Method of synthesizing phosphorothioate oligonucleotides

Abstract

This invention presents novel methods for synthesizing phosphorothioate oligonucleotides, using support-bound phosphoramidites. Novel intermediates useful in the methods are also provided.

What is claimed is:  
     
         1 . A method for synthesizing a phosphorothioate oligonucleotides on a solid phase support comprising the steps of: 
 reacting a phosphordiamidite of formula:                          wherein:    R 1  is a protecting group;    R 2  and R 3  are dialkylamino or morpholino;    B is a nucleosidic base;    R 6  is halogen, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, or a polyether of the formula (O-alkyl) m , where m is 1 to about 10;    with a support-bound synthon of formula:                          wherein:    R 4  is a linker connected to a solid support;    R 5  is a phosphoryl protecting group;    n is 0 to 100;    to form a support-bound phosphoramidite of formula:                          protecting the support-bound phosphoramidite, thereby forming a support-bound phosphite of formula:                          sulfurizing the support-bound phosphite to form a protected phosphorothioate; and    deprotecting the protected phosphorothioate to form a further support-bound synthon wherein n is increased by 1.    
     
     
         2 . The method of  claim 1  wherein the the support-bound phosphoramidite is protected by reaction with an alcohol of formula R 5 —OH, where R 5  is a phosphoryl protecting group.  
     
     
         3 . The method of  claim 1  wherein at least one of R 2  and R 3  is diisopropylamino.  
     
     
         4 . The method of  claim 1  wherein R 2  and R 3  are diisopropylamino.  
     
     
         5 . The method of  claim 1  wherein R 5  is 2-cyanoethyl.  
     
     
         6 . The method of  claim 1  wherein R 5  is 4-cyano-2-butenyl or diphenylmethylsilylethyl.  
     
     
         7 . The method of  claim 1  wherein the reaction of the phosphite compound with the support-bound synthon is preformed in the presence of an organic base.  
     
     
         8 . The method of  claim 7  wherein the organic base is a tetrazole.  
     
     
         9 . The method of  claim 1  wherein the sulfurization reagent is Beaucage reagent, tetraethylthiuram disulfide, dibenzoyl tetrasulfide, phenacetyl disilfide, 1,2,4-dithiuazoline-5-one, 3-ethoxy-1,2,4-dithiuazoline-5-one, a disulfide of a sulfonic acid, sulfur, or sulfur in combination with a ligand.  
     
     
         10 . The method of  claim 1  wherein the sulfurization reagent is Beaucage reagent, tetraethylthiuram disulfide, dibenzoyl tetrasulfide, phenacetyl disilfide, 1,2,4-dithiuazoline-5-one, or 3-ethoxy-1,2,4-dithiuazoline-5-one.  
     
     
         11 . The method of claims  1  wherein the nucleosidic base is adenine, guanine, cytosine, thymine, or uracil.  
     
     
         12 . The method of  claim 1  wherein n is from 10 to about 30 nucleotide units.  
     
     
         13 . The method of  claim 1  wherein n is from 15 to about 25 nucleotide units.  
     
     
         14 . A compound of formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 B is a nucleosidic base;  
 R 1  is a protecting group;  
 R 2  is dialkylamino or morpholino;  
 R 4  is a linker connected to a solid support;  
 R 5  is a phosphoryl protecting group;  
 R 6  is halogen, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkylimidazole, or a polyether of the formula (O-alkyl) m , where m is 1 to about 10; and  
 n is 1 to about 100.  
 
     
     
         15 . The compound of  claim 14  wherein R 2  is diisopropylamino or morpholino.  
     
     
         16 . The compound of  claim 15  wherein the phosphoryl protecting group is cyanoethyl.  
     
     
         17 . The compound of  claim 14  wherein the phosphoryl protecting group is 4-cyano-2-butenyl or diphenylmethylsilylethyl.  
     
     
         18 . The compound of  claim 14  wherein n is 1 to 30.  
     
     
         19 . The compound of  claim 14  wherein n is from 15 to 25.