Hydrolyzable fluorescent substrates and analytical determinations using same
First Claim
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1. A hydrolyzable compound represented by the formula:
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space="preserve" listing-type="equation">BLOCK--X--R.sup.f --Lwherein BLOCK is a hydrolyzable group selected from the group consisting of;
--COR1 ;
a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide;
or a monovalent moiety derived by removal of a hydroxy group from a mono- or polysaccharide,R1 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group,X is --O--, --S-- or --NR-- and is attached to BLOCK at the open valence on BLOCK wherein R is hydrogen, alkyl, cycloalkyl, phenyl or a heterocyclic group,Rf is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR6## provided that when released as --X--Rf --L, --X--Rf --L exhibits maximum fluorescent emission at least about 580 nm at a pH of 9 or less, and L is hydrogen or a specific binding ligand.
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Abstract
Hydrolyzable substrates comprise blocked dye moieties which, when cleaved from the substrate during hydrolysis, provide fluorescent dyes having maximum absorptions above about 530 nm and maximum emissions at least about 580 nm at physiological pH. These substrates can be used in analytical determinations of hydrolytic substances including hydrolytic enzymes or biological cells containing such enzymes.
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Citations
18 Claims
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1. A hydrolyzable compound represented by the formula:
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space="preserve" listing-type="equation">BLOCK--X--R.sup.f --Lwherein BLOCK is a hydrolyzable group selected from the group consisting of; --COR1 ;
a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide;
or a monovalent moiety derived by removal of a hydroxy group from a mono- or polysaccharide,R1 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group, X is --O--, --S-- or --NR-- and is attached to BLOCK at the open valence on BLOCK wherein R is hydrogen, alkyl, cycloalkyl, phenyl or a heterocyclic group, Rf is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR6## provided that when released as --X--Rf --L, --X--Rf --L exhibits maximum fluorescent emission at least about 580 nm at a pH of 9 or less, and L is hydrogen or a specific binding ligand. - View Dependent Claims (2, 3, 4, 5)
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6. An analytical composition comprising an solution buffered to a pH of 9 or less and containing a hydrolyzable compound represented by the formula:
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space="preserve" listing-type="equation">BLOCK--X--R.sup.f --Lwherein BLOCK is a hydrolyzable group selected from the group consisting of; --COR1 ;
a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide;
or a monovalent moiety derived by removal of a hydroxy group from a mono- or polysaccharide,R1 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group, X is --O--, --S-- or --NR-- and is attached to BLOCK at the open valence on BLOCK, wherein R is hydrogen, alkyl, cycloalkyl, phenyl or a heterocyclic group, Rf is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR7## provided that when released as --X--Rf --L, --X--Rf --L exhibits maximum fluorescent emission at least about 580 nm and maximum absorption above about 540 nm at a pH of 9 or less, and L is hydrogen or a specific binding ligand. - View Dependent Claims (7, 8)
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9. An analytical element comprising an absorbent carrier material and containing a hydrolyzable compound represented by the formula:
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space="preserve" listing-type="equation">BLOCK--X--R.sup.f --Lwherein BLOCK is a hydrolyzable group selected from the group consisting of; --COR1 ;
a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide;
or a monovalent moiety derived by removal of a hydroxy group from a mono- or polysaccharide,R1 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group, X is --O--, --S-- or --NR-- and is attached to BLOCK at the open valence on BLOCK, wherein R is hydrogen, alkyl, cycloalkyl, phenyl or a heterocyclic group, Rf is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR8## provided that when released as --X--Rf --L, --X--Rf --L exhibits maximum fluorescent emission at least about 580 nm and maximum absorption above about 530 nm at a pH of 9 or less, and L is hydrogen or a specific binding ligand. - View Dependent Claims (10, 11, 12)
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13. A method for the determination of a hydrolytic analyte comprising the steps of:
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A. under hydrolyzing conditions, contacting a sample of a liquid suspected of containing a hydrolytic analyte with a hydrolyzable compound represented by the formula;
space="preserve" listing-type="equation">BLOCK--X--R.sup.f --Lwherein BLOCK is a hydrolyzable group selected from the group consisting of; --COR1 ;
a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an amino acid or peptide;
or a monovalent moiety derived by removal of a hydroxy group from a mono- or polysaccharide,R1 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl or a heterocyclic group, X is --O--, --S-- or --NR-- and is attached to BLOCK at the open valence on BLOCK, wherein R is hydrogen, alkyl, cycloalkyl, phenyl or a heterocyclic group, Rf is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR9## provided that when released as --X--Rf --L, --X--Rf --L exhibits maximum fluorescent emission at least about 580 nm and maximum absorption above about 530 nm at a pH of 9 or less, and L is hydrogen or a specific binding ligand, and B. determining the fluorescent moiety released from said compound by hydrolysis as a result of the presence of said hydrolytic analyte at a wavelength at least about 580 nm after excitation at a wavelength above about 530 nm. - View Dependent Claims (14, 15, 16, 17, 18)
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Specification