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 .- 13 . (canceled) 
     
     
         14 . A reagent useful for labeling an oligonucleotide, comprising a label moiety that comprises an N-protected NH-rhodamine moiety, a phosphate ester precursor group, and a suitably protected synthesis handle, which is a compound according to structural formula (IX.1): 
       
         
           
           
               
               
           
         
         wherein LM represents the label moiety, —Z— represents a portion of a linkage contributed by a functional group F z , Sp 1 , Sp 2  and Sp 3 , which can be the same or different, each represent spacing moieties, G represents CH, N, or a group comprising an arylene, phenylene, heteroarylene, lower cycloalkylene, cyclohexylene, and/or a lower cycloheteroalkylene, R e  represents an acid-labile protecting group, and PEP represents the phosphate ester precursor group. 
       
     
     
         15 . The reagent of  claim 14  in which Sp 1 , Sp 2  and Sp 3  are each, independently of one another, selected from an alkylene chain containing from 1 to 10 carbon atoms, —(CH 2 ) a —[(Ar) b —(CH 2 ) a ] c —, —(CH 2 ) a —[C≡C—(CH 2 ) a ] c —, —(CH 2 ) a —[C≡C—(Ar) b ] c —(CH 2 ) a —, —(C H 2 ) d —NH—C(O)—[(CH 2 ) a —(Ar)—(CH 2 ) a —C(O)—NH] c —(CH 2 ) d — and —[CH 2 (CH 2 ) e O] f —CH 2 (CH 2 )—, where a, b, c, d, e, f and Ar are as defined in claim  4 . 
     
     
         16 . The reagent of  claim 14  which is a compound according to structural formula (IX.2), (IX.3), (IX.4) or (IX.5): 
       
         
           
           
               
               
           
         
         wherein 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. 
       
     
     
         17 . The reagent of  claim 16  in which the nucleobase is selected from adenine, 7-deazaguanine, guanine, 7-deazaguanine, cytosine, uracil, thymine, inosine, xanthene and hypoxanthene. 
     
     
         18 . The reagent of  claim 17  in which B is selected from A iBu , A Pac , C Ac , G iPr-Pac , T and U. 
     
     
         19 . The reagent of  claim 16  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 C H 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 Ar is as defined in claim  4 . 
     
     
         20 . The reagent of  claim 16  in which -B-L 2 - is selected from 
       
         
           
           
               
               
           
         
       
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . A reagent useful for labeling an oligonucleotide, comprising a label moiety that comprises an N-protected NH-rhodamine moiety, a solid support, and a suitably protected synthesis handle. 
     
     
         25 . The reagent of  claim 24 , which is a compound according to structural formula (X): 
       
         
           
           
               
               
           
         
         wherein LM represents the label moiety, L represents an optional selectively cleavable linker and R e  represents an acid-labile protecting group. 
       
     
     
         26 . The reagent of  claim 25  which is a compound according to structural formula (XI.1): 
       
         
           
           
               
               
           
         
         wherein Z— represents a portion of a linkage contributed by a functional group F z , Sp 1 , Sp 2  and Sp 3 , which can be the same or different, each represent spacing moieties, G represents CH, N, or a group comprising an arylene, phenylene, heteroarylene, lower cycloalkylene, cyclohexylene, and/or a lower cycloheteroalkylene, R e  represents an acid-labile protecting group, and PEP represents the phosphate ester precursor group, and Sp 4  represents a selectively cleavable spacing moiety. 
       
     
     
         27 .- 36 . (canceled) 
     
     
         37 . A reagent useful for labeling an oligonucleotide, comprising a label moiety that comprises an N-protected NH-rhodamine moiety, 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 aryl group of the formula —C(O)R 10 , where R 10  is selected from hydrogen, lower alkyl, methyl, —CX 3 , —CHX 2 , —CH 2 X, —CH 2 —OR d  and phenyl optionally mono-substituted with a lower alkyl, methyl, —X, —OR d , cyano or nitro group, where R d  is selected from lower alkyl, phenyl and pyridyl, and each X is a halo group, typically fluoro, or chloro; 
         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 intger ranging from 1 to 10 and R b  is selected from —X, —OH, —OR a , —SH, —SR a , —NH 2 , —NHR 1 , —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 , —O (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 − , —O P(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)N HR 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 substituted 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 . 
 
       
     
     
         38 .- 66 . (canceled)