PNAs not deriving from such partial modification of 1 have
been evaluated for base-pairing specificity in detail.7 Fur-
thermore, none of these appeared to exhibit a comparable
level of binding affinity and specificity toward DNA and
RNA as 1.
Scheme 1. Synthesis and Structures of PNA Monomersa
Proline is often used as a basic structural motif in the
design of new PNA because of the possibility of conforma-
tional restriction of the PNA through a limited flexibility of
the pyrrolidine ring.6 We have recently reported a series of
new â-pyrrolidinyl PNA (2) consisting of an alternating
sequence of D-proline and a cyclic â-amino acid spacer
(Figure 1).8 The homothymine decamer of 2 with a (1S,2S)-
a Abbreviations: Boc, tert-butoxycarbonyl; Bz, benzoyl; Dpm,
diphenylmethyl; EDC, N-ethyl-N′′-(3-dimethylaminopropyl)carbo-
diimide; Ibu, isobutyryl; Fmoc, fluoren-9-ylmethoxycarbonyl; Pfp,
pentafluorophenyl; Su, succinimidyl.
of the monomers (4) and Pfp-activated ACPC spacer (5)10
using a standard Fmoc solid-phase peptide synthesis
protocol.8,9b After the desired sequence was obtained, the
N-terminal amino group was capped by acetylation. A lysine
residue was also incorporated into the PNA sequence, usually
at the C-terminus, to improve solubility and reduce nonspe-
cific aggregration.1a The PNA was obtained after on-resin
nucleobase deprotection (1:1 aq NH3-dioxane, 55 °C),
cleavage from the resin (TFA, rt), and reverse-phase HPLC
purification. They were characterized by MALDI-TOF mass
spectrometry. The sequences of PNAs synthesized and used
in this study are as shown in Figure 2.
Figure 1. Structure of PNA 1 and 2.
2-amino-1-cyclopentanecarboxylic acid (SS-ACPC) spacer
was shown to bind with its complementary DNA to form a
1:1 hybrid with high affinity and sequence specificity.8c In
this paper, we demonstrate that 2 incorporating all four DNA
nucleobases (A, T, C, and G) exhibits a remarkable fidelity
in recognition of DNA following Watson-Crick base-pairing
rules (A‚T, C‚G). Furthermore, the PNA 2 with asymmetric
sequences also shows a strong preference for binding to DNA
in only one specific orientation. Finally, we disclose a
preliminary result on an unusual preference of 2 in binding
to DNA over RNA and over self pairing.
Figure 2. Sequences and abbreviations of PNA synthesized and
used in this study. The base sequences are written from the N- to
C-terminus. All PNA were N-acetylated and modified with L-
LysNH2 at C-termini except 2g where NR-acetyl-L-lysine was
included at the N-terminus to facilitate purification by HPLC.
The four pentafluorophenyl (Pfp)-activated, Fmoc-pro-
tected proline nucleotide equivalents 4 were synthesized from
N-Boc C-diphenylmethyl (Dpm)-protected monomers 39
(Scheme 1). The protection scheme for nucleobases was
chosen in accordance with standard oligonucleotide synthesis,
i.e., none for thymine, benzoyl (Bz) for adenine (A) and
cytosine (C), and isobutyryl (Ibu) for guanine (G). The
mixed-sequence PNA was synthesized by stepwise coupling
To investigate the base-pairing specificity between 2 and
DNA, each of the four PNAs 2b-e with one nucleobase
variation at the central position was hybridized with one of
the four DNA probes AAAAYAAAA (Y ) A, T, C, G).
The highest four Tm values out of the possible sixteen pairs
of PNA-DNA hybrids corresponded to the Watson-Crick-
type base pairing (A‚T, C‚G) (Figure 3). The remaining
mismatched hybrids had a Tm between 15 and 48 °C lower.
Percent hyperchromicity for the mismatched hybrids was also
significantly decreased (about 25-37% for a perfect match
and 7-27% for a single mismatch) indicating a less favorable
base stacking. Because 2 was shown to form only a 1:1
hybrid with DNA even with a thymine-rich sequence,8c
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