Processes for the preparation of oligonucleotides

Abstract

The present invention discloses methods for synthesizing oligomeric compounds. The methods include a modified phosphoramidite protocol wherein the oxidation and capping steps are combined into a single step. The methods result in increased efficiency and are especially amenable to the large scale synthesis of oligomeric compounds.

1 . A method of preparing an oligomeric compound having at least one moiety of formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 X 2  is O or S;  
 X 1  is Pg—O—, Pg—S—, C 1 -C 10  straight or branched chain alkyl, CH 3 (CH 2 ) n —O—, R 2 R 3 N— or a group remaining from coupling a chiral auxiliary;  
 nn is from 0 to 10;  
 Pg is CH 3 , —CH 2 CH 2 CN, —C(CH 3 )(CH 3 )—CCl 3 , —CH 2 —CCl 3 , —CH 2 CH═CH 2 , CH 2 CH 2 SiCH 3 , 2-yl-ethyl phenylsulfonate, δ-cyanobutenyl, cyano p-xylyl, diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl, or a blocking group;  
 each R 2  and R 3  is, independently, hydrogen, C 1 -C 10  alkyl, cycloalkyl or aryl;  
 or optionally, R 2  and R 3 , together with the nitrogen atom to which they are attached form a cyclic moiety;  
 each Bx is, independently, a heterocyclic base moiety; and  
 each R 1  is, independently, H, a blocked hydroxyl group, or a sugar substituent group;  
 comprising the steps of:  
 (a) providing a 5′-O-protected compound of the formula:  
                     
 wherein:  
 T 1  is a hydroxylprotecting group; and  
 T 2  is a covalent attachment to a support media, a nucleoside bound to a support media, a nucleotide, an oligonucleoside or an oligonucleotide;  
 (b) treating said 5′-O-protected compound with a deprotecting reagent for a time and under conditions effective to form a 5′-O-deprotected compound;  
 (c) coupling said 5′-O-deprotected compound with an activated phosphorus composition of the formula:  
                     
 wherein:  
 T 3  is a hydroxylprotecting group, a nucleoside, a nucleotide, an oligonucleoside or an oligonucleotide;  
 R 4  is N(L 1 )L 2 ;  
 each L 1  and L 2  is, independently, C 1-6  straight or branched alkyl, or a C 5-7  cyclic aliphatic ring system;  
 or L 1  and L 2  are joined together to form a 4- to 13-membered heterocyclic ring system including the nitrogen atom to which L 1  and L 2  are attached; and  
 R 5  is X 1 ;  
 or R 4  and R 5  together with the phosphorus atom to which R 4  and R 5  are attached form a chiral auxiliary;  
 for a time and under conditions effective to form an extended compound having the formula:  
                     
 (d) treating said extended compound with a mixture comprising an oxidizing reagent and a capping reagent for a time and under conditions effective to form said oligomeric compound.  
 
     
     
         2 . The method of  claim 1  further comprising treating said oligomeric compound with a reagent for a time and under conditions effective to remove said blocking groups thereby forming a deblocked oligomeric compound.  
     
     
         3 . The method of  claim 2  wherein said reagent is effective to cleave the oligomeric compound from the support media.  
     
     
         4 . The method of  claim 3  wherein said reagent is aqueous ammonium hydroxide.  
     
     
         5 . The method of  claim 2  further comprising treating said oligomeric compound with a further reagent for a time and under conditions effective to cleave the oligomeric compound from the support media.  
     
     
         6 . The method of  claim 1  further comprising treating said oligomeric compound with a deprotecting reagent for a time and under conditions effective to deprotect the T 3  hydroxylprotecting group.  
     
     
         7 . The method of  claim 1  wherein said mixture comprises from 0.02M to 0.2M oxidizing reagent.  
     
     
         8 . The method of  claim 7  wherein said mixture comprises from 0.1M to 0.2M oxidizing reagent.  
     
     
         9 . The method of  claim 1  wherein said oxidizing reagent transfers an oxygen atom.  
     
     
         10 . The method of  claim 9  wherein said oxidizing reagent is iodine, m-chloroperbenzoic acid, iodobenzene diacetate, tetra-n-butylammonium periodate, tert-butyl hydroperoxide, di-tert-butyl hydroperoxide, cumene hydroperoxide, hydrogen peroxide; bis-trimethylsilyl peroxide; dinitrogen tetroxide, oxone, molecular oxygen, (1S)-(+)-(10-camphorsulfonyl)oxaziridine or a peracid.  
     
     
         11 . The method of  claim 10  wherein said oxidizing reagent is iodine, m-chloroperbenzoic acid, iodobenzene diacetate, tert-butyl hydroperoxide, di-tert-butyl hydroperoxide, hydrogen peroxide, oxone, molecular oxygen or a peracid.  
     
     
         12 . The method of  claim 1  wherein said oxidizing reagent transfers a sulfur atom.  
     
     
         13 . The method of  claim 12  wherein said oxidizing reagent is 3-amino-1,2,4-dithiazole-5-thione; 3-ethoxy-1,2,4-dithiazoline-5-one; 1,2,4-dithiazolidine-3,5-dione; 3-methyl-1,2,4-dithiazolin-5-one; or dimethylthiuram disulfide.  
     
     
         14 . The method of  claim 13  wherein said oxidizing reagent is dimethylthiuram disulfide.  
     
     
         15 . The method of  claim 1  wherein said capping reagent comprises about one part by volume of either acetic anhydride in acetonitrile or tetrahydrofuran; or chloroacetic anhydride in acetonitrile or tetrahydrofuran; added to about one part by volume of either N-methylimidazole and pyridine in acetonitrile or tetrahydrofuran; or t-butylphenoxyacetic anhydride in acetonitrile or tetrahydrofuran.  
     
     
         16 . The method of  claim 15  wherein said capping reagent comprises about one part by volume of 20% acetic anhydride in acetonitrile mixed with about one part by volume of a solution having 20% N-methylimidazole, 30% pyridine and 50% acetonitrile.  
     
     
         17 . The method of  claim 1  wherein said mixture comprises dimethylthiuram disulfide, acetic anhydride, acetonitrile, N-methyl imidazole and pyridine.  
     
     
         18 . The method of  claim 1  wherein said mixture comprises from about 0.05M to 0.2M dimethylthiuram disulfide, about 10% acetic anhydride, about 10% N-methyl imidazole and about 15% pyridine in a suitable solvent.  
     
     
         19 . The method of  claim 18  wherein said solvent is acetonitrile, toluene, ethyl acetate, tetrahydrofuran, dichloromethane, dichloroethane, dioxane, dimethylacetamide and dimethylformamide.  
     
     
         20 . The method of  claim 1  wherein said coupling of the 5′-O-deprotected compound with the activated phosphorus composition is performed in the presence of an activating agent.  
     
     
         21 - 78 . (canceled)