Non-fluorescent quencher compounds and biomolecular assays

Abstract

Bis-diazo,triaryl and aryldiazo-N-arylphenazonium quencher moieties, substituted with electron-withdrawing and electron-donating substituents which induce polarity in the delocalized aryl/diazo ring systems, are useful as labels when attached to biomolecules such as polynucleotides, nucleosides, nucleotides, and polypeptides. The quencher moieties are non-fluorescent and accept energy from fluorescent reporter labels by any energy-transfer mechanism, such as FRET. Fluorescence quencher compositions are useful in preparing quencher labelled biomolecules for various molecular biology assays based on fluorescence detection.

We claim:  
     
         1 . A fluorescence quencher composition having the structure:  
       
         
           
           
               
               
           
         
         wherein Y is selected from N and CR, where R is H, C 1 -C 6  alkyl or C 5 -C 14  aryl;  
         L 1 , L 2 , and L 3  are independently selected from a bond, C 1 -C 12  alkyldiyl, C 1 -C 12  alkoxyldiyl, C 1 -C 12  alkylaminodiyl, C 1 -C 12  alkylamidediyl, C 5 -C 14  aryldiyl, and 1-20 ethyleneoxy units;  
         X is an amino acid, a polypeptide, a nucleoside, a nucleotide, a polynucleotide, or a protected form thereof; or X is an acid-labile protecting group;  
         Z is selected from H, CO 2 H, OH, NH 2 , NHR, NR 2 , SH, an ester, a cleavable linker, a solid support, a reactive linking group, and a label selected from a fluorescent dye, a hybridization-stabilizing moiety, a chemiluminescent dye, and an affinity ligand; and  
         Q is selected from the diazo structures:  
         
           
             
             
                 
                 
             
           
           wherein Ar is C 5 -C 14  aryl; one of the aryl carbons of the diazo structures is the site of attachment to L 1 ; at least one aryl carbon of each diazo structure is substituted with an electron-withdrawing group and at least one aryl carbon of each diazo structure is substituted with an electron-donating group.