REVERSIBLE MODIFICATION OF NUCLEOTIDES
Disclosed herein, inter alia, are methods for modifying a nucleotide, for example including reacting a nucleotide having a 3′-O-oxime moiety such as with a reagent having an —ONH 2 moiety to produce a nucleotide having a 3′-O—NH 2 moiety such as wherein the reagent having the —ONH 2 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: