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 transfer from fluorescent reporter labels by any energy-transfer mechanism, such as FRET. Fluorescence quencher compositions are useful in preparing quencher labeled biomolecules for various molecular biology assays based on fluorescence detection.

1 - 54 . (canceled) 
     
     
         55 . A compound comprising:
 a quencher moiety comprising structure (Q1) linked to a reactive linking group, wherein Q1 comprises the following structure:   
       
         
           
           
               
               
           
         
       
       wherein:
 R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13  and R 14  are each, independently of one another, selected from hydrogen, an electron-withdrawing group and an electron-donating group, or, alternatively R 4  may be taken together with R 5  or R 6  may be taken together with R 7  to form a benzo group, 
 wherein: (i) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13  or R 14  is an electron-withdrawing group; and (ii) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13  or R 14  is an electron-donating group. 
 
     
     
         56 . The compound of  claim 55 , wherein the electron-withdrawing groups are each, independently of one another, selected from NO 2 , CN, CF 3 , CO 2 H, CO 2 R′, C(O)NH 2 , N(O)NHR′, C(O)NR′R′, CHO, C(O)R′, SO 2 R′, SO 2 CF 3 , SO 2 OR′, SO 3 H, NO and C 5 -C 14  aryl, wherein each R′ is independently H, C 1 -C 12  alkyl or C 5 -C 14  aryl. 
     
     
         57 . The compound of  claim 55 , wherein the electron-donating groups are each, independently of one another, selected from O − , S − , NR″R″, NHR″, NH 2 , NHC(O)R″, OR″, OH, OC(O)R″, SR″, SH, Br, I, Cl, F, R″ and C 5 -C 14  aryl, wherein each R″ is independently H, C 1 -C 12  alkyl or C 5 -C 14  aryl. 
     
     
         58 . The compound of  claim 55 , wherein the reactive linking group comprises an electrophile. 
     
     
         59 . The compound of  claim 58 , wherein the electrophile of the reactive linking group is a carboxylic acid or an ester. 
     
     
         60 . The compound of  claim 59 , wherein the reactive linking group linked to the quencher moiety Q1 via an NR″R″ group, where each R″ is independently C 1 -C 12  alkyl. 
     
     
         61 . The compound of  claim 60 , wherein one R″ is substituted C 1 -C 12  alkyl and the other R″ is unsubstituted C 1 -C 12  alkyl. 
     
     
         62 . The compound of  claim 61 , wherein the one R″ that is unsubstituted C 1 -C 12  alkyl is CH 2 CH 3 . 
     
     
         63 . The compound of  claim 61  which is selected from compounds 30, 31, 32, 34, 35, 36, 40, 41, 58, 59, 60, 63, 64, 70 and 71. 
     
     
         64 . The compound of  claim 58 , wherein the reactive linking group is linked to the quencher moiety Q1, either directly or by way of a linker moiety. 
     
     
         65 . The compound of  claim 58 , wherein the reactive linking group further comprises a protecting group. 
     
     
         66 . The compound of  claim 58  which has the structure: 
       
         
           
           
               
               
           
         
         wherein:
 L 1  and L 3  are each, independently of one another, 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; 
 L 2  is a C 1 -C 12  alkoxyldiyl; 
 X is an acid-labile protecting group; 
 Y is selected from N and CR, where R is selected from H, C 1 -C 6  alkyl and C 5 -C 14  aryl; and 
 Z is a reactive linking group. 
 
       
     
     
         67 . The compound of  claim 66 , wherein Z is a phosphoramidite of the formula —O—P(NR 21 R 22 )(OR 23 ) where R 21  and R 22  are each, independently of one another, selected from C 1 -C 12  alkyl, C 5 -C 14  aryl and a saturated ring comprising up to 10 ring atoms or, alternatively, R 21  and R 22  are taken together with the nitrogen atom to which they are bonded form a saturated ring comprising from 5 to 12 ring toms and R 23  is a phosphate ester protecting group. 
     
     
         68 . The compound of  claim 66 , wherein Z is a carboxylic acid or an ester. 
     
     
         69 . The compound of  claim 66 , wherein X is selected from DMT, MMT, trityl, substituted trityl, pixyl and trialkylsilyl. 
     
     
         70 . The compound of  claim 66 , wherein R 23  is selected from C 1 -C 6  alkyl, cyanoethyl, C 5 -C 14  aryl, phenyl and 2-(4-nitrophenyl)ethyl. 
     
     
         71 . The compound of  claim 66 , wherein R 21  and R 22  are each isopropyl and R 23  is cyanoethyl. 
     
     
         72 . A method of attaching a quencher moiety to a polynucleotide, comprising contacting the polynucleotide with a compound according to  claim 63  under conditions in which the reactive linking group forms a covalent attachment to a group on the polynucleotide. 
     
     
         73 . A method of attaching a quencher moiety to a polypeptide, comprising contacting the polypeptide with a compound according to  claim 63  under conditions in which the reactive linking group forms a covalent attachment to a group on the polypeptide.