Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 145

nucleoside attached to a solid support. Since the 5
-hydroxyl group is a more effective nucleophile than
the secondary 3
-hydroxyl group, the phosphate/phosphite group is best placed on the 3
-position. To achieve
this selectively it is necessary to protect the nucleobase exocyclic amino groups and the 5
-hydroxyl group.

4.1.2.1 Nucleobases. Permanent protecting groups for the exocyclic amino groups of adenine, cyto-

sine and guanine have been used for many years in oligonucleotide synthesis.^1 Acyl protecting groups were
chosen, since they are stable for long periods during mildly basic and acidic conditions used during
oligonucleotide synthesis, and are removed with concentrated ammonia at the end of the synthesis
(Section 4.1.4). The benzoyl group is used to protect both adenine and cytosine, while isobutyryl is used
to protect guanine (Figure 4.2). Thymidine does not require protection since it does not have an exocyclic
amino group. While these acyl protecting groups are still suitable for oligonucleotide synthesis today, new
chemistries and new nucleoside building blocks have been introduced, which require milder deprotection
conditions at the end of the synthesis. For example, a matched set of phenoxyacetyl (PAC) for dA,
isopropylphenoxyacetyl for dG and acetyl for dC can be removed by treatment with 0.05 M potassium car-
bonate in methanol at room temperature within a few hours.
When nucleosides are prepared for incorporation into oligonucleotides, it is usual to protect nucleobase
exocyclic amino groups first. There are two common methods for the synthesis of acylated nucleosides,
per-acylationand transient protection(Figure 4.3). The per-acylation method involves use of an excess of
acylating agent such that the hydroxyl groups and the exocyclic amino groups are each acylated (bis-acylated
in the case of the amino groups), and then the hydroxylic and one of the amino acyl groups are removed
selectively under mild basic conditions. The selectivity arises because of the greater stability of amides
compared to esters (and bis-amides) at high pH. In the transient protection route, the nucleoside is treated
with trimethylsilyl chloride (TMSCl), which reacts selectively with the hydroxyl groups. Treatment with
benzoyl chloride is then selective for the exocyclic amino group (again the bis-acylated product may
be formed). The silyl protecting groups are removed under basic conditions to give the desired N^6 -
benzoyl-2
-deoxyadenosine. This method may also be used for protection of 2
-deoxycytidine.
In the case of 2
-deoxyguanosine protection, the reaction may be carried out with isobutyric anhydride by
either per-acylation or the transient protection route. However, in the case of dG, the O^6 -position is susceptible
to reaction under certain conditions, particularly with coupling agents and phosphorylating agents, or in
the synthesis of G-rich oligonucleotides. Under these conditions it is necessary to protect the O^6 -position
using alkyl or aryl protecting groups. However, such protection is not necessary in the case of the phos-
phoramidite method (Section 4.1.3). Another common protecting group for dG is the dimethylformami-
dine group that is readily introduced using dimethylformamide dimethylacetal.

4.1.2.2 5
-Hydroxyl Group. By far the most common protecting group for the 5
-hydroxyl group is the

4,4
-dimethoxytrityl group (DMT) (Figure 4.4). The DMT group is readily removed under acidic conditions.
It is introduced onto the 5
-hydroxyl group of N-acylated nucleosides with DMT–Cl in the presence of a base
such as pyridine or 4-dimethylaminopyridine. Reaction occurs principally at the 5
-hydroxyl rather than at
the secondary 3
-hydroxyl group because of steric effects. The DMT group is removed during oligonucleotide

O

HO

HO N

N

O

HN

O

Ph

O

HO

HO

N

NH

N

N

O

HN

O

O

HO

HO

N

N

N

N

HN

O

Ph

dABz dGiB dCBz

Figure 4.2 Common protecting groups for the heterocyclic bases of dA, dG and dC