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Fluorescent haloalkyl derivatives of reporter molecules well retained in cells

  • US 5,362,628 A
  • Filed: 03/05/1993
  • Issued: 11/08/1994
  • Est. Priority Date: 08/23/1991
  • Status: Expired due to Term
First Claim
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1. 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;

    
    
    space="preserve" listing-type="equation">XR-REPORTER-BLOCKwherein 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.

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