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Chapter 1
PNAs including monomers containing D- or L-Lysine side chains in position 2 and 5 were
also synthesized and studied during the hybridization of complementary sequences. As
predictable, the best configurations turned out to be 2D-5L, and the affinity and the selectivity
where the highest for these probes.
The reasons for the high performances showed by chiral PNAs were investigated by studying
chiral PNA-PNA duplexes by circular dichroism, also by using cyanine dyes such as Disc 2 (5)
dye 82 , which is able to form aggregates into the minor groove of a duplex PNA-PNA or PNA-
DNA. The formation of such complexes can be detected by UV-Vis spectrophotometry, due
to an ipsochromic effect, or by circular dichroism, due to the exciton coupling effect of the
aggregate, indicative of its handedness, which is dictated by that of the double helix. These
data clearly indicated that, while achiral PNA-PNA duplexes are an equimolar mixture of
right-handed and left-handed helices, 2D-PNA and 5L-PNA preferentially formed righthanded
helices when hybridized to complementary achiral PNAs. Thus, 2-D and 5L-PNAs
prefer the same handedness as DNA. This preference makes the formation of the duplex with
complementary DNA easier and more stable 83 .
Since chirality is of crucial importance in these probes, the eventual racemization has to be
checked during the synthesis of such probes. Since the chiral center in position 2 can undergo
racemization during standard peptide synthesis, as it is known for amino acids, a new
strategy, called submonomeric strategy, was developed in order to preserve the enantiomeric
purity. This synthetic approach allowed to reduce the racemization in this position from 30-
50% to 2-6%. This strategy consists in the synthesis of a submonomer that bears a Fmoc
protecting group on the nitrogen in position 3 instead of the nucleobase. The carbammate
formed with the Fmoc group is less electron withdrawing than the amide formed with the
nucleobase, and this allows to reduce the acidity of the hydrogen in position 2 and thus the
racemization during the carboxylic group activation. The Fmoc group can be removed after
the coupling and the nucleobase is introduced by a standard coupling directly on the resin
(Figure 1-11) 84 .
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