Evidences for complex formation between l-dabPNA and aegPNA

Article abstract of DOI:10.1016/j.bmcl.2008.08.005

Continuing our research on the development of nucleopeptides as ODN analogs for biomedical and bioengineering applications, here we report the synthesis and the chemical-physical characterization of a homoadenine hexamer based on a l-diaminobutyric acid (l-DABA) backbone (dabPNA), and its binding studies with a complementary aegPNA. We demonstrated by CD and UV experiments that the l-dabPNA binds the aegPNA forming a complex with good thermal stability, that we identified as a left-handed triplex.

Full text of DOI:10.1016/j.bmcl.2008.08.005

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4757–4760
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evidences for complex formation between L-dabPNA and aegPNA
c
a
Giovanni N. Roviello a, Domenica Musumeci b, , Enrico M. Bucci , Mariangela Castiglione ,
*
Annalisa Cesarani a, Carlo Pedone a, Gennaro Piccialli d
a Istituto di Biostrutture e Bioimmagini—CNR, via Mezzocannone 16, I-80134 Napoli, Italy
b Bionucleon srl, via D. Montesano 49, I-80131 Napoli, Italy
c Bioindustrypark del Canavese, via Ribes 5, I-10010 Colleretto Giacosa (TO), Italy
d Dipartimento di Chimica delle Sostanze Naturali, Università ‘‘Federico II”, via D. Montesano 49, I-80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Continuing our research on the development of nucleopeptides as ODN analogs for biomedical and bio-
engineering applications, here we report the synthesis and the chemical–physical characterization of a
Received 24 June 2008
Revised 31 July 2008
Accepted 1 August 2008
Available online 6 August 2008
homoadenine hexamer based on a
L-diaminobutyric acid (
L-DABA) backbone (dabPNA), and its binding
studies with a complementary aegPNA. We demonstrated by CD and UV experiments that the
L
-dabPNA
binds the aegPNA forming a complex with good thermal stability, that we identified as a left-handed
triplex.
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
L
-dabPNA
aegPNA
Triplex
CD
Chirality
The simple four-base recognition in nucleic acids has inspired
anchored through a carboxymethylene linker.6 These analogs were
found to possess remarkable properties such as high nuclease
resistance as well as good affinity and specificity for complemen-
tary natural nucleic acids and for complementary aegPNAs them-
selves.6 However, some drawbacks, such as low water solubility,
tendency to aggregate, and costly precursors, limit their use in var-
ious applications.6b,6c In order to improve the water solubility and
to decrease their aggregation, positive charges, generally coming
from basic aminoacid moieties, were inserted in the pseudopeptide
backbone of aegPNAs.6b,7 Furthermore, the introduction of chiral
centers into aegPNAs induced preferential orientation (parallel or
antiparallel) of the PNA relative to the complementary strand
increasing the specificity for the target.6b,8
for decades chemists and biologists to develop natural oligonucle-
otides (ODNs) as therapeutic and diagnostic tools or as new
nanomaterials in biomedical and bioengineering applications.1,2
However, the use of natural ODNs is limited by several factors
including poor cellular uptake, a relatively short half-life in physi-
ological conditions due to nucleases, and various non-specific ef-
fects relative to ODN–protein interactions.1,2 In order to
overcome these problems, the development of new ODN analogs
has been widely explored for decades and many modified ODNs
were designed and tested.1–3 One of the most conservative ODN
modification is contained in phosphorothioate (PS) ODNs, in which
a non-bridging oxygen atom in the phosphate group is replaced by
sulfur.4 PS-ODNs showed high affinity to nucleic acid targets, good
nuclease resistance, and remarkable ability to cross the lipid
bilayer. Despite these positive characteristics, which allowed the
entrance of PS-ODNs in advanced phases of clinical trials, the effect
of their backbone stereogenicity on target binding and the toxicity
connected with their non-specific binding to several proteins are
still subjects of studies.4,5 On the other hand, one of the most
dramatic deviations from the natural DNA structure is represented
by aminoethylglycylPNAs (aegPNAs), introduced in 1991 by
Nielsen,6a which contain an achiral pseudopeptide backbone, in-
stead of the sugar-phosphate one, on which nucleobases are
In order to develop new nucleopeptides as ODN analogs for bio-
medical and bioengineering applications, recently we began to
study chiral dabPNAs, reporting for the first time the synthesis of
a new homothymine L-DABA-based dodecamer, its characteriza-
tion, and hybridization studies with natural nucleic acids.9 Contin-
uing our research in this field, here we describe the synthesis and
the characterization of a novel adenine L-DABA monomer suitably
protected for solid phase assembly, its oligomerization to the cor-
responding hexamer, and binding studies with a complementary
aegPNA in order to explore the potential use of these nucleopep-
tides as new materials.
The synthesis of the adenine
Scheme 1) was performed starting from the commercially available
Boc- -DAB(Fmoc)-OH diaminoacid with a procedure that allowed
L
-DABA monomer (aL
2,
-dab,
* Corresponding author. Fax: +39 081 7486552.
E-mail address: dmusumeci@bionucleon.com (D. Musumeci).
L
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.005

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G. N. Roviello et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4757–4760
peptide-like protocol and Fmoc-chemistry (see Supplementary
Material for details). Firstly, the solid support was functionalized
with a -lysine by reaction of deprotected Rink-amide resin (0.5
mmol NH2/g) with Fmoc- -Lys(Boc)-OH following a standard pro-
L
L
cedure (PyBOP/DIEA in DMF), reducing the resin functionalization
to 0.25 mmol/g. This value is generally appropriate for the synthe-
sis of PNAs in order to avoid aggregation effects during chain elon-
gation. Then, we performed six coupling steps with dabPNA
monomer 2 using a procedure, reported in the literature,8d,9 that
minimizes racemization (HATU/TMP without preactivation). Cou-
pling yields were checked spectrophotometrically on solid phase
by UV Fmoc test and found to be in the range of 70–75%. A glycine
was added, as the last residue, at the N-terminus to prevent side
reactions (N-acyl transfer or loss of last residue through cycliza-
tion) that occur, as for aegPNAs, when the N-terminal amino group
of dabPNAs is free in basic or neutral medium.6b A 20% overall yield
for 3 was estimated on the final Fmoc test with respect to the ini-
tial functionalization of the Rink-Lys-NH2 resin. The L-lysine and
glycine residues, incorporated in the strand at C- and N-terminus,
respectively, are useful to improve the solubility in water of the
free homoadenine oligomer. Deprotection and cleavage from the
solid support were achieved by TFA treatment, followed by precip-
itation in cold diethyl ether. Crude dabPNA was purified by reverse
phase HPLC, and the pure product 3 was quantified by UV and
characterized by LC-ESIMS (Fig. 1b).
Scheme 1. Synthesis of aL
monomer 2.
-dab
us to obtain the desired building block in only two synthetic steps
(see Supplementary Material for details). Firstly, the Boc was selec-
Subsequently, we studied the CD behavior of the homoadenine
tively removed from the a-amino group of DABA by TFA treatment
L
-dabPNA single strand. In particular, the CD spectrum of nucleo-
peptide 3 (Fig. 2, solid line) presented a profile similar to that we
recently published for t12
-dabPNA9 (Fig. 2, dashed line) with a
giving compound 1 in almost quantitative yield. The successive
coupling of 1 with the Bhoc-protected (adenin-9-yl)acetic acid
was performed using HATU/DIEA/TMP in DMF as the activating
L
shift of the negative band minimum from 281 to 269 nm. It is
interesting to underline that the observed CD profile is analogous
system, leading to the orthogonally protected aL
nucleoamino-
-dab
acid 2 (Scheme 1). After removal of DMF followed by precipitation
in cold water and centrifugation, the crude pellet was purified by
RP-HPLC, using TFA-free eluents to prevent loss of the Bhoc group,
and pure product 2 was obtained in 51% yield.
to that reported for other peptide nucleic acids carrying
acid residues in the backbone, such as homothymine
oligomers.8d
L
-amino-
L-ornPNA
In order to explore the ability of L-dabPNA to bind aegPNA, we
performed CD studies using a tandem cell. For this purpose, a t12
The new adenine DABA-based monomer was characterized by
NMR and LC-ESIMS (Fig. 1a) and oligomerized on solid phase to
aegPNA was assembled on an automatic synthesizer using stan-
the corresponding dabPNA hexamer
3 (Scheme 2), using a
Figure 1. LC-ESIMS profiles for aL
monomer 2 (a) and hexamer 3 (b).
-dab

G. N. Roviello et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4757–4760
4759
Scheme 2. Synthesis of the homoadenine DABA-based oligomer 3.
Figure 3. Overlapped sum (solid line), mix after 5 min (dotted line) and mix after
60 min (dashed line) CD spectra of 1:1 (aL-dab)6/t12 aegPNA (8
mixing, 2 Â 0.4375 cm cell path length).
lM each strand before
Figure 2. Overlapped CD spectra of (aL
8 lM in 10 mM phosphate buffer, pH = 7.5 (1 cm cell path length).
)
6 3 (solid line) and (tL
)12 (dashed line),
-dab
-dab
dard protocol and Fmoc solid phase chemistry. The sum CD spec-
trum of t12 aegPNA and nucleopeptide 3, contained in two sepa-
rated compartments of the tandem cell at the same
concentration (2:1 ratio in bases) in phosphate buffer (pH = 7.5)
at 20 °C, is reported in Figure 3 (solid line). As expected, the sum
spectrum was essentially due to the contribution of the chiral
reported in the literature for an a10(t10)2
PNA–PNA–PNA triplex10
presenting the same positive maxima at 220, 260, and 280 nm.
Thus, we suggest the formation of a triple helix also in our case,
of the type aegPNA–dabPNA–aegPNA based on T–A–T triplets. More
particularly, the helix structure should have a left-handed winding
in accord with previous studies in which it was shown that com-
plexes between PNAs containing S stereocenters, or equivalently
nucleopeptide because the achiral aegPNA, even with a L-lysine
at its C-terminus, do not furnish a CD signal, as we verified by per-
forming the CD spectrum of the t12 aegPNA alone (data not shown)
and in agreement with literature data.8d On the other hand, the mix
CD spectrum, recorded 5 min after the two components were
mixed (dotted line, Fig. 3), evidenced a significant change with re-
spect to the sum, with the appearance of three main positive bands
centered at 220, 260, and 280 nm, whose intensity increased and
stabilized after approximately 60 min (dashed line).
In other words, the mixture of the complementary chiral
dabPNA 3 and achiral aegPNA strands gave a strong CD signal, sug-
gesting the formation of a complex (aL-dab)6/t12 aegPNA adopting a
well-defined structure of stacked base pairs. More particularly, the
CD spectrum of the complex showed a pattern very similar to that
L-aminoacids, in one of the PNA strands were characterized by
left-handed helices, predominant in solution.8c,11 However, this
hypothesis should be confirmed by NMR spectroscopy.
Thermal stability for the complex (aL-dab)6/t12 aegPNA (1:1 in
phosphate buffer, pH = 7.5) was then studied by UV spectroscopy.
The UV melting curve, reported in Figure 4, exhibited a transition
at 43.8 °C (Tm). Assuming the formation of an a6/(t6)2 triplex, a con-
tribution to the Tm of 7.3 °C per T–A–T triplet can be deduced. This
value is essentially identical to that obtained for pure aegPNA tri-
plexes (7.6 °C per triplet).10 The presence of only one transition
in the melting curve suggests that no duplex intermediate was
effectively formed, in perfect agreement to the reported behavior
of PNA triplexes.10 The sigmoidal profile of the UV melting curve

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G. N. Roviello et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4757–4760
DABA,13a together with nucleobases,13b in the extraterrestrial soil
of Murchison meteorite. Furthermore, both aegPNA and dabPNA
submonomeric units were obtained in simulated stellar conditions
involving electric discharge reactions from CH4, N2, NH3, and
H2O.14 Thus, the formation of a complex dabPNA/aegPNA, evi-
denced by this work, is a question that should be deeply investi-
gated to ascertain if this recognition could have been involved in
the already hypothesized prebiotic PNA world.12,13a,15
Acknowledgments
This work has been funded by the Italian MIUR (FIRB-Contract
number RBRN07BMCT). We gratefully thank Dr. Giuseppe Perretta
for his invaluable technical assistance.
Figure 4. UV melting of (aL-dab)6/t12 aegPNA complex (0.55 lM each strand,
A:T = 1:2) in phosphate buffer (pH = 7.5, heating rate 0.5 °C/min).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.005.
suggested the formation of a complex based on cooperative hydro-
gen bonds and base-stacking. The process was reversible and the
pairing was completed over a temperature change of approxi-
mately 35 °C (70 min at 0.5 °C/min).
In this work we have demonstrated, for the first time, the for-
mation of a complex between complementary achiral aegPNA
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and chiral
synthesized starting from the Fmoc/Bhoc-protected adenine
-DABA-based monomer, and characterized by CD and LC-ESIMS,
L-dabPNA molecules. In particular, the (aL-dab)6 oligomer,
L
was shown to bind a t12 aegPNA forming a complex of high thermal
stability (Tm = 43.8 °C). On the basis of the CD spectrum profile,
similar to those reported in the literature for (homoadenine)/
(homothymine)2 PNA triplexes, we suggest a triple helical struc-
ture also for the a6 L-dabPNA/t12 aegPNA system, hypothesis to be
validated by further studies based on NMR spectroscopy. More-
over, UV melting experiments evidenced that this complex denatu-
rates in one step without involving a duplex intermediate, as
already reported also for other PNA triplexes.
From a biotechnological point of view, the results obtained in
this work provide important direction for the development of no-
vel self-assembling materials based on mixed structures of aeg/
dabPNA. The chirality of these mixed systems, due to the presence
of dabPNA, could confer remarkable properties to the resulting
bio-inspired material, such as preferential strand orientation and
helical handedness, providing highly ordered three-dimensional
networks. Thus, we suggest the combined utilization of dabPNA
and aegPNA in order to realize chiral self-assembling systems,
characterized by nucleobase-directed recognition, thermal
stability, as well as chemical and enzymatic resistance, which
could be useful tools, for example, as hydrogels for the controlled
drug delivery.
10. Wittung, P.; Nielsen, P. E.; Norden, B. J. Am. Chem. Soc. 1997, 119, 3189.
11. Lagriffoule, P.; Wittung, P.; Jensen, K.; Eriksson, M.; Norden, B.; Buchardt, O.;
Nielsen, P. E. Chem. Eur. J. 1997, 3, 912.
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(b) Wolman, Y.; Haverland, W. J.; Miller, S. L. Proc. Nat. Acad. Sci. U.S.A. 1972, 69,
809.
Nevertheless, it is worth to underline that both aegPNAs and
dabPNAs were proposed as ancestors of ribonucleic acid during
the evolution of life on Earth, firstly by Nielsen12 in 1993 and, sub-
sequently, by Meierhenrich13a in 2004 following the recovery of
15. Strasdeit, H. Chembiochem 2005, 6, 801.