Haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells

Abstract

The subject invention provides substrates useful 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 substrates have the form XR-SPACER-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 properties different from those of the substrate, -SPACER- is a covalent linkage, 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.

1. A substrate having the formula:
   XR-SPACER-REPORTER-BLOCK  
 
       wherein BLOCK is a monovalent moiety derived by removal of a hydroxy group from phosphate or from sulfate, or biologically compatible salt thereof; or a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an aromatic carboxylic acid or of an amino acid, protected amino acid, peptide, or protected peptide; or a monovalent moiety derived by removal of a hydroxy group from an alcohol or from a mono- or polysaccharide, where said BLOCK is selected to be removable from -REPORTER by action of an enzyme; -REPORTER- is an anthracene, a benzphenalenone, an acridinone , a coumarin , a fluorescein, a naphthalene, a phenalenone, a pyrene, a resorufin, a rhodol, or a rhodamine, that when no longer bound to BLOCK by a BLOCK-REPORTER bond, is fluorescent; -SPACER- is a single covalent bond, or a covalent linkage having 0-8 carbon atoms and 1-4 heteroatoms, wherein said heteroatoms, which may be the same or different, are selected from the group consisting of O, N and S; and XR— is a haloalkyl moiety, where X is a single Cl, I, or Br, and R is an alkyl linkage having 1-4 carbons. 
     
     
       2. A substrate, as claimed in  claim 1 , wherein -SPACER-XR is one of —(C═O)—XR, —(SO 2 )—XR, XR—C 6 H 4 —(C═O)—, or XR—C 6 H 4 —(SO 2 )—. 
     
     
       3. A substrate, as claimed in  claim 1 , 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. 
     
     
       4. A substrate, as claimed in  claim 1 , 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. 
     
     
       5. A substrate, as claimed in  claim 1 , 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. 
     
     
       6. A substrate, as claimed in  claim 1 , that is an ether of either a fluorescein or a rhodol and a lower alcohol having 6 or fewer carbon atoms, or a steriodal steroidal alcohol, or of a mono- or polysaccharide, and XR— is XCH 2 —. 
     
     
       7. A substrate, as claimed in  claim 6 , where the monosaccharide is β-D-galactose, β-D-glucose or β-D-glucuronic acid and XR— is ClCH 2 —. 
     
     
       8. A substrate, as claimed in claim  1   25 , that is an ether of a 7-hydroxycoumarin and a mono- or polysaccharide, or a lower alcohol having 6 or fewer  2 - 6  carbon atoms, or a steroidal alcohol, and XR- is XCH 2 —. 
     
     
       9. A substrate, as claimed in  claim 8 , where the monosaccharide is β-D-galactose, β-D-glucose or β-D-glucuronic acid and XR— is ClCH 2 —. 
     
     
       10. A substrate, as claimed in  claim 1 , that is an ether of a fluorescein and a mono- or polysaccharide, or a lower alcohol having 6 or fewer carbon atoms, and XR— is XCH 2 —. 
     
     
       11. A substrate, as claimed in  claim 10 , where the monosaccharide is β-D-galactose, β-D-glucose or β-D-glucuronic acid and XR— is CHCl 2 —. 
     
     
       12. A substrate, as claimed in  claim 1 , that is an amide of a 7-aminocoumarin and an amino acid or peptide or a biologically compatible salt of an amino acid or peptide and XR— is XCH 2 —. 
     
     
       13. A substrate, as claimed in  claim 1 , that is an amide of rhodamine 110 and an amino acid, protected amino acid, or peptide, or a biologically compatible salt of an amino acid or peptide, and XR— is XCH 2 —. 
     
     
       14. A substrate, as claimed in  claim 1 , that is an amide of rhodamine 110 and an amino acid, protected ammo acid or peptide or biologically compatible salt of an amino acid, protected amino acid or peptide, and -SPACER- is a carboxamide or sulfonamide bound to the second rhodamine nitrogen. 
     
     
       15. A substrate, as claimed in  claim 1 , having the formula 
                 
 
     
     
       16. A substrate, as claimed in  claim 1 , having the formula 
                 
 
     
     
       17. A substrate, as claimed in  claim 16 , wherein XR-SPACER- is one of XR—(C═O)—, XR—(SO 2 )—, XR—C 6 H 4 —(C═O), or XR—C 6 H 4 —(SO 2 )—. 
     
     
       18. A substrate, as claimed in  claim 1 , having the formula 
                 
 
     
     
       19. A substrate, as claimed in  claim 1 , having the formula 
                 
 
     
     
       20. A substrate, as claimed in claim  1   25 , having the formula 
                 
 
     
     
       21. A substrate, as claimed in claim  1   25 , having the formula 
                 
 
     
     
       22. A fluorescent enzymatic reaction producing product having the formula 
                 
 where REPORTER is an anthracene, a benzphenalenone, an acridinone, a coumarin, a fluorescein, a naphthalene, phenalenone, a pyrene, a resorufin, a rhodol, or a rhodamine;  
 -SPACER- is a single covalent bond, or a covalent linkage having 0-8 carbon atoms and 1-4 heteroatoms, wherein said heteroatoms, which may be the same or different, are selected from the group consisting of O, N and S; and  
 R is an alkyl linker having 1-4 carbon atoms;  
 wherein, in the presence of reduced glutathione, said enzymatic reaction product is the result of the action of an analyte enzyme on the substrate 
   XR-SPACER-REPORTER-BLOCK  
 
 wherein BLOCK is a monovalent moiety derived by removal of a hydroxy group from phosphate or from sulfate, or a biologically compatible salt thereof; or a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an aromatic carboxylic acid or of an amino acid or of a peptide; or a monovalent moiety derived by removal of a hydroxy group from an alcohol or from a mono- or polysaccharide, where said BLOCK is selected to be removable from -REPORTER by action of an analyte enzyme; and X is a single Cl, I, or Br.  
 
     
     
       23. A fluorescent enzymatic reaction product, as claimed in  claim 22 , wherein said analyte enzyme is present intracellularly. 
     
     
       24. A fluorescent enzymatic reaction product, as claimed in  claim 22 , where REPORTER is a coumarin, a fluorescein, a resorufin, a rhodol, or a rhodamine. 
     
     
       25. A substrate having the formula:
   XR-SPACER-REPORTER-BLOCK    
       wherein BLOCK is a monovalent moiety derived by removal of a hydroxy group from phosphate or from sulfate, or a biologically compatible salt thereof; or a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an aromatic carboxylic acid or of an amino acid, protected amino acid, peptide, or protected peptide; or a monovalent moiety derived by removal of a hydroxy group from an alcohol having  2 - 6  carbons, or from a steroidal alcohol, or from a mono-or polysaccharide, where said BLOCK is selected to be removable from-REPORTER by action of an enzyme; -REPORTER-is a coumarin, that when no longer bound to BLOCK by a BLOCK-REPORTER bond, is fluorescent; -SPACER-is a single covalent bond, or a covalent linkage having  0 - 8  carbon atoms and  1 - 4  heteroatoms, wherein said heteroatoms, which may be the same or different, are selected from the group consisting of O, N and S; and XR— is a haloalkyl moiety, where X is a single Cl, I, or Br, and R is an alkyl linkage having  1 - 4  carbons.