3'-OH RNA-CONTAINING OLIGONUCLEOTIDE DEPROTECTION
The disclosure relates to a method of deprotecting a 3′-hydroxyl of an oligonucleotide having the 3′-hydroxyl protected by a silyl protecting group, comprising contacting the oligonucleotide with a mixture comprising DMSO and a deprotecting reagent. The disclosure also relates to a method of recovering a synthesized oligonucleotide from a solid support based on the method of deprotecting a 3′-hydroxyl of an oligonucleotide. The disclosure also relates to a 3′-hydroxyl protected oligonucleotide intermediate involved during the method of deprotecting the oligonucleotide.
1 . A 3′-hydroxyl protected oligonucleotide intermediate having the structure of formula (I):
A-[C] n -E (I), or a salt thereof,
wherein:
A is
C is
E is
provided that the oligonucleotide intermediate contains at least one (A-3′), (C-3′), or (E-3′);
n is 1-50;
B is a modified or unmodified nucleobase;
J is
or a phosphorous-containing group or moiety;
R 1 is H, a hydroxyl protecting group, or a phosphorous-containing group or moiety;
R 2 is H, halo, OR 5 , or NR 6 R 7 ;
R 3 is —(CH 2 ) m Si(R 4 ) 3 ;
each R 4 is independently optionally substituted alkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl;
m is 0, 1, 2, or 3;
R 5 , R 6 , and R 7 are each independently H or optionally substituted alkyl, alkoxyalkyl, aryl, aralkyl, alkaryl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl or cycloalkynyl; or R 6 and R 7 are linked to form a heterocyclyl; and
X and Y are each independently O or S.
2 . The oligonucleotide intermediate of claim 1 , wherein n is 16 to 23.
3 . The oligonucleotide intermediate of claim 1 , wherein A is A-3′.
4 . The oligonucleotide intermediate of claim 1 , wherein at least one C is C-3′.
5 . The oligonucleotide intermediate of claim 1 , wherein E is E-3′.
6 . The oligonucleotide intermediate of claim 1 , wherein m is 0 or 1, and each R 4 is isopropyl.
7 . The oligonucleotide intermediate of claim 1 , wherein R 2 is H, F, OCH 3 , or OCH 2 CH 2 OCH 3 .
8 . A method of deprotecting a 3′-hydroxyl of an oligonucleotide having the 3′-hydroxyl protected by a silyl protecting group, comprising:
contacting the oligonucleotide with a mixture comprising DMSO and a deprotecting reagent having a formula of (R) 4 N + F − , wherein each R is independently alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, under conditions sufficient for the deprotecting reagent to remove the silyl protecting group.
9 . The method of claim 8 , wherein the silyl protecting group is trialkylsilyl.
10 . The method of claim 8 , wherein the silyl protecting group is triisopropylsilyl (TIPS) or tert-butyldimethylsilyl (TBDMS).
11 . The method of claim 8 , wherein the deprotecting reagent is tetraethylammonium fluoride (TEAF) or tetra-n-butylammonium fluoride (TBAF).
12 . The method of claim 11 , wherein the deprotecting reagent is tetraethylammonium fluoride hydrate (TEAF⋅xH 2 O).
13 . The method of claim 11 , wherein the mixture is 0.9 M TEAF in aqueous DMSO solution.
14 . The method of claim 11 , wherein the contacting step is carried out at 60° C. or higher.
15 . The method of claim 11 , wherein the contacting step is carried out for 30 minutes or longer.
16 . The method of claim 15 , wherein the contacting step is carried out for 2 hours or longer.
17 . The method of claim 8 , further comprising, prior to removing the silyl protecting group:
contacting a nucleobase-protected oligonucleotide having a 3′-hydroxyl protected by a silyl protecting group bound to a solid support with a base under conditions suitable to cleave the oligonucleotide from the solid support and to remove amino protecting groups from the nucleobase-protected oligonucleotide, to provide the oligonucleotide having the 3′-hydroxyl protected by a silyl protecting group.
18 . The method of claim 17 , wherein the base is ammonia in a solvent.
19 . The method of claim 18 , wherein the amino protecting groups are exocyclic amino (nucleobase) protecting groups, and the base is a mixture of 30% ammonia in a solvent at a v/v ratio of no more than 3:1.
20 . The method of claim 17 , further comprising, prior to the base treatment step, contacting a fully-protected oligonucleotide having a 3′-hydroxyl protected by a silyl protecting group bound to a solid support with a base under conditions suitable to remove a phosphate protecting group of the oligonucleotide, to provide the nucleobase-protected oligonucleotide having a 3′-hydroxyl protected by a silyl protecting group.
21 . The method of claim 20 , wherein the base is a weak base.
22 . The method of claim 21 , wherein the weak base is diethyl amine (DEA).
23 . The method of claim 22 , wherein the phosphate protecting group is cyanoethyl, and the weak base is 20% DEA in acetonitrile.
24 . The method of claim 17 , further comprising the step of purifying the oligonucleotide via a chromatographic purification.