Synthesis of Oligonucleotides 155
can be removed by treatment with fluoride ion at the end of assembly (Section 4.2.2). TBDMS chemistry
is useful both on small and larger production scale (Figure 4.13a).^8
4.2.1.2.2 TOM. Triisopropylsilyloxymethyl (TOM) is a silyl-protected acetal, which has the advan-
tage that no 2↔ 3 migration occurs under the usual basic conditions of introduction.^9 The nucleobase
protecting groups are N-acetyl and the 5-hydroxyl group is protected with DMT. TOM chemistry may
have an advantage over TBDMS of slightly improved overall yields in RNA synthesis, but this view is not
universally held (Figure 4.13b).
4.2.1.2.3 ACE. The 2-O-[bis[2-(acetyloxy)ethoxy]methyl] (ACE) protecting group is entirely different
from the other two RNA chemistries in being a protected protecting group. It is a protected orthoester.^10
The nucleobases are protected by acyl groups (N^4 -acetyl-C, N^6 -benzoyl-A and N^2 -isopropyl-G), but at the
5 -position there is a cyclododecyloxy-bis(trimethylsiloxy)silyl (SIL) group rather than the usual acid-
labile DMT group (Figure 4.13c). The ACE protecting group becomes acid-labile once the two acetyl
groups of ACE have been removed (Section 4.2.2). The 3-phosphoramidite has a methyl protecting group
instead of the usual 2-cyanoethyl group, because the latter is unstable under the fluoride ion conditions
needed to remove the 5-SIL protecting group. ACE chemistry is currently only used for relatively small-
scale syntheses on a polystyrene support (glass or silica is not compatible with the 5-deprotection condi-
tions) but has become particularly popular for siRNA synthesis (see Section 5.7.2).
4.2.2 Oligoribonucleotide Synthesis
4.2.2.1 Assembly. The assembly cycle for each of the three different RNA chemistries follows a simi-
lar overall route to that for DNA assembly (Section 4.1.4) and involves
(i) Deprotection of the 5-protecting group. For TBDMS and TOM chemistry, this involves removal
of DMT groups with di- or trichloroacetic acid, but for ACE chemistry, deprotection of the 5-silyl
group (SIL) is accomplished with triethylamine.3HF.
(ii) Coupling of the 3-phosphoramidite to the free 5-hydroxyl group usingS-ethylthio tetrazole as
activator.
(iii) Capping of any unreacted 5-hydroxyl groups (acetic anhydride/2,6-lutidine and N-methylimida-
zole in MeCN, same reagents as for DNA assembly).
(iv) Oxidation with iodine/pyridine/water (same reagent as for DNA assembly).4.2.2.2 Deprotection and Purification. Deprotection and removal of oligonucleotides from the
solid support also uses procedures similar to those described for DNA synthesis (Section 4.1.4), but the
reagents and conditions depend on the choice of protection strategy.
4.2.2.2.1 TBDMS Chemistry. 5 -Deprotection of DMT groups is usually carried out first by acidic
treatment, while oligoribonucleotides are still attached to the support. Subsequent ammonia treatment then
results in cleavage of the linkage of the oligonucleotide to the solid support simultaneously with the
removal of nucleobase and phosphate (2-cyanoethyl) protecting groups. Mild methanolic ammonia (PAC
protection) or aqueous ammonia (dimethylaminomethylene protection) at room temperature is suitable.
Lastly, removal of the 2-TBDMS group is effected by treatment with 1 M tetrabutylammonium fluoride
(TBAF) in THF for 16–24 h or with triethylamine(3HF). Final purification of deprotected oligoribonu-
cleotides is carried out by polyacrylamide gel electrophoresis or by HPLC on ion exchange columns (Section
4.1.4). For ‘DMT-on’ purification, steps two and three are performed and the DMT-protected oligoribonu-
cleotide is purified by HPLC and stored. The DMT group is removed by mild acid treatment before use.
4.2.2.2.2 TOM Chemistry. 5 -Deprotection of DMT groups is the same as for the TBDMS route.
Cleavage from the solid support and removal of nucleobase protecting groups is effected with a 1:1 mix-
ture of 40% aqueous methylamine and 33% ethanolic methylamine at room temperature overnight or 6 h