Ionic tags for synthesis of oligoribonucleotides

Abstract

The invention relates to the chemical synthesis of oligonucleotides, e.g., oligoribonucleotides. In another aspect, the invention relates to compounds of formula (II):processes for making these compounds, and the use thereof in the chemical synthesis of oligonucleotides, e.g., oligoribonucleotides. The invention also relates to methods of synthesis of oligomers, including but not limited to oligopeptides, oligosaccharides and oligonucleotides, particularly oligoribonucleotides and also oligodeoxyribonucleotides, in solution systems, and ionic tag linkers for use in methods provided herein.

What is claimed is: 
     
         1 . A compound of formula (II): 
       
         
           
           
               
               
           
         
         wherein: 
         n is an integer from 1 to 19; 
         R 1  is a protecting group; 
         R 3  is selected from H, a protecting group, 
       
       
         
           
           
               
               
           
         
         and an ionic tag linker; 
         R 5  is selected from H, and a protecting group; 
         R p  is a protecting group; 
         R is lower alkyl, or the N(R) 2  moiety is a cyclic alkylamine or a substituted cyclic alkylamine, preferably morpholine; and 
         B is a nitrogen-containing base; 
         wherein each B, R 1  and R p  may be the same or different from any other B, R 1  and R p , respectively. 
       
     
     
         2 . The compound of  claim 1 , wherein the ionic linker comprises:
 a photolabile moiety, an ionic moiety and a linker, wherein the photolabile moiety is a nitrobenzyl derivative, and wherein the ionic tag linker has the structure of formula (P):   
       
         
           
           
               
               
           
         
         wherein X is N or O, and wherein C3′ is the terminal 3′-hydroxyl of an oligoribonucleotide or oligodeoxiribonucleotide. 
       
     
     
         3 . The compound of  claim 1 , wherein:
 R 3  is   
       
         
           
           
               
               
           
         
         R 5  is selected from DMTr or MMTr; 
         R p  is selected from methyl (Me), 2-cyanoethyl (CNEt), ortho-chlorophenyl (o-ClPh), and para-chlorophenyl (p-ClPh); 
         R is selected from isopropyl, methyl, and ethyl; and 
         B is a nucleobase protected on at least one nitrogen by a suitable N-protecting group. 
       
     
     
         4 . The compound of  claim 3 , wherein the N-protecting group is selected from levulinyl, acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, N,N-diphenyl carbamate and an ionic tag linker. 
     
     
         5 . The compound of  claim 1 , wherein R 3  is 
       
         
           
           
               
               
           
         
       
       or a protecting group. 
     
     
         6 . The compound of  claim 1 , wherein the protecting group is a levulinyl group (Lev) or an ionic tag linker. 
     
     
         7 . The compound of  claim 6 , wherein the ionic tag linker comprises:
 a photolabile moiety, an ionic moiety and a linker, wherein the photolabile moiety is a nitrobenzyl derivative, and wherein the ionic tag linker has the structure of formula (P):   
       
         
           
           
               
               
           
         
         wherein X is N or O, and wherein C3′ is the terminal 3′-hydroxyl of an oligoribonucleotide or oligodeoxiribonucleotide. 
       
     
     
         8 . A process for preparing the compound of  claim 1 , wherein the compound has the structure: 
       
         
           
           
               
               
           
         
         wherein: 
         n is selected from 1, 2, and 3; 
         R 1  is a protecting group; 
         R 3  is selected from H, a protecting group and an ionic tag linker; 
         R 5  is a protecting group; 
         R p  is a protecting group; and 
         B is a nitrogen-containing base; 
         wherein each B, R 1  and R p  may be the same or different from any other B, R 1  and R p , respectively; 
         the process comprising the steps of:
 (a) condensing a phosphoramidite of formula (III): 
 
       
       
         
           
           
               
               
           
         
         
           
             wherein B, R 1 , R 5 , and R p  are as defined above; and 
             R is lower alkyl, or the N(R) 2  moiety is a cyclic alkylamine, or a substituted cyclic alkylamine, preferably morpholine; 
           
           with a nucleoside of formula (IV): 
         
       
       
         
           
           
               
               
           
         
         
           
             wherein B and R 3  are as defined above; and 
           
           (b) oxidizing the product of step (a) to produce the compound of formula (II) where n is 1, and B, R 1 , R 3 , R 5 , and R p  are as defined above; and 
           (c) where n>1, the process further comprising:
 (i) deprotecting the terminal —OR 5  group of the product of the previous step to form a free 5′-OH group; 
 (ii) condensing the product of step (i) with a phosphoramidite of formula (III), wherein B, R 1 , R 5 , R p  and R are as defined above, and each B, R 1 , R 5 , R p  and R may be the same or different from any other B, R 1 , R 5 , R p  and R, respectively; 
 (iii) oxidizing the product of step (ii); and 
 (iv) repeating steps (i)-(iii) n−2 times; 
 
         
         to form the compound of formula (II). 
       
     
     
         9 . The process of  claim 8 , wherein R 3  is H. 
     
     
         10 . The process of  claim 8 , wherein R 3  is a protecting group, the process further comprising:
 (d) removal of the R 3  protecting group.   
     
     
         11 . The process of  claim 10 , wherein the protecting group is a levulinyl (Lev) group or an ionic linker. 
     
     
         12 . The process of  claim 11 , wherein the ionic tag linker comprises:
 a photolabile moiety, an ionic moiety and a linker, wherein the photolabile moiety is a nitrobenzyl derivative, and wherein the ionic tag linker has the structure of formula (P):   
       
         
           
           
               
               
           
         
         wherein X is N or O, and wherein C3′ is the terminal 3′-hydroxyl of an oligoribonucleotide or oligodeoxiribonucleotide. 
       
     
     
         13 . The process of  claim 8 , further comprising phosphitylation of the product of steps (b) or (c) to form a compound of formula (IIa): 
       
         
           
           
               
               
           
         
         wherein n, B, R 1 , R 5 , R p  and R are as previously defined. 
       
     
     
         14 . The process of  claim 8 , further comprising phosphitylation of the product of step (d) to form a compound of formula (IIa): 
       
         
           
           
               
               
           
         
         wherein n, B, R 1 , R 5 , R p  and R are as previously defined. 
       
     
     
         15 . The process of  claim 8 , wherein:
 R 1  is TBDMS;   R 5  is selected from DMTr and MMTr;   R p  is selected from methyl (Me), 2-cyanoethyl (CNEt), ortho-chlorophenyl (o-ClPh), and para-chlorophenyl (p-ClPh);   R is selected from isopropyl, methyl, and ethyl; and   B is a nucleobase protected on at least one nitrogen by a suitable N-protecting group, wherein the N-protecting group is selected from levulinyl, acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, and N,N-diphenyl carbamate.   
     
     
         16 . A method for synthesizing an oligoribonucleotide or an oligodeoxyribonucleotide, the method comprising:
 (a) attaching an ionic tag linker to a first ribonucleoside or deoxyribonucleoside at the terminal 3′-hydroxyl;   (b) contacting the first ribonucleoside or deoxyribonucleoside with at least one further ribonucleoside or deoxyribonucleoside at reaction conditions to provide an oligoribonucleotide or a deoxyribonucleotide comprising from 2 to 30 ribonucleosides or deoxyribonucleosides; and   (c) cleaving the ionic tag linker from the oligoribonucleotide or oligodeoxyribonucleotide to provide the free oligoribonucleotide or oligodeoxyribonucleotide.   
     
     
         17 . The method of  claim 16 , further comprising a step of isolating the oligoribonucleotide or the oligodeoxyribonucleotide before cleaving the ionic tag linker in step (c). 
     
     
         18 . The method of  claim 16 , wherein the ionic tag linker comprises:
 a photolabile moiety, an ionic moiety and a linker, wherein the photolabile moiety is a nitrobenzyl derivative, and wherein the ionic tag linker has the structure of formula (P):   
       
         
           
           
               
               
           
         
         wherein X is N or O, and wherein C3′ is the terminal 3′-hydroxyl of an oligoribonucleotide or oligodeoxiribonucleotide. 
       
     
     
         19 . The method of  claim 16 , wherein the oligoribonucleotide or the oligodeoxyribonucleotide is isolated by precipitation. 
     
     
         20 . The method of  claim 19 , wherein the precipitation is based on the ionic properties of the ionic tag linker.