Reversible modification of nucleotides

Abstract

Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3′-O-oxime moiety such aswith a reagent having an —ONH2 moiety to produce a nucleotide having a 3′-O—NH2 moiety such aswherein the reagent having the —ONH2 moiety further comprises alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, or (heteroalicyclyl)alkyl.

1 .- 30 . (canceled) 
     
     
         31 . A method for modifying a nucleotide, comprising reacting a nucleotide comprising a 3′-O-oxime moiety with a reagent to produce a nucleotide comprising a 3′-O—NH 2  moiety,
 wherein the nucleotide that comprises the 3′-O-oxime moiety is: 
 
       
         
           
           
               
               
           
         
         wherein the nucleotide that comprises the 3′-O—NH 2  moiety is: 
       
       
         
           
           
               
               
           
         
         wherein B is a nucleobase; R1 is halogen, OCH 3 , H or OH; Q is monophosphate, diphosphate, or triphosphate; and R3 and R4 are H, CH 3 , alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, or (heteroalicyclyl)alkyl, wherein either R3 or R4, or both R3 and R4 are not H; and; 
         wherein the reagent is an alkoxyamine of formula R2-ONH 2 , 
         wherein R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted variant thereof, 
         wherein R2 has a molecular weight greater than 36 g/mol. 
       
     
     
         32 . The method of  claim 31 , wherein R3 is CH 3 . 
     
     
         33 . The method of  claim 31 , wherein R4 is CH 3 . 
     
     
         34 . The method of  claim 31 , wherein Q is triphosphate. 
     
     
         35 . The method of  claim 31 , wherein the reagent is: 
       
         
           
           
               
               
           
         
       
     
     
         36 . The method of  claim 35 , wherein the reagent is: 
       
         
           
           
               
               
           
         
       
     
     
         37 . The method of  claim 35 , wherein the reagent is: 
       
         
           
           
               
               
           
         
       
     
     
         38 . The method of  claim 31 , wherein R2 has a molecular weight greater than 57 g/mol. 
     
     
         39 . The method of  claim 38 , wherein R2 has a molecular weight greater than 100 g/mol. 
     
     
         40 . The method of  claim 38 , wherein R2 has a molecular weight greater than 200 g/mol. 
     
     
         41 . The method of  claim 31 , wherein R2 comprises a hydrocarbon ring. 
     
     
         42 . The method of  claim 31 , wherein R2 comprises a heterocyclic ring. 
     
     
         43 . The method of  claim 31 , wherein no more than 5% of the nucleotide is degraded. 
     
     
         44 . The method of  claim 31 , wherein no more than 3% of the nucleotide is degraded. 
     
     
         45 . The method of  claim 31 , wherein no more than 2% or no more than 1% of the nucleotide is degraded. 
     
     
         46 . The method of  claim 31 , wherein no more than 3% of the nucleotide is degraded when the nucleotide comprises a cytosine nucleobase. 
     
     
         47 . The method of  claim 31 , wherein the degradation of the nucleotide is less than half the degradation that would be obtained if R2 was a methyl group. 
     
     
         48 . The method of  claim 31  or  43 , wherein at least 95% of the nucleotide comprising a 3′-O-oxime moiety is consumed. 
     
     
         49 . The method of  claim 31 , wherein at least 97% or at least 98% of the nucleotide comprising a 3′-O-oxime moiety is consumed. 
     
     
         50 . The method of  claim 49 , wherein the method adds a blocking group in place of a 3′ protecting group to at least 98% of a population of nucleotides having a 3′ protecting group in no more than 3 hours. 
     
     
         51 . The method of  claim 31 , wherein R2-ONH 2  has a formula selected from the group consisting of the following: