US5362628A
Expired / Lapsed
US
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Fluorescent haloalkyl derivatives of reporter molecules well retained in cells
Assignee: Molecular Probes Inc
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Filing: 1993-03-05
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Granted: 1994-11-08
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Expires: 2013-03-05
Abstract
Abstract The subject invention provides a method for analyzing the metabolic activity in cells by improving the retention of a detectable reporter molecule only in intact cells where a particular enzyme is present. In particular, improved retention results from a two part process involving conjugation of haloalkyl-substituted derivatives of a reporter molecule with intracellular cysteine-containing peptides while unblocking the reporter molecule. The method for analyzing metabolic activity of cells involves the use of a substrate having the form XR-REPORTER-BLOCK wherein -BLOCK is a group selected to be removable by action of a specific analyte, to give REPORTER spectral properties different from those of the substrate, -REPORTER- is a molecule that, when no longer bound to BLOCK by a BLOCK-REPORTER bond, has spectral properities different from those of the substrate, and XR-- is a haloalkyl moiety that can covalently react with an intracellular thiol (Z--S--H) to form a thioether conjugate (Z--S--R--). After the substrate enters the cells, the analyte removes BLOCK to make REPORTER detectable by the change in spectral properties, and the haloalkyl XR reacts with the intracellular thiol to form the thioether conjugate inside the cells, which is well-retained in the cells.
Inventors
- 👤 Richard P. Haugland
- 👤 Yu-zhong Zhang
- 👤 Ram Sabnis
- 👤 Nels A. Olson
- 👤 John J. Naleway
- 👤 Rosaria P. Haugland
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 20:37
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/02; C12Q1/04; C12Q1/34; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/535; C12Q2334/00; C12Q2334/20; C12Q2334/40; C12Q2337/00; C12Q2337/20; G01N2333/00; G01N2333/90; G01N2333/914; G01N2333/924; Y; Y10; Y10S; Y10S530/00; Y10S530/802
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
303 words
A method for retaining a fluorescent reaction product of enzyme activity in a cell for more than one hour to accomplish relevant analysis, comprising: a) preparing a biocompatible solution of a substrate having the form: XR-REPORTER-BLOCK wherein BLOCK is selected to be removable from REPORTER by enzyme activity to give a fluorescent REPORTER; -REPORTER- is a molecule that is bound to BLOCK by a BLOCK-REPORTER bond that is an amide bond that results from removal of a hydroxy group from a carboxylic acid of an amino acid or peptide and a hydrogen atom from an amino moiety on REPORTER; or an ether bond formed by removal of a hydroxy group from a lower alcohol having 6 or less carbon atoms or of hydroxy from a mono- or polysaccharide and a hydrogen atom from a phenolic moiety on REPORTER; or an ester bond, where the ester is an ester of an aliphatic or aromatic carboxylic acid or of phosphoric or sulfuric acid, or a biologically compatible salt thereof; such that REPORTER, when bound to BLOCK is virtually non-fluorescent and when BLOCK is removed, REPORTER becomes fluorescent; and XR-- is a haloalkyl moiety, where X is a single Cl, I, or Br, and R contains 1-4 carbons, where XR-- can covalently react with an intracellular thiol (Z--S--H) to form a thioether conjugate (Z--S--R-REPORTER); b) bringing the biocompatible solution containing the substrate into contact with the cell under conditions where the substrate can readily enter intracellular areas of the cell that may contain the enzyme activity; c) allowing sufficient time for the enzyme activity to remove BLOCK to make REPORTER fluorescent, and for the haloalkyl XR to react with said intracellular thiol to form the thioether conjugate inside the cell; and d) preparing the cell for detection or measurement of fluorescence of REPORTER inside the cell.
Claim 2
Independent Claim
303 words
A method for retaining a fluorescent reaction product of enzyme activity in a cell for more than one hour to accomplish relevant analysis, comprising: a) preparing a biocompatible solution of a substrate having the form: XR-REPORTER-BLOCK wherein BLOCK is selected to be removable from REPORTER by enzyme activity to give a fluorescent REPORTER; -REPORTER- is a molecule that is bound to BLOCK by a BLOCK-REPORTER bond that is an amide bond that results from removal of a hydroxy group from a carboxylic acid of an amino acid or peptide and a hydrogen atom from an amino moiety on REPORTER; or an ether bond formed by removal of a hydroxy group from a lower alcohol having 6 or less carbon atoms or of hydroxy from a mono- or polysaccharide and a hydrogen atom from a phenolic moiety on REPORTER; or an ester bond, where the ester is an ester of an aliphatic or aromatic carboxylic acid or of phosphoric or sulfuric acid, or a biologically compatible salt thereof; such that REPORTER, when bound to BLOCK is virtually non-fluorescent and when BLOCK is removed, REPORTER becomes fluorescent; and XR-- is a haloalkyl moiety, where X is a single Cl, I, or Br, and R contains 1-4 carbons, where XR-- can covalently react with an intracellular thiol (Z--S--H) to form a thioether conjugate (Z--S--R-REPORTER); b) bringing the biocompatible solution containing the substrate into contact with the cell under conditions where the substrate can readily enter intracellular areas of the cell that may contain the enzyme activity; c) allowing sufficient time for the enzyme activity to remove BLOCK to make REPORTER fluorescent, and for the haloalkyl XR to react with said intracellular thiol to form the thioether conjugate inside the cell; and d) preparing the cell for detection or measurement of fluorescence of REPORTER inside the cell.
Claim 3
Dependent Claim
28 words
A method, as claimed in claim 1, wherein REPORTER an anthracene, a benzphenalenone, a coumarin, a fluorescein, a naphthalene, a phenalenone, a pyrene, a resorufin, or a rhodamine.
Claim 4
Dependent Claim
39 words
A method, as claimed in claim 2, wherein REPORTER is a coumarin, fluorescein, resorufin or rhodamine; and fluorescence of REPORTER is detected in the cell about 15 minutes after incubating the cell with the biocompatible solution under physiological conditions.
Claim 5
Dependent Claim
27 words
A method, as claimed in claim 2, wherein XR is XCH 2 and the thioether conjugate is retained inside the cell through at least one cell division.
Claim 6
Dependent Claim
39 words
A method, as claimed in claim 2, where preparing the cell for detection or measurement of fluorescence of REPORTER includes fixing the cell with an aldehyde-containing fixative in a manner so as to retain REPORTER inside the fixed cell.
Claim 7
Independent Claim
313 words
A method for retaining a fluorescent reaction product formed by activity of an enzyme in a cell for analysis of such activity, comprising: a) preparing a biocompatible solution of a substrate having the form: XR-REPORTER-BLOCK wherein BLOCK is selected to be removable from REPORTER by activity of the enzyme to give REPORTER spectral properties different from those of the substrate; -REPORTER- is a molecule that is bound to BLOCK by a BLOCK-REPORTER bond that is an amide bond that results from removal of a hydroxy group from a carboxylic acid of an amino acid or peptide and a hydrogen atom from an amino moiety on REPORTER; or an ether or thioether bond formed by removal of a hydroxy group from a lower alcohol having 6 or less carbon atoms or of hydroxy from a mono- or polysaccharide and a hydrogen atom from a phenolic or thiophenolic moiety on REPORTER; or an ester bond where the ester is an ester of an aromatic carboxylic acid or of phosphoric or sulfuric acid, or a biologically compatible silt thereof; such that when REPORTER is no longer bound to BLOCK by a BLOCK-REPORTER bond, REPORTER has spectral properties different from those of the substrate; and XR-- is a haloalkyl moiety, where X is a single Cl, I, or Br, and R contains 1-4 carbons, where XR-- can covalently react with an intracellular thiol (Z--S--H) to form a thioether conjugate (Z--S--R-REPORTER); b) introducing the substrate into the cell under conditions where the substrate can enter intracellular areas of the cell that may contain the enzyme; c) allowing sufficient time for activity of the enzyme to remove BLOCK to give REPORTER different spectral properties, and for the haloalkyl XR to react with said intracellular thiol to form the thioether conjugate inside the cell; and d) preparing the cell for detection or measurement of fluorescence of REPORTER inside the cell.
Claim 8
Independent Claim
313 words
A method for retaining a fluorescent reaction product formed by activity of an enzyme in a cell for analysis of such activity, comprising: a) preparing a biocompatible solution of a substrate having the form: XR-REPORTER-BLOCK wherein BLOCK is selected to be removable from REPORTER by activity of the enzyme to give REPORTER spectral properties different from those of the substrate; -REPORTER- is a molecule that is bound to BLOCK by a BLOCK-REPORTER bond that is an amide bond that results from removal of a hydroxy group from a carboxylic acid of an amino acid or peptide and a hydrogen atom from an amino moiety on REPORTER; or an ether or thioether bond formed by removal of a hydroxy group from a lower alcohol having 6 or less carbon atoms or of hydroxy from a mono- or polysaccharide and a hydrogen atom from a phenolic or thiophenolic moiety on REPORTER; or an ester bond where the ester is an ester of an aromatic carboxylic acid or of phosphoric or sulfuric acid, or a biologically compatible silt thereof; such that when REPORTER is no longer bound to BLOCK by a BLOCK-REPORTER bond, REPORTER has spectral properties different from those of the substrate; and XR-- is a haloalkyl moiety, where X is a single Cl, I, or Br, and R contains 1-4 carbons, where XR-- can covalently react with an intracellular thiol (Z--S--H) to form a thioether conjugate (Z--S--R-REPORTER); b) introducing the substrate into the cell under conditions where the substrate can enter intracellular areas of the cell that may contain the enzyme; c) allowing sufficient time for activity of the enzyme to remove BLOCK to give REPORTER different spectral properties, and for the haloalkyl XR to react with said intracellular thiol to form the thioether conjugate inside the cell; and d) preparing the cell for detection or measurement of fluorescence of REPORTER inside the cell.
Claim 9
Dependent Claim
39 words
A method, as claimed in claim 6, where preparing the cell for detection or measurement of fluorescence of REPORTER includes fixing the cell with an aldehyde-containing fixative in a manner so as to retain REPORTER inside the fixed cell.
Claim 10
Dependent Claim
22 words
A method, as claimed in claim 6, wherein the thioether conjugate is retained inside the cell through at least one cell division.
Claim 11
Dependent Claim
29 words
A method, as claimed in claim 6, wherein REPORTER is an anthracene, a benzphenalenone, a coumarin, a fluorescein, a naphthalene, a phenalenone, a pyrene, a resorufin, or a rhodamine.
Claim 12
Dependent Claim
37 words
A method, as claimed in claim 6, wherein REPORTER is a coumarin, a fluorescein, a resorufin, or a rhodamine, such that REPORTER, when bound to BLOCK is virtually nonfluorescent and when BLOCK is removed, REPORTER becomes fluorescent.
Claim 13
Dependent Claim
50 words
A method, as claimed in claim 6, wherein the BLOCK-REPORTER bond is an ether bond formed by removal of a hydroxy group from a lower alcohol having 6 or less carbon atoms or of hydroxy from a mono- or polysaccharide and a hydrogen atom from a phenolic moiety on REPORTER.
Claim 14
Dependent Claim
38 words
A method, as claimed in claim 6, wherein the substrate is an ether of a fluorescein and a lower alcohol having 6 or fewer carbon atoms or of a mono- or polysaccharide and XR is XCH 2 .
Claim 15
Dependent Claim
39 words
A method, as claimed in claim 6, wherein the ether bond is an ether of a 7-hydroxycoumarin and a lower alcohol having 6 or fewer carbon atoms or of a mono- or polysaccharide and XR is XCH 2 .
Claim 16
Dependent Claim
34 words
A method, as claimed in claim 6, wherein the BLOCK-REPORTER bond is an ester bond, wherein the ester is an ester of a guanidinobenzoic, phosphoric, or sulfuric acid, or a biologically compatible salt thereof.
Claim 17
Dependent Claim
31 words
A method, as claimed in claim 6, wherein the substrate is an ester of a 7-hydroxycoumarin and phosphate or a biologically compatible salt of phosphate and XR is XCH 2 .
Claim 18
Dependent Claim
43 words
A method, as claimed in claim 6, wherein the BLOCK-REPORTER bond is an amide bond that results from removal of a hydroxy group from a carboxylic acid of an amino acid or peptide and a hydrogen atom from an amino moiety on REPORTER.
Claim 19
Dependent Claim
28 words
A method, as claimed in claim 6, wherein the substrate is an amide of a 7-aminocoumarin and an amino acid or peptide and XR is XCH 2 .
Claim 20
Dependent Claim
15 words
A method, as claimed in claim 6, wherein the enzyme is a β-galactosidase or β-glucuronidase.
Claim 21
Dependent Claim
20 words
A method, as claimed in claim 6, wherein the enzyme is an alkaline phosphatase, acid phosphatase, aryl sulfatase, or guanidinobenzoatase.
Claim 22
Dependent Claim
13 words
A method, as claimed in claim 6, wherein the enzyme is a peptidase.
Claim 23
Dependent Claim
35 words
A method, as claimed in claim 6, for improving retention of a fluorescent reaction product formed by activity of an enzyme in a cell, the presence of which enzyme indicates expression of a marker gene.
Claim 24
Dependent Claim
102 words
A method, as claimed in claim 6, wherein BLOCK is a monovalent moiety derived by removal of a hydroxy group from phosphate or a biologically compatible salt thereof; or a monovalent moiety derived by removal of a hydroxy group from a mono- or polysaccharide; where BLOCK is selected to be removable from REPORTER by activity of an enzyme that indicates the presence of a marker gene; -REPORTER- is a coumarin or a fluorescein that is bound to BLOCK, such that REPORTER, when bound to BLOCK is virtually non-fluorescent and when BLOCK is removed, REPORTER becomes fluorescent; and XR-- is --ClCH 2 .
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-11-07 | 1998-05-09 | 1998-11-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-11-07 | 2002-05-09 | 2002-11-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-11-07 | 2006-05-09 | 2006-11-08 | $7700.00 | Window Closed / Lapsed |