Protected monomer and method of final deprotection for rna synthesis

Abstract

A nucleoside monomer that is protected by a thionocarbamate protecting group is provided, as well as a method for making a polynucleotide that uses the same. Also provided is a polynucleotide synthesis method that employs a diamine to deprotect a protected polynucleotide.

1 . A compound of the structure: 
       
         
           
           
               
               
           
         
       
       wherein:
 B P  is a protected or unprotected heterocycle; 
 R 1  and R 2  are each independently selected from hydrogen, a protecting group, and a group comprising a phosphorus; and 
 PG is a thionocarbamate protecting group. 
 
     
     
         2 . The compound of  claim 1  wherein:
 one of R 1  and R 2  is selected from a phosphoramidite group and a H-phosphonate group; and   one of R 1  and R 2  is a protecting group.   
     
     
         3 . The compound of  claim 2 , wherein said thionocarbamate protecting group (PG) is selected from one of the structures: 
       
         
           
           
               
               
           
         
         wherein R 3 , R 4  and R 5  are independently selected from a hydrocarbyl, a substituted hydrocarbyl, an aryl, and a substituted aryl, and wherein optionally R 4  and R 5  can be cyclically linked. 
       
     
     
         4 . The compound of  claim 2 , wherein said thionocarbamate protecting group (PG) is selected from one of the structures: 
       
         
           
           
               
               
           
         
       
     
     
         5 . The compound of  claim 2 , wherein said thionocarbamate protecting group (PG) is of the structure: 
       
         
           
           
               
               
           
         
       
     
     
         6 . The compound of  claim 5  wherein, R 1  is DMT, R 2  is beta-cyanoethyl-N,N-diisopropylphosphoramidite and B P  is selected from the group consisting of U, N 6 -benzoyl-A, N 6 -isobutyryl-A, N 6 —(N,N)-dimethylacetamidine-A, N 6 —(N,N)-dibutylformamidine-A, N 6 -phenoxyacetyl-A, N 6 -4-tert-butylphenoxyacetyl-A, N 4 -acetyl-C, N 4 -isobutyryl-C, N 4 -phenoxyacetyl-C, N 4 -4-tert-butylphenoxyacetyl-C, N 2 -isobutyryl-G, N 2 —(N,N)-dibutylformamidine-G, N 2 —(N,N)-dimethylformamidine-G, N 2 -phenoxyacetyl-G and N 2 -4-tert-butylphenoxyacetyl-G. 
     
     
         7 . A method of synthesizing a polynucleotide comprising at least one ribonucleotide residue, said method comprising:
 contacting a nucleotide residue or a nucleoside monomer having an unprotected hydroxyl group with;   a compound of  claim 2  under conditions sufficient to covalently bond said compound to said nucleotide residue or said nucleoside monomer and produce said polynucleotide.   
     
     
         8 . The method of  claim 7  further comprising:
 contacting said polynucleotide with a composition comprising a sulfurization agent to produce an oxidized polynucleotide.   
     
     
         9 . The method of  claim 7  wherein said nucleotide residue or said nucleoside monomer is bound directly or indirectly to a solid support. 
     
     
         10 . The method of  claim 9  wherein said solid support is selected from a CPG support and a polystyrene support. 
     
     
         11 . The method of  claim 9  wherein said solid support is selected from a bead and an array substrate. 
     
     
         12 . The method according to  claim 7 , further comprising:
 cleaving said polynucleotide from a solid support to produce a free polynucleotide.   
     
     
         13 . The method according to  claim 12  wherein said free polynucleotide is retained on said solid support. 
     
     
         14 . The method according to  claim 12 , further comprising:
 chemically modifying said free polynucleotide to produce a modified polynucleotide.   
     
     
         15 . A polynucleotide product produced by the method of  claim 7 . 
     
     
         16 . A polynucleotide product produced by the method of  claim 8 . 
     
     
         17 . A polynucleotide comprising:
 a ribonucleotide residue comprising the structure:   
       
         
           
           
               
               
           
         
       
       wherein:
 B P  is a protected or unprotected heterocycle; and 
 R 12  is selected from hydrogen, a hydrocarbyl, a substituted hydrocarbyl, an aryl, and a substituted aryl; and 
 X is O or S; and 
 PG is a thionocarbamate protecting group. 
 
     
     
         18 . The polynucleotide of  claim 17  wherein said thionocarbamate protecting group (PG) is selected from one of the structures: 
       
         
           
           
               
               
           
         
       
       wherein:
 wherein R 3 , R 4  and R 5  are independently selected from a hydrocarbyl, a substituted hydrocarbyl, an aryl, and a substituted aryl, and wherein optionally R 4  and R 5  can be cyclically linked. 
 
     
     
         19 . The polynucleotide of  claim 17 , wherein said thionocarbamate protecting group (PG) is selected from one of the structures: 
       
         
           
           
               
               
           
         
       
     
     
         20 . The polynucleotide of  claim 17 , wherein said thionocarbamate protecting group (PG) is of the structure: 
       
         
           
           
               
               
           
         
       
       and
 B P  is selected from the group consisting of U, N 6 -benzoyl-A, N 6 -isobutyryl-A, N 6 —(N,N)-dimethylacetamidine-A, N 6 —(N,N)-dibutylformamidine-A, N 6 -phenoxyacetyl-A, N 6 -4-tert-butylphenoxyacetyl-A, N 4 -acetyl-C, N 4 -isobutyryl-C, N 4 -phenoxyacetyl-C, N 4 -4-tert-butylphenoxyacetyl-C, N 2 -isobutyryl-G, N 2 —(N,N)-dibutylformamidine-G, N 2 —(N,N)-dimethylformamidine-G, N 2 -phenoxyacetyl-G and N 2 -4-tert-butylphenoxyacetyl-G; and 
 R 12  is selected from beta-cyanoethyl, and methyl; and 
 X is O or S. 
 
     
     
         21 . A method of deprotecting a solid support bound polynucleotide comprising at least one 2′-protected ribonucleotide residue, wherein said residue is not a 2′-ester protected ribonucleotide, said method comprising:
 contacting said polynucleotide with a composition comprising a diamine under conditions sufficient to deprotect said 2′-protected ribonucleotide residue.   
     
     
         22 . The method of  claim 21  wherein said 2′-protected ribonucleotide residue comprises the structure: 
       
         
           
           
               
               
           
         
       
       wherein:
 B P  is a protected or unprotected heterocycle; and 
 R 12  is a protecting group selected from a hydrocarbyl, a substituted hydrocarbyl, an aryl, and a substituted aryl; and 
 X is O or S; and 
 PG is a thionocarbamate protecting group. 
 
     
     
         23 . The method of  claim 22  wherein said thionocarbamate protecting group (PG) is selected from one of the structures: 
       
         
           
           
               
               
           
         
         wherein R 3 , R 4  and R 5  are independently selected from a hydrocarbyl, a substituted hydrocarbyl, an aryl, and a substituted aryl, and wherein optionally R 4  and R 5  can be cyclically linked. 
       
     
     
         24 . The method of  claim 22  wherein said thionocarbamate protecting group (PG) is selected from one of the structures: 
       
         
           
           
               
               
           
         
       
     
     
         25 . The method of  claim 22 , wherein said thionocarbamate protecting group (PG) is of the structure: 
       
         
           
           
               
               
           
         
       
       and
 B P  is selected from the group consisting of U, N 6 -benzoyl-A, N 6 -isobutyryl-A, N 6 —(N,N)-dimethylacetamidine-A, N 6 —(N,N)-dibutylformamidine-A, N 6 -phenoxyacetyl-A, N 6 -4-tert-butylphenoxyacetyl-A, N 4 -acetyl-C, N 4 -isobutyryl-C, N 4 -phenoxyacetyl-C, N 4 -4-tert-butylphenoxyacetyl-C, N 2 -isobutyryl-G, N 2 —(N,N)-dibutylformamidine-G, N 2 —(N,N)-dimethylformamidine-G, N 2 -phenoxyacetyl-G and N 2 -4-tert-butylphenoxyacetyl-G; and 
 R 12  is selected from beta-cyanoethyl, and methyl; and 
 X is O or S. 
 
     
     
         26 . The method of  claim 21  wherein said diamine reagent comprises two primary amino groups connected by a linker of about 2 to 12 atoms in length. 
     
     
         27 . The method of  claim 26  wherein said linker is of about 2 to 6 atoms in length. 
     
     
         28 . The method of  claim 21  wherein said diamine is selected from 1,2-diaminoethane, 1,2-diaminopropane, 1,3-diaminopropane, 1,4-diaminobutane, 2,2′-diaminodiethylamine, and substituted versions thereof. 
     
     
         29 . The method of  claim 21  wherein said diamine is 1,2-diaminoethane. 
     
     
         30 . The method of  claim 21  wherein said composition comprises at least 50% by volume 1,2-diaminoethane. 
     
     
         31 . The method of  claim 21  wherein said composition comprises 1,2-diaminoethane and a solvent. 
     
     
         32 . A method comprising:
 (a) contacting a solid support bound polynucleotide comprising:   a ribonucleotide residue comprising a 2′-protecting group, a phosphate protecting group, and optionally a nucleobase protecting group;   with a first composition comprising a phosphate deprotection reagent, to remove said phosphate protecting group and produce a first deprotected polynucleotide that remains bound to said solid support;   (b) contacting said first deprotected polynucleotide with a second composition comprising a diamine to remove said 2′-protecting group and removing the nucleobase protecting group, if present, to produce a second deprotected polynucleotide; and   (c) or (d) wherein:   (c) comprises simultaneously cleaving said second deprotected polynucleotide from said solid support; and   (d) comprises contacting said second deprotected polynucleotide with a third composition comprising a linker cleaving reagent to cleave said second deprotected polynucleotide from said solid support,   to produce a deprotected, cleaved polynucleotide.   
     
     
         33 . The method of  claim 32  wherein:
 said phosphate protecting group is a 2-cyanoethyl group or a methyl group.   
     
     
         34 . The method of  claim 33  wherein:
 said phosphate deprotection reagent is selected from diethylamine, t-butylamine, diaza(1,3)bicyclo[5.4.0]undecane (DBU), thiophenol and disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate; and   wherein said diamine is 1,2-diaminoethane.   
     
     
         35 . A method of deprotecting a polynucleotide comprising a nucleobase protecting group; and a ribonucleotide residue comprising a 2′-protecting group selected from tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM) and 2′-O-bis(2-acetoxyethoxy)methyl (ACE); said method comprising:
 (a) contacting said polynucleotide with a first composition comprising a 2′-deprotection reagent, under conditions sufficient to remove said 2′-protecting group and produce a first deprotected polynucleotide;   (b) contacting said first deprotected polynucleotide with a second composition comprising a diamine, under conditions sufficient to remove said nucleobase protecting group and produce a fully deprotected polynucleotide.   
     
     
         36 . A method of deprotecting a solid support bound polynucleotide comprising a phosphate protecting group, a nucleobase protecting group; and a ribonucleotide residue comprising 2′-protecting group; said method comprising:
 (a) contacting said polynucleotide with a first composition comprising a phosphate deprotection reagent, under conditions sufficient to remove said phosphate protecting group and produce a first deprotected polynucleotide;   (b) contacting said first deprotected polynucleotide with a second composition comprising a 2′-deprotection reagent under conditions sufficient to remove said 2′-protecting group and produce a second deprotected polynucleotide;   (c) contacting said second deprotected polynucleotide with a third composition comprising a diamine, under conditions sufficient to remove said nucleobase protecting group and produce a fully deprotected polynucleotide.   
     
     
         37 . The method of  claim 36  wherein said 2′-protecting group is selected from tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM) and 2′-O-bis(2-acetoxyethoxy)methyl (ACE). 
     
     
         38 . A method of deprotecting a solid support bound polynucleotide comprising a nucleobase protecting group and a ribonucleotide residue comprising a thionocarbamate protecting group, said method comprising:
 (a) contacting said polynucleotide with a composition comprising a diamine, under conditions sufficient to remove said protecting groups and cleave said polynucleotide from said solid support, and produce a cleaved polynucleotide; wherein said cleaved polynucleotide is retained on the solid support;   (b) washing the solid support and said cleaved polynucleotide; and   (c) eluting said cleaved polynucleotide from said solid support.   
     
     
         39 . A polynucleotide produced by the method of  claim 21 . 
     
     
         40 . A kit for deprotecting a polynucleotide comprising a 2′ protected ribonucleotide residue, said kit comprising:
 a composition comprising 1,2-diaminoethane, or a derivative thereof.