US 5,362,628 AGranted

Fluorescent haloalkyl derivatives of reporter molecules well retained in cells

AssigneeMolecular Probes, Inc.

InventorsRichard P. Haugland, Yu-Zhong Zhang, Ram Sabnis, Nels A. Olson, John J. Naleway, Rosaria P. Haugland

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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.

Claims22

Dependent claims

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.

10 more elements

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.

10 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/026,633Filed 5 Mar 1993 · continuation
Priority: 23 Aug 1991
Granted: 8 Nov 1994
Term ends: Not estimatedThe source records don’t support an estimate.

Classification12 codes

C12Q 1/04
Involving viable microorganisms: determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides;…
C12Q 1/34
Measuring or testing processes involving enzymes, nucleic acids or microorganisms ; Compositions therefor; Processes of preparing such…
G01N 33/535
Production of labelled immunochemicals: with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
C12Q 2334/00
O-linked chromogens for determinations of hydrolase enzymes