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PNAs: from birth to adulthood
repetitive and easy protocols used, the synthesis can be implemented on an automated
synthesizer for peptide chemistry, making these molecules more affordable 34 .
Many efforts have been done for finding alternatives to the standard synthetic strategies, in
order to make PNA synthesis cheaper, or to use milder conditions for the linking of acidsensitive
groups. This led to the development of other protecting groups: dithiasuccinoyl (Dts)
(Figure 1-7) has been used because this group can be removed under mild deprotecting
conditions by treatment with a thiol in slightly acidic conditions. The deprotection of Dts can
be carried out by using dithiothreitol (DTT) in acetic acid-CH 2 Cl 2 , followed by neutralization
with diisopropylethylamine (DIEA)-CH 2 Cl 2 and subsequent coupling of Dts-PNA-monomer
by using HBTU/DIEA in NMP 35 . The monomethoxytrityl (Mmt) protecting group (Figure 1-
7) strategy has been developed for the same reason, in order to use milder conditions during
the synthesis 36 , since the Mmt group can be removed by treatment with 3% trichloroacetic
acid in CH 2 Cl 2 . In order to ensure mild synthetic conditions, a new solid phase linker based
on an aminohexyl spacer and a base cleavable succinyl linker have also been developed,
which allow to perform the cleavage by treatment with an ammonia solution, with the
simultaneous deprotection of the acyl-protected nucleobases. The four monomers have been
synthesized and a PNA probe was produced by using this technique, obtaining a molecule of
purity comparable with those produced by standard technique procedures.
Other examples of different synthetic approaches can be found in the literature, such as the
use of the azide groups (Figure 1-7) in order to mask primary amines; these groups protect the
amine during the synthesis and can be reduced by phosphine after the coupling 37 . The
synthesis of PNAs using 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde) protected
monomers has been demonstrated as well 38 (Figure 1-7). This group represents an alternative
to Fmoc group, since it can be removed under mild slightly basic conditions, using a
hydrazine solution, that removes also the Fmoc group; lately a deprotection step has been
optimized, using hydroxylamine (mixture of NH 2 OH·HCl and imidazole) under slightly
acidic conditions which is fully orthogonal to the Fmoc group.
Another strategy is the so called “fully protected backbone approach”, in which a backbone
containing protecting groups in place of the nucleobase is synthesized. The selective
deprotection of the protecting groups and coupling with the correspondent nucleobase gives
the complete PNA 39 . PNAs were synthesized by using this technique, in order to verify the
quality of the technique, only containing cytosine and thymine; more complex sequences still
have to be synthesized. A challenging but charming approach is the template synthesis 40 ,
which implies the synthesis of a PNA backbone without nucleobases, and the use of a
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