid or a biologically compatible salt of a carboxylic acid; R8 is H, halogen or SO3 X; X is H, or a biologically compatible cation; R3 is H, SO3 X, halogen, CN, NO2, or --L--SC ; R4 is H, CN, C1 -C18 alkyl, C1 -C18 perfluoroalkyl, CH2 SO3 X;
or --L--SC ;
whereineach L is a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O 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 and carbon-sulfur bonds; and SC is a conjugated substance that is said first member of a specific binding pair; provided that at least one of R3, R6 or R8 is SO3 X; and provided that one of R3 and R4 is --L--SC ; b) allowing sufficient time for the dye-conjugate to form a complex with the complementary member, said complex exhibiting a detectable optical response; and c) detecting said complex to locate the complementary member. - View Dependent Claims (18, 19, 20, 21, 22)
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23. A method of staining a sample, comprising:
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a) adding to said sample a compound of the formula ##STR49## wherein R5 is H; R6 is H, methyl, halogen, or SO3 X; R7 is NR1 R2 ;
whereinR1 and R2 are independently H, C1 -C18 alkyl;
or R1 in combination with R2 forms a saturated 5- or 6-membered heterocycle that is a piperidine, a morpholine, a pyrrolidine or a piperazine;or R2 is H and R1 is a radical formed by a removal of the hydroxyl group from the carboxylic acid of an amino acid or from the carboxylic acid of the carboxy-terminal amino acid of a linear peptide consisting of 2-6 amino acids; or R1 is a 2-nitrobenzyloxycarbonyl of the formula ##STR50## wherein R9 and R10 are H, C1 -C6 alkoxy, or R9 and R10 taken in combination are --O--CH2 --O--; and R11 is H, CH3, a carboxylic acid or a biologically compatible salt of a carboxylic acid; R8 is H, halogen or SO3 X; X is H, or a biologically compatible cation; R3 is H, SO3 X, halogen, CN, NO2, or --L--SC ; R4 is H, CN, C1 -C18 alkyl, C1 -C18 perfluoroalkyl, CH2 SO3 X;
or --L--SC ;
whereineach L is a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O 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 and carbon-sulfur bonds; and SC is a conjugated substance; provided that at least one of R3, R6 or R8 is SO3 X; in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength. - View Dependent Claims (24, 25, 26, 27)
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Specification