Reagents useful for synthesizing rhodamine-labeled oligonucleotides

Abstract

The present disclosure provides reagents that can be used to label synthetic oligonucleotides with rhodamine dyes or dye networks that contain rhodamine dyes.

1 - 66 . (canceled) 
     
     
         67 . An oligonucleotide comprising a label moiety that comprises an N-protected NH-rhodamine moiety. 
     
     
         68 . The oligonucleotide of  claim 67  in which the N-protected NH-rhodamine moiety comprises a structure selected from: 
       
         
           
           
               
               
           
         
         wherein LM represents a label moiety, PEP represents the phosphate ester precursor group, B represents a suitably protected nucleobase, L 2  represents a linker linking label moiety LM to nucleobase B and, in structure (IX.4), R 11  represents a protecting group. 
       
     
     
         69 . The oligonucleotide of  claim 68  in which L 2  is selected from —C≡C—CH 2 —NH—, —C≡C—C(O)—, —CH═CH—NH—, —CH═CH—C(O)—, —C≡C—CH 2 —NH—C(O)—(CH 2 ) 1-6 —NH—, —CH═CH—C(O)—NH—(CH 2 ) 1-6 —NH—C(O)—, —C≡CH—CH 2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH—, —C≡C—C≡C—CH 2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH—, —C≡C—(Ar) 1-2 —C≡C—CH 2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH—, —C≡C—(Ar) 1-2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH— and —C≡C—(Ar) 1-2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH—, where each Ar represents, independently of the others, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene or heteroarylene group. 
     
     
         69 . The oligonucleotide of  claim 68  in which —B-L 2 - is selected from 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         70 . The oligonucleotide of  claim 68  in which the N-protected NH-rhodamine moiety comprises a structure selected from structural formulae (IIIc), (IIIb) and (IIIc): 
       
         
           
           
               
               
           
         
         wherein:
 R′ is selected from R 3′  and hydrogen; 
 R″ is selected from R 6′  and hydrogen; 
 R 9  is an acyl protecting group; 
 R 1′ , R 2′ , R 2″ , R 4′ , R 4″ , R 5′ , R 5″ , R 7′ , R 7″ , R 8′ , R 4 , R 5 , R 6 , and R 7 , when taken alone, are each, independently of one another, selected from hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, —R b  and —(CH 2 ) x —R b , where x is an integer ranging from 1 to 10 and R b  is selected from —X, —OH, —OR a , —SH, —SR a , —NH 2 , —NHR a , —NR c R c , —N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, —B(OH) 3 , —B(OR a ) 3 , —B(OH)O − , —B(OR a ) 2 O − , —B(OH)(O − ) 2 , —B(OR a )(O − ) 2 , —P(OH) 2 , —P(OH)O − , —P(OR a ) 2 , —P(OR a )O − , —P(O)(OH) 2 , —P(O)(OH)O − , —P(O)(O − ) 2 , —P(O)(OR a ) 2 , —P(O)(OR a )O − , —P(O)(OH)(OR a ), —OP(OH) 2 , —OP(OH)O − , —OP(OR a ) 2 , —OP(OR a )O − , —OP(O)(OH) 2 , —OP(O)(OH)O − , —OP(O)(O − ) 2 , −OP(O)(OR a ) 2 , —OP(O)(OR a )O − , —OP(O)(OR a )(OH), —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R a , —C(O)H, —C(O)R a , —C(S)X, —C(O)O − , —C(O)OH, —C(O)NH 2 , —C(O)NHR a , —C(O)NR c R c , —C(S)NH 2 , —C(O)NHR a , —C(O)NR c R c , —C(NH)NH 2 , —C(NH)NHR a , and —C(NH)NR c R c , where X is halo, each R a  is, independently of the others, selected from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, and each R c  is, independently of the others, an R a , or, alternatively, two R c  bonded to the same nitrogen atom may be taken together with that nitrogen atom to form a 5- to 8-membered saturated or unsaturated ring that may optionally include one or more of the same or different ring heteroatoms, which are typically selected from O, N and S,
 or, alternatively, R 1′  and R 2′  or R 7′  and R 8′  are taken together with the carbon atoms to which they are bonded to form an optionally substituted (C6-C14) aryl bridge and/or R 4′  and R 4″  and/or R 5′  and R 5″  are taken together with the carbon atoms to which they are bonded to form a benzo group; and 
 
 R 3′  and R 6′ , when taken alone, are each, independently of one another, selected from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, or alternatively, R 3′  and R 2′  or R 4′  and/or R 6′  and R 5′  or R 7′  in the compounds of structural formula (IIIa), R 3′  and R 2′  or R 4′  and/or R 6′  and R 5′  or R 7″  in the compounds of structural formula (III6), or R 3′  and R 2″  or R 4′  and/or R 6′  and R 5′  or R 7″  in the compounds of structural formula (IIIc) are taken together with the atoms to which they are bonded to form a 5- or 6-membered saturated or unsaturated ring which is optionally sutstituted with one or more of the same or different lower alkyl, benzo or pyrido groups, 
 
         with the proviso that at least one of R 2′ , R 4′ , R 5′ , R 7′ , R 5  or R 6  in the compounds structural formula (IIIa), at least one of R 2′ , R 4′ , R 5′ , R 7″ , R 5  or R 6  in the compounds of structural formula (IIIb) and at least one of R 2″ , R 4′ , R 5′ , R 7″ , R 5  or R 6  in the compounds of structural formula (IIIc) comprises a group of the formula —Y—, where Y represents a portion of a linkage contributed by a functional group F y . 
       
     
     
         71 . The oligonucleotide of  claim 70 , in which the N-protected NH-rhodamine moiety comprises a structure selected from structural formulae (IIIa.1), (IIIa.2), (IIIb.1), (IIIb.2), (IIIc.1) and (IIIc.2): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein R′, R″, R 1′ , R 2′ , R 2″ , R 3′ , R 4′ , R 4″ , R 5′ , R 5″ , R 6′ , R 7′ , R 7″ , R 4 , R 5 , R 6 , R 7 , R 8′ , R 9  and Y are as previously defined in  claim 70 . 
       
     
     
         72 . The oligonucleotide of  claim 70  in which the N-protected NH-rhodamine moiety has one or more applicable features selected from:
 (i) Y is selected from —C(O)—, —S(O) 2 —, —S— and —NH—; 
 (ii) R 4  and R 7  are each chloro; 
 (iii) R 1′  and R 8′  are each hydrogen; 
 (iv) R 1′  and R 2′  or R 7′  and R 8′  are taken together to form a benzo group; 
 (v) R 2′  and R 7′  are each hydrogen or lower alkyl; 
 (vi) R′ is R 3′  and R″ is R 6′ ; and 
 (vii) R′ is R 3′ , R″ is R 6′ , and R 3′  and R 6′  are taken together with a substituent group on an adjacent carbon atom to form a group selected from —CH 2 CH 2 —, —CH 2 CH 2 CH 2 —, —C(CH 3 ) 2 CH═C(CH 3 )—, —C(CH 3 ) 2 CH═CH—, —CH 2 —C(CH 3 ) 2 — and 
 
       
         
           
           
               
               
           
         
       
     
     
         73 . The oligonucleotide of  claim 67  in which the label moiety is linked to the 3′- or 5′-hydroxyl of the oligonucleotide. 
     
     
         74 . The oligonucleotide of  claim 67  in which the label moiety is linked to a nucleobase of the oligonucleotide. 
     
     
         75 . The oligonucleotide of  claim 67  in which the oligonucleotide is further labeled with a donor and/or acceptor moiety for the N-protected NH-rhodamine moiety. 
     
     
         76 . The oligonucleotide of  claim 75  in which the label moiety comprises structural formula (VI):
   A-Z 1 -Sp-Z 2 -D   (VI)
 
 wherein A represents the N-protected NH-rhodamine moiety, D represents the donor moitey, Z 1  and Z 2 , which may be the same or different, represent portions of linkages provided by linking moieties comprising a functional group F z , and Sp represents a spacing moiety. 
 
     
     
         77 . The oligonucleotide of  claim 76  in which A is selected from structural formulae A.1, A.2, A.3, A.4, A.5 and A.6 and D is selected from structural formulae D.1, D.2, D.3, D.4, D.5 and D.6, or A is selected from structural formulae A.7, A.8, A.9, A.10, A.11 and A.12 and D is selected from structural formulae D.7, D.8, D.9, D.10, D.11 and D.12; 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein:
 E 1  is selected from —NHR 9 , —NR 3′ R 9  and —OR 9b ; 
 E 2  is selected from —NHR 9 , —NR 6′ R 9  and —OR 9b ; 
 R 9b  is R 9 ; 
 Y 1a , Y 1b , Y 2a , Y 2b , Y 3a , and Y 3b  are each, independently of one another, selected from —O—, —S—, —NH—, —C(O—) and —S(O) 2 —; and 
 R′, R″, R 1′ , R 2″ , R 3′ , R 4′ , R 4″ , R 5′ , R 5″ , R6′, R 7′ , R 7″ , R 8′ , R 4 , R 5 , R 6 , R 7 , and R 9  are as previously defined in claim  34 , with the proviso that when E 1  and E 2  are —OR 9b , then R 1′  and R 2′  and/or R 7′  and R 8′  may only be taken together with the carbon atoms to which they are bound to form an optionally substituted (C6-C14) aryl bridge. 
 
       
     
     
         78 . The oligonucleotide of  claim 67  in which the oligonucleotide is further labeled with a quencher moiety. 
     
     
         79 . The oligonucleotide of  claim 67  in which the oligonucleotide is further labeled with a minor groove binding moiety.