Patent US4812409A successfully refreshed from Google Patents.
US4812409A
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US
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Hydrolyzable fluorescent substrates and analytical determinations using same
Assignee: Clinical Diagnostic Systems Inc
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Filing: 1986-01-31
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Granted: 1989-03-14
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Expires: 2006-01-31
Abstract
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.
Inventors
- 👤 Bruce E. Babb
- 👤 Robert T. Belly
- 👤 Patricia M. Scensny
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 16:54
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/52; C; C09; C09B; C09B3/00; C09B57/00; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/44; C12Q2334/00; Y; Y10; Y10S; Y10S436/00; Y10S436/80
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
153 words
A hydrolyzable compound represented by the formula: BLOCK--X--R.sup.f --L wherein BLOCK is a hydrolyzable group selected from the group consisting of: --COR 1 ; 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, R 1 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, R f is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR6## provided that when released as --X--R f --L, --X--R f --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.
Claim 2
Dependent Claim
153 words
A hydrolyzable compound represented by the formula: BLOCK--X--R.sup.f --L wherein BLOCK is a hydrolyzable group selected from the group consisting of: --COR 1 ; 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, R 1 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, R f is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR6## provided that when released as --X--R f --L, --X--R f --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.
Claim 3
Dependent Claim
17 words
The compound of claim 1 wherein BLOCK is --CO--R 1 wherein R 1 is hydrogen or alkyl.
Claim 4
Dependent Claim
9 words
The compound of claim 1 wherein L is hydrogen.
Claim 5
Dependent Claim
12 words
The compound of claim 1 wherein L is a specific binding ligand.
Claim 6
Dependent Claim
11 words
The compound of claim 1 wherein X is --O-- or --NR--.
Claim 7
Dependent Claim
176 words
An analytical composition comprising an solution buffered to a pH of 9 or less and containing a hydrolyzable compound represented by the formula: BLOCK--X--R.sup.f --L wherein BLOCK is a hydrolyzable group selected from the group consisting of: --COR 1 ; 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, R 1 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, R f is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR7## provided that when released as --X--R f --L, --X--R f --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.
Claim 8
Dependent Claim
176 words
An analytical composition comprising an solution buffered to a pH of 9 or less and containing a hydrolyzable compound represented by the formula: BLOCK--X--R.sup.f --L wherein BLOCK is a hydrolyzable group selected from the group consisting of: --COR 1 ; 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, R 1 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, R f is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR7## provided that when released as --X--R f --L, --X--R f --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.
Claim 9
Dependent Claim
13 words
The composition of claim 6 further comprising a water-miscible solvent and a surfactant.
Claim 10
Dependent Claim
27 words
The composition of claim 6 wherein BLOCK is --CO--R 1 wherein R 1 is independently hydrogen or alkyl, L is hydrogen and X is --O-- or --NR--.
Claim 11
Dependent Claim
170 words
An analytical element comprising an absorbent carrier material and containing a hydrolyzable compound represented by the formula: BLOCK--X--R.sup.f --L wherein BLOCK is a hydrolyzable group selected from the group consisting of: --COR 1 ; 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, R 1 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, R f is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR8## provided that when released as --X--R f --L, --X--R f --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.
Claim 12
Dependent Claim
170 words
An analytical element comprising an absorbent carrier material and containing a hydrolyzable compound represented by the formula: BLOCK--X--R.sup.f --L wherein BLOCK is a hydrolyzable group selected from the group consisting of: --COR 1 ; 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, R 1 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, R f is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR8## provided that when released as --X--R f --L, --X--R f --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.
Claim 13
Dependent Claim
20 words
The element of claim 9 further containing a buffer which maintains the pH at 9 or less during an assay.
Claim 14
Dependent Claim
20 words
The element of claim 9 comprising a nonporous support having therein a porous spreading zone as said absorbent carrier material.
Claim 15
Dependent Claim
26 words
The element of claim 9 wherein BLOCK is --CO--R 1 wherein R 1 is hydrogen or alkyl, L is hydrogen, and X is --O-- or --NR--.
Claim 16
Independent Claim
229 words
A method for the determination of a hydrolytic analyte comprising the steps of: A. under hydrolyzing conditions, contacting a sample of a liquid suspected of containing a hydrolytic analyte with a hydrolyzable compound represented by the formula: BLOCK--X--R.sup.f --L wherein BLOCK is a hydrolyzable group selected from the group consisting of: --COR 1 ; 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, R 1 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, R f is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR9## provided that when released as --X--R f --L, --X--R f --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.
Claim 17
Independent Claim
229 words
A method for the determination of a hydrolytic analyte comprising the steps of: A. under hydrolyzing conditions, contacting a sample of a liquid suspected of containing a hydrolytic analyte with a hydrolyzable compound represented by the formula: BLOCK--X--R.sup.f --L wherein BLOCK is a hydrolyzable group selected from the group consisting of: --COR 1 ; 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, R 1 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, R f is a phenalenone or benzphenalenone fluorescent moiety selected from the group consisting essentially of ##STR9## provided that when released as --X--R f --L, --X--R f --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.
Claim 18
Dependent Claim
14 words
The method of claim 13 carried out at a pH of 8 or less.
Claim 19
Dependent Claim
12 words
The method of claim 13 for the determination of a hydrolytic enzyme.
Claim 20
Dependent Claim
11 words
The method of claim 13 which is a competitive binding assay.
Claim 21
Dependent Claim
26 words
The method of claim 13 wherein BLOCK is --CO--R 1 wherein R 1 is hydrogen or alkyl, L is hydrogen, and X is --O-- or --NR--.
Claim 22
Dependent Claim
21 words
The method of claim 13 for the determination of Klebsiella pneumoniae or Enterobacter cloacae wherein BLOCK is derived from a monosaccharide.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-03-13 | 1992-09-12 | 1993-03-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-03-13 | 1996-09-12 | 1997-03-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-03-13 | 2000-09-12 | 2001-03-14 | $7700.00 | Window Closed / Lapsed |