Quenched Fluorophores Conjugated to Peptides Via Linkers Containing Dithio Groups

Abstract

Disclosed are dithio compounds that include a quenched fluorophore and a non-fluorophore peptide linked via a dithio bond to the fluorophore. The dithio compounds may be used in methods for detecting thiol-containing compounds or dithio-containing compounds. The dithio compounds also may be used as cellular probes where the peptide portion of the compounds targets the compounds to a specific cellular location.

1 . A dithio compound having a formula F—S—S—P wherein F comprises a quenched fluorophore and P comprises a peptide. 
     
     
         2 . The dithio compound of  claim 1  having a formula: 
       
         
           
           
               
               
           
         
       
     
     
         3 . The dithio compound of  claim 1  having a formula: 
       
         
           
           
               
               
           
         
       
     
     
         4 . The dithio compound of  claim 1  having a formula: 
       
         
           
           
               
               
           
         
       
     
     
         5 . The dithio compound of  claim 1 , wherein the peptide comprises a signal peptide sequence. 
     
     
         6 . The dithio compound of  claim 1 , wherein the peptide comprises the arginine-rich RNA-binding motif (ARM) of the human immunodeficiency virus Tat peptide. 
     
     
         7 . The dithio compound of  claim 1 , wherein the peptide comprises an RGD sequence. 
     
     
         8 . The dithio compound of claim, wherein the peptide comprises a hormone ligand for a G-coupled cell surface receptor. 
     
     
         9 . The dithio compound of  claim 1 , wherein the peptide comprises an amino acid targeting sequence for a cellular nucleus. 
     
     
         10 . The dithio compound of  claim 1 , wherein the peptide comprises an amino acid targeting sequence for a cellular endoplasmic reticulum. 
     
     
         11 . The dithio compound of  claim 1 , wherein the peptide comprises an amino acid targeting sequence for a cellular mitochondria. 
     
     
         12 . The dithio compound of  claim 1  having a formula F—X 1 —S—S—X 2 —P, wherein at least one of X 1  and X 2  comprises an aryl group. 
     
     
         13 . The dithio compound of  claim 1  having a formula: 
       
         
           
           
               
               
           
         
       
       wherein R 1  comprises a fluorophore and R 2  comprises a peptide. 
     
     
         14 . The dithio compound of  claim 1  having a formula: 
       
         
           
           
               
               
           
         
       
       wherein R 1  comprises a fluorophore and R 2  comprises a peptide. 
     
     
         15 . A method for preparing a dithio compound having a formula F—S—S—P wherein F comprises a quenched fluorophore and P comprises a peptide, the method comprising reacting precursors that include: (A) a first precursor that comprises a fluorophore; (B) a second precursor that comprises a peptide; and (C) a dithio reagent having a formula X 1 —S—S—X 2 , wherein X 1  and X 2  may be the same or different and each comprise reactive groups capable of reacting with the first precursor and the second precursor. 
     
     
         16 . The method of  claim 15 , further comprising adding one or more cysteine residues to the N-terminus or C-terminus of the peptide in order to prepare the second precursor. 
     
     
         17 . A method for detecting a thiol- or dithio-containing compound, the method comprising: (A) reacting a dithio compound having a formula F—S—S—P wherein F comprises a quenched fluorophore and P comprises a peptide with the thiol- or dithio-containing compound to form at least one reaction product; and (B) detecting the at least one reaction product. 
     
     
         18 . The method of  claim 17 , wherein the thiol- or dithio-containing compound is a protein. 
     
     
         19 . A method of preparing a dithio compound having a formula F—S—S—P wherein F comprises a quenched fluorophore and P comprises a peptide, the method comprising reacting precursors that include: (A) a first precursor having a formula F—S—S—F, and (B) a second precursor having a formula P-S—S—P. 
     
     
         20 . A kit comprising: (a) a reagent having a formula F—S—S—F; and (b) a reagent having a formula P-S—S—P; wherein F is a fluorophore and P is an aliphatic peptide.