Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 163

ribose conformation (see Section 2.1.1). In contrast to RNA, there is a considerable increase in the stability
of the oligonucleotide towards exonuclease degradation. Thus, 2
-O-methyl modifications are particularly
useful in the 3
- and 5
-flanking regions of oligonucleotide gapmers and in steric block applications
(see Section 5.7.1), with or without additional phosphorothioate modifications. The 2
-O-methoxyethyl
(MOE) modification has similar uses.
2
-Deoxy-2
-fluoro--D-ribofuranosides can be considered close analogues of the natural -D-ribose found
in RNA since the sugar favours a C3
-endopucker and A-type conformation when hybridised with RNA.
However, such 2
-fluoro-containing oligonucleotides are not substrates for RNase H in duplexes with RNA.
However, 2
-fluoronucleotides are one of a number of analogues being explored for use in synthetic siRNA
(see Section 5.7.2) and in aptamer applications (see Section 5.7.3). By contrast, the 2
-deoxy-2
-fluoro--D-
arabinonucleoside oligomers (2
-F-ANA, Figure 4.24) are substrates to direct cleavage by RNase H.^36
Since modifications at the 2
-position are generally very well tolerated in oligonucleotide duplexes, the
2
-position has been widely used to attach a large variety of substituents, such as fluorophores, into
oligonucleotides, either using the 2
-hydroxyl group or viaa 2
-amino-2
-deoxy modification.

4.4.3.7 Locked Nucleic Acids (LNA). LNAs, also known as BNA (Figure 4.24), were first

described by Takeshi Imanishi^37 and Jesper Wengel,^38 LNA has a methylene bridge between the 2
-oxygen
and the C4
-carbon, which results in a locked 3
-endosugar conformation, reduced conformational flexi-
bility of the ribose ring and an increase in the local organisation of the phosphate backbone. The entropic
constraint in LNA results in significantly stronger binding of LNA to complementary DNA and RNA.
LNA-modified oligonucleotides have considerably enhanced resistance to nuclease degradation and they
have proven to be effective in antisense strategies when used in flanking regions of gapmers or in steric
block applications (see Section 5.7.1).

4.4.3.8 Peptide Nucleic Acids (PNA). PNAs were first introduced by Peter Nielsen and have normal

nucleobases attached to a peptide-like backbone that is built from 2-aminoethylglycine units (Figure 4.25).
As a result, PNA is electrically neutral but has excellent natural DNA and RNA recognition properties.39,40
PNA is synthesised by sequential solid phase synthesissimilar to the methods employed in peptide
synthesis and using protected PNA building blocks. In one system, the PNA unit has an acid-labile
t-butyloxycarbonyl (tBoc) group for N-protection and an active ester activation of the carboxylic group.
Additional benzyloxycarbonyl protecting groups are removed at the end of PNA assembly by treatment
with HF. In a second method, involving milder chemistry, a 9-fluorenylmethoxycarbonyl (Fmoc) amino
protecting groupis removed by treatment with 20% piperidine/DMF while nucleobase protection uses a

O

OMe

HO Base

HO

O

HO Base

HO

F

O

HO Base

OH

O

HO Base

HO O

O

O

HO Base

HO

2'-O-Methyl nucleoside 2'-Fluoro-arabinonucleoside (2'-F-ANA)

Locked nucleic acid (LNA) DNA C2'-endo LNA C3'-endo

Figure 4.24 Structures of sugar modifications: 2
-O-methyl, 2
-O-F-ANA and locked nucleic acids (LNA). The
conformation of the LNA sugar is compared to that of DNA