Chemical modifications of monomers and oligonucleotides with cycloaddition

Abstract

The invention features compounds of formula I or II: In one embodiment, the invention relates compounds and processes for conjugating ligand to oligonucleotide. The invention further relates to methods for treating various disorders and diseases such as viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies and immunosuppression.

1 . An oligonucleotide comprising at least one subunit of formula (I) at one or more positions that occur at 1-6 nucleotides from either end of the oligonucleotide: 
       
         
           
           
               
               
           
         
         wherein: 
         X is O, S, NR N  or CR P   2 ; 
         B is hydrogen, optionally substituted natural or non-natural nucleobase, optionally substituted triazole, or optionally substituted tetrazole, NH—C(O)—O—C(CH 2 B 1 ) 3 , NH—C(O)—NH—C(CH 2 B 1 ) 3 ; where B 1  is independently halogen, mesylate, N 3 , CN, optionally substituted triazole or optionally substituted tetrazole, and where the nucleobase may further be substituted by -J-linker-N 3 , -J-linker-CN, -J-linker-cycloalkyne, -J-linker-R L , -Linker-Q-R L , or -J-linker-Q-linker-R L ; 
         R 1 , R 2 , R 3 , R 4  and R 5  are each independently H, OR 6 , F, N(R N ) 2 , N 3 , CN, -J-linker-N 3 , -J-linker-CN, -J-linker-C≡R 8 , -J-linker-cycloalkyne, -J-linker-R L , -Linker-Q-R L , or -J-linker-Q-linker-R L ; 
         J is absent, O, S, NR N , OC(O)NH, NHC(O)O, C(O)NH, NHC(O), NHSO, NHSO 2 , NHSO 2 NH, OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R P ) 2 )O, or OP(N(R P ) 2 ); 
         R 6  is hydrogen, hydroxyl protecting group, optionally substituted alkyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted alkenyl, optionally substituted heteroaryl, polyethyleneglycol (PEG), a phosphate, a diphosphate, a triphosphate, a phosphonate, a phosphonothioate, a phosphonodithioate, a phosphorothioate, a phosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, a phosphodiester, a phosphotriester, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z 1 )(Z 2 )—O-nucleoside, —P(Z 1 )(Z 2 )—O-oligonucleotide, —P(Z 1 )(Z 2 )-formula (I), —P(Z 1 )(O-linker-Q-linker-R L )—O-nucleoside, P(Z 1 )(O-linker-R L )—O-nucleoside, —P(Z 1 )(O-linker-N 3 )—O-nucleoside, P(Z′)(O-linker-CN)—O-nucleoside, P(Z 1 )(O-linker-C≡R 8 )—O-nucleoside, P(Z 1 )(O-linker-cycloalkyne)-O-nucleoside, —P(Z 1 )(O-linker-Q-linker-R L )—O-oligonucleotide, P(Z 1 )(O-linker-R L )—O-oligonucleotide, P(Z 1 )(O-linker-N 3 )—O-oligonucleotide, —P(Z 1 )(O-linker-CN)—O-oligonucleotide, P(Z 1 )(O-linker-C≡R 8 )—O-oligonucleotide, P(Z 1 )(O-linker-cycloalkyne)-O-oligonucleotide, —P(Z 1 )(-linker-Q-linker-R L )—O-nucleoside, —P(Z 1 )(-linker-Q-R L )—O-nucleoside, —P(Z 1 )(-linker-N 3 )—O-nucleoside, P(Z 1 )(-linker-CN)—O-nucleoside, P(Z 1 )(-linker-C≡R 8 )—O-nucleoside, P(Z 1 )(-linker-cycloalkyne)-O-nucleoside, —P(Z 1 )(-linker-Q-linker-R L )—O-oligonucleotide, —P(Z 1 )(-linker-R L )—O-oligonucleotide, P(Z 1 )(-linker-N 3 )—O-oligonucleotide, —P(Z 1 )(-linker-CN)—O-oligonucleotide, P(Z 1 )(-linker-C≡R 8 )—O-oligonucleotide or P(Z 1 )(-linker-cycloalkyne)-O-oligonucleotide; 
         R N  is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted heteroaryl, or an amino protecting group; 
         R P  is independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, or optionally substituted heteroaryl; 
         Q is 
       
       
         
           
           
               
               
           
         
         R L  is hydrogen or a ligand; 
         R 8  is N or CR 9 ; 
         R 9  is H, optionally substituted alkyl, or silyl; 
         Z 1  and Z 2  are each independently O, S, or optionally substituted alkyl; 
       
       provided that Q, —N 3 , —CN, —C≡R 8 , an optionally substituted triazole, or an optionally substituted tetrazole is present at least once in the compound. 
     
     
         2 . (canceled) 
     
     
         3 . The oligonucleotide of  claim 1 , comprising at least one subunit represented by formula (III): 
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , R 4 , R 5  and B are as previously defined in  claim 1 . 
     
     
         4 . The oligonucleotide of  claim 1 , comprising at least one subunit represented by formula (IV): 
       
         
           
           
               
               
           
         
       
       wherein R 10  and R 20  are each independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, or optionally substituted heteroaryl; and wherein R 1 , R 2 , R 3 , R 4 , R 5 , and X are as defined in  claim 1 . 
     
     
         5 . The oligonucleotide of  claim 1 , comprising at least one subunit represented by formula (V): 
       
         
           
           
               
               
           
         
       
       wherein B 1  is halogen, N 3 , CN, optionally substituted triazole, or optionally substituted tetrazole; R 1 , R 2 , R 3 , R 4 , and R 5  are as defined in  claim 1 . 
     
     
         6 . The oligonucleotide of  claim 1 , wherein the ligand is selected from the group consisting of thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, multivalent N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent N-acetyl glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, mannose, lactose, galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, and an RGD peptide. 
     
     
         7 . (canceled) 
     
     
         8 . The oligonucleotide of  claim 1 , wherein the linker is represented by structure
   —[P-Q 1 -R] q -T-,
   
       wherein:
 P, R and T are each independently absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH, CH 2 O; NHCH(R a )C(O), —C(O)—CH(R a )—NH—, —C(O)-(optionally substituted alkyl)-NH—, CH═N—O, 
 
       
         
           
           
               
               
           
         
         Q 1  is absent, —(CH 2 ) n —, —C(R 100 )(R 200 )(CH 2 ) n —, —(CH 2 ) n C(R 100 )(R 200 )—, —(CH 2 CH 2 O) m CH 2 CH 2 —, or —(CH 2 CH 2 O) m CH 2 CH 2 NH—; 
         R a  is H or an amino acid side chain; 
         R 100  and R 200  are each independently H, CH 3 , OH, SH or N(R X ) 2 ; 
         R X  is independently H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; 
         q is an integer from 0-20; 
         n is an integer from 1-20; and 
         m is an integer from 0-50; 
       
       provided that at least one of P, R, T, and Q 1  is present in the linker. 
     
     
         9 - 11 . (canceled) 
     
     
         12 . The oligonucleotide of  claim 1 , wherein the oligonucleotide is a single-stranded oligonucleotide. 
     
     
         13 . The oligonucleotide of  claim 12 , wherein the single-stranded oligonucleotide is a single-stranded siRNA. 
     
     
         14 . The oligonucleotide of  claim 12 , wherein the single-stranded oligonucleotide is a microRNA. 
     
     
         15 . The oligonucleotide of  claim 1 , wherein the oligonucleotide is a double-stranded oligonucleotide. 
     
     
         16 . The oligonucleotide of  claim 15 , wherein the double-stranded oligonucleotide is a double-stranded siRNA. 
     
     
         17 - 20 . (canceled) 
     
     
         21 . A pharmaceutical composition comprising the oligonucleotide of  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         22 - 24 . (canceled) 
     
     
         25 . The oligonucleotide of  claim 1 , wherein R 1  and R 4  are H. 
     
     
         26 . The oligonucleotide of  claim 1 , wherein R 5  is OR 6 . 
     
     
         27 . The oligonucleotide of  claim 1 , wherein R 3  is OR 6 ; and R 2  is —O-Linker-Q-Linker-R L , —OC(O)N(H)-Linker-Q-Linker-R L , or -Linker-Q-Linker-R L . 
     
     
         28 . The oligonucleotide of  claim 27 , wherein R 2  is 
       
         
           
           
               
               
           
         
       
       and f is 1-20. 
     
     
         29 . The oligonucleotide of  claim 28 , wherein R 2  is 
       
         
           
           
               
               
           
         
       
     
     
         30 . The oligonucleotide of  claim 6 , wherein the ligand is mannose, lactose, galactose, N-acetyl-galactosamine, N-acetyl glucosamine, multivalent lactose, multivalent galactose, multivalent mannose, multivalent fucose, multivalent N-acetyl-galactosamine, or multivalent N-acetyl glucosamine. 
     
     
         31 . The oligonucleotide of  claim 1 , wherein at least two subunits of formula I is incorporated at positions that occur at 1-6 nucleotides from either end of the oligonucleotide. 
     
     
         32 . The oligonucleotide of  claim 1 , wherein at least three subunits of formula I is incorporated at positions that occur at 1-6 nucleotides from either end of the oligonucleotide.