Design and synthesis of intrinsically cell-penetrating nucleopeptides

Article abstract of DOI:10.1039/b811639c

Nucleopeptides, which are constituted of α-amino acids bearing nucleobases at their side chains, are able to penetrate into cells and to reach the nucleus without cytotoxic effects. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b811639c

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Design and synthesis of intrinsically cell-penetrating nucleopeptides†
Piero Geotti-Bianchini,a,b Julien Beyrath,a Olivier Chaloin,a Fernando Formaggio*b and Alberto Bianco*a
Received 8th July 2008, Accepted 5th September 2008
First published as an Advance Article on the web 17th September 2008
DOI: 10.1039/b811639c
Nucleopeptides, which are constituted of a-amino acids
bearing nucleobases at their side chains, are able to penetrate
into cells and to reach the nucleus without cytotoxic effects.
an easy, robust and efficient method to deliver nucleobase-rich
oligomers into cells is very much needed.
To address this issue, we have designed a series of a-
nucleopeptides based on the sequence Ala-AlaT-Ala [AlaT, b-
(thymin-1-yl)alanine] repeated four times (Fig. 1). The nucle-
opeptides contain four nucleobases which were previously found
sufficient for a considerable duplex stability.11 Moreover, the
polyalanine scaffold should allow enough structural flexibility for
a possible interaction with complementary DNA or RNA strands.
The total sequence of the Ala-rich 14-mers comprises also two Lys
residues at both the N- and C-terminal ends of the nucleopeptides.
The insertion of Lys has a dual purpose: i) to improve the solubility
in physiological conditions; and ii) to allow the introduction of
the fluorescent probe FITC (fluorescein isothiocyanate) or biotin
(Fig. 1, T4FIT and T4Bt, respectively) for cell penetration and
surface plasmon resonance studies.
Nucleopeptides were first introduced at the beginning of the
70’s,1,2 but only recently have they emerged as a promising
alternative to peptide nucleic acids (PNAs).3–5 In 1991 the group
of Nielsen demonstrated the ability of PNAs to strongly interact
with DNA and RNA thus paving the way to their potential
biomedical applications.3–5 The PNAs may be constituted of
many structural pseudopeptide motifs.5 The most important
and exploited scaffolds are based on the repetition of N-(2-
aminoethyl)glycine residues containing the nucleobase linked to
the central nitrogen via an amide bond.3,4 Nucleopeptides, less
explored than PNAs, are instead constituted of sequences of
amino acids bearing nucleobases covalently bound to their side
chains.6 Typically, the nucleobase is linked in the b-position of
the alanyl side chain.7,8 The a- and b-nucleopeptides are able
to form rigid and well defined double strands stabilized by H-
bonding with complementary sequences.9–12 The capability of these
synthetic hybrids to form stable base-pairing might be useful
for DNA displacement, specific recognition and modulation of
the interactions with the natural nucleic acids. Alternatively, the
nucleoamino acids were introduced at defined positions within
a synthetic peptide backbone, constituted of protein a-amino
acids, to take advantage of the conformational changes of the
peptide during its interaction with complementary single-strand
DNA/RNA sequences.13,14 Nucleoamino acids are also versatile
building blocks for the preparation of nucleobase substituted
analogues of natural products as DNA intercalating agents,15 or
for the synthesis of novel and efficient double strand DNA binding
ligands,16 or new opioid selective mimetics.17
In the context of the biological applications, including gene
regulation, one of the disadvantages of PNAs, the closest to drug
development, is their poor cellular uptake.5,18 Indeed, very few
examples have shown that PNAs penetrate efficiently into the
cells.19,20 Cellular uptake can be facilitated by conjugating the
PNAs to a cell penetrating peptide or to other carrier systems.21–26
On the contrary the a- and b-nucleopeptides have never been
studied for their capacity to cross the cell membranes without
provoking undesired effects in terms of cell viability. Indeed,
Fig. 1 Molecular structures of the nucleopeptides.
Initially, the nucleoamino acid AlaT was prepared following the
approach of nucleophilic ring opening of the lactone of the Boc
protected serine using the thymine base.27,28 The nucleopeptides
were subsequently prepared by solid-phase synthesis using the
Merrifield strategy (see Supporting Information†).29 Following the
cleavage from the resin, the crude compounds were purified by
HPLC to afford the desired products in a fairly good yield and
purity higher than 94%.
aCNRS, Institut de Biologie Mole´culaire et Cellulaire, Laboratoire
d’Immunologie et Chimie The´rapeutiques, 15 Rue Rene´ Descartes, 67000,
Strasbourg, France. E-mail: A.Bianco@ibmc.u-strasbg.fr
bDipartimento di Chimica, Universita` di Padova, via Marzolo 1, 35131,
Padova, Italy. E-mail: fernando.formaggio@unipd.it
† Electronic supplementary information (ESI) available: Details on
the solid phase synthesis, characterization, cell uptake and viability
tests, and surface plasmon resonance of the nucleopeptides. See DOI:
10.1039/b811639c
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To assess the biological behaviour of our nucleopeptides, we
initially studied their ability to penetrate into cells. RENCA cells
were incubated for times between 5 and 60 min and up to 12 hours
at 37 ◦C with concentrations of T4FIT and T4Bt ranging from
0.5 to 50 mM. The cells treated with T4FIT were directly analyzed
using fluorescence microscopy and flow cytometry. As shown in
Fig. 2A the nucleopeptide rapidly penetrates into the cells. We have
found that this happens in a time (Fig. 2B) and dose dependent
manner (Fig. S2–S4). At the highest concentration of T4FIT, more
than half of the cells are stained. The fluorescent oligomers seem
to be confined into vesicular compartments, particularly abundant
in the perinuclear region. Cellular uptake of T4FIT is energy-
dependent since the internalization is significantly reduced treating
the cells with sodium azide and 2-deoxy-D-glucose (Fig. 2B).30
the cells, and particularly to enter the nucleus, is an extremely
relevant finding, as we may envisage using such conjugates to bind
RNA or DNA and modulate their activity. Preliminary binding
studies, using surface plasmon resonance, of the thyminyl-based
oligomer T4H to its complementary sequence containing four
adenylyl-nucleoamino acids (details on the synthesis are reported
in Supporting Information) allowed us to calculate an apparent
equilibrium dissociation constant in the micromolar range (Fig. 3).
For this purpose, nucleopeptide A4Bt was immobilized on the
sensor chip via streptavin previously covalently fixed to the
dextran carboxylic functions on the chip gold surface, activated
in turn with carbodiimide and N-hydroxysuccinimide. After this
coupling, increasing concentrations of T4H from 12.5 to 50 mM
were injected to evaluate the interaction capability of the two
complementary nucleopeptides. Nucleopeptide T4H gradually
complexed A4Bt as evidenced by an increase of the signal during
the association phase. During the dissociation phase, T4H was
very slowly released. The (apparent) association rate constant (ka)
and the (apparent) dissociation rate constant (kd) corresponded
to 3.18 ¥ 102 M-1 s-1 and 1.09 ¥ 10-2 s-1, respectively. The
resulting dissociation constant at the equilibrium (KD) is 34 mM,
which represents the affinity between the two complementary
nucleopeptide chains. Comparable results were obtained using
two synthetic complementary oligonucleotide sequences (dT8
and dT10) (Fig. S7). To prove that the recognition between the
complementary bases on the nucleopeptides and DNA follows
the Watson–Crick rules, and it is not only due to non specific
base-stacking, we immobilized on the gold chip nucleopeptides
containing four thymines or four cytosines (details on the synthesis
are reported in Supporting Information). Such nucleopeptide
sequences did not show any interaction either with T4H or with
thymine-based DNA sequences dT8 and dT10 (Fig. S8 and S9).
This supports the capacity of nucleopeptides to specifically bind
nucleotide sequences.
Fig. 2 (A) Confocal microscopy image of RENCA cells incubated with
50 mM of T4FIT for 12 h. (B) Time dependent uptake of 50 mM of T4FIT
by Raji cells at 37 ◦C in the absence (blue bars) or in the presence (red
bars) of endocytosis inhibitors NaN3 and 2-deoxy-D-glucose, quantified
by flow cytometry. Plots display the mean fluorescence signal of three
experiments the standard error. (C) Confocal microscopy images of
RENCA cells co-incubated with 50 mM of T4FIT (green) and 20 mg
mL-1 of transferrin-Alexa-546 (red) at 37 ◦C for 30 min. Overlapping
of the images evidences endosomal co-localization (yellow). (D) Confocal
microscopy image of RENCA cells incubated with 50 mM of T4Bt at 37 ◦C
for 12 h. Nucleopeptide was revealed with streptavidin-FITC (green). The
cell nucleus was labelled with DAPI (blue).
Confocal images of the cells incubated with transferrin, a
marker of endocytosis, confirmed a substantial co-localization
with T4FIT (Fig. 2C).31 This type of entrapment into endosomal
vesicles was found also for the fluorescent PNAs.19 However,
our images show a diffusion of the nucleopeptide into the
cytoplasm, suggesting either an endosomal escape or a possible
contribution of an alternative route of cellular entry. In addition,
nucleopeptides were found into the nucleus (Fig. 2A and D).
The confocal analysis confirmed that they are inside and not
simply associated to the nuclear membrane (see also Fig. S6). This
behaviour is reminiscent of cationic cell penetrating peptides, as
our nucleopeptides contain two positively charged residues which
impart high water solubility.30,31
Fig. 3 Sensorgrams obtained by allowing various concentrations of
nucleopeptide T4H to interact with the nucleopeptide A4Bt immobilized
onto sensor chip. RU corresponds to the resonance unit (1000 RU =
1 ng mm-2 of analyte) [12.5 mM (magenta), 25 mM (cyan), 50 mM (black)].
To further investigate the impact of the nucleopeptides on cell
behaviour, cell viability was evaluated using three different cell
lines. Increasing doses of nucleopeptides T4H and T4Bt were
added to the cell cultures. The cells were analyzed after 24 hour
incubation and no significant decrease in the number of living cells
was observed in comparison to the doxorubicin-treated control
(Fig. 4). The viability was the same for Raji, Jurkat and BL41
cell lines (see also Fig. S10 and S11). This is another interesting
Similarly, the biotinylated nucleopeptide T4Bt, which was
detected using a fluorescent streptavidin, presents the same
behaviour (Fig. 2D). The capability of our nucleopeptides to enter
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Fig. 4 Effect of T4H and T4Bt on the viability of Raji cell line.
Concentration–response curves: Raji cells were treated with increasing
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In summary, we have designed and prepared a series of highly
water-soluble nucleopeptides which are easily taken up by the
cells without toxic effects. Very importantly, they are able to
pass the nuclear membrane thus paving the way for their use as
DNA modulators. The improvement of cell penetration is certainly
extremely beneficial for increasing the diagnostic and therapeutic
efficacy of nucleopeptide-based drugs.
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This work has been supported by the CNRS. P. G.-B. is grate-
ful to Universita` italo-francese/Universite´ franco-italienne for
supporting his international PhD (VINCI program 2005). The
authors wish to thank Marion Decossas and Ste´phanie Lacotte
for her precious help and fruitful discussions. Confocal images
were recorded at RIO Cellular Imaging Platform of Esplanade
(Strasbourg, France).
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The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 3661–3663 | 3663
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