Long wavelength fluorogenic intracellular ion indicators that are well retained in the cytosol

Abstract

Cell permeable metal ion indicator compounds and methods of their use and synthesis are described. The compound comprises a metal chelating moiety (M c ), a reporter molecule and two or more lipophilic groups (G L ) covalently bonded through a linker to the reporter molecule, wherein the lipophilic groups, when present in a live cell, are cleaved resulting in two or more negatively charged groups.

1 .- 45 . (canceled) 
     
     
         46 . A method for identifying intracellular metal ions in a sample, comprising:
 providing a sample comprising live cells;   contacting the sample with a intracellular metal ion indicator compound, wherein the compound comprises a metal ion chelating moiety, a reporter molecule and one or more lipophilic groups covalently bonded to the reporter molecule, wherein the lipophilic groups, when present in a live cell, are cleaved resulting in one or more negatively charged groups;   incubating the sample for a period of time sufficient for the compound to enter the cell;   cleaving the one or more lipophilic groups; and   illuminating the sample with an appropriate wavelength to generate an optical signal, whereby the intracellular metal ions are identified.   
     
     
         47 . The method of  claim 46 , wherein the metal ion is thallium, rubidium or lithium. 
     
     
         48 . The method of  claim 46 , wherein the metal ion is a surrogate for a physiologically relevant ion selected from a calcium, magnesium, zinc and potassium ion. 
     
     
         49 . The method of  claim 46 , wherein the reporter molecule is a fluorescent dye. 
     
     
         50 . The method of  claim 49 , wherein the fluorescent dye is selected from the group consisting of dansyl, xanthene, cyanine, borapolyazaindacene, pyrene, naphthalene, coumarin, oxazine and derivatives thereof. 
     
     
         51 . The method of  claim 49 , wherein the fluorescent dye is a xanthene or a borapolyazaindacene. 
     
     
         52 . The method of  claim 46 , wherein the metal ion chelating moiety is selected from the group consisting of BAPTA, crown ether, cryptan, APTRA, FluoZin-1, FluoZin-2, FluoZin-3 or phenanthroline chelating moieties. 
     
     
         53 . The method of  claim 46 , wherein the thallium ion chelating moiety is BAPTA or APTRA. 
     
     
         54 . The method of  claim 46 , wherein the thallium ion chelating moiety is FluoZin-1 or FluoZin-2. 
     
     
         55 . The method of  claim 46 , wherein the reporter molecule is a xanthene and the metal ion chelating moiety is FluoZin-1 or FluoZin-2. 
     
     
         56 . The method of  claim 46 , wherein the lipophilic groups comprise an ester. 
     
     
         57 . The method of  claim 46 , wherein the lipophilic groups are selected from the group consisting of C 1 -C 6 carboxyalkyl, —(CH 2 ) 16 COOCR 15   2 OC(═O)(CH 2 ) 0-4 CH 3 , and alpha-acyloxyalkyl; wherein R 15  is H, alkyl or substituted alkyl. 
     
     
         58 . The compound of  claim 46 , wherein the one or more lipophilic groups is —CH 2 CH 2 COOCH 2 OC(—O)CH 3 . 
     
     
         59 . The method of  claim 46 , wherein illuminating the sample with an appropriate wavelength results in change in the optical signal of the reporter molecule. 
     
     
         60 . The method of  claim 46 , further comprising incubating the sample with the compound for a time sufficient for the compound to bind the intracellular metal ion in the cell.