Evidences for supramolecular organization of nucleopeptides: Synthesis, spectroscopic and biological studies of a novel dithymine l-serine tetrapeptide

Article abstract of DOI:10.1039/c0mb00214c

This work concerns a dithymine tetrapeptide, which can be seen as a new analogue of a dinucleoside monophosphate, made of both unfunctionalized and thymine-containing l-serine units alternated in the sequence. The new nucleopeptide was obtained on the solid phase by two different synthetic strategies. The first one is suitable to easily realize nucleopeptides with homonucleobase sequences, obtained by assembling an oligoserine backbone and then simultaneously coupling the free serine hydroxyl groups with the carboxymethylated nucleobase. The other strategy, which makes use of a Fmoc-protected nucleo-l-serine monomer, allows for the obtainment of nucleopeptides with mixed nucleobase sequences. CD spectroscopic studies and laser light scattering experiments, performed on solutions of the novel nucleopeptide, suggested the formation of supramolecular networks based on the self-assembly of the dithymine tetrapeptide molecules. Furthermore, CD binding studies with natural nucleic acids revealed a very weak interaction between the nucleopeptide and DNA (but not RNA). Molecular networks based on this biodegradable and water-soluble nucleopeptide, which is more resistant in plasma than standard tetrapeptides (and oligopeptides), contain a hydrophobic core which could provide the necessary environment to incorporate poorly water-soluble drugs, as evidenced by fluorescence spectroscopy. Furthermore, our studies evidenced that the structure of the tetrapeptide-based supramolecular assembly can be modified by metal ions as evidenced by UV interaction studies with Cu²⁺. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0mb00214c

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Molecular
BioSystems
Cite this: Mol. BioSyst., 2011, 7, 1073–1080
www.rsc.org/molecularbiosystems
PAPER
Evidences for supramolecular organization of nucleopeptides: synthesis,
spectroscopic and biological studies of a novel dithymine L-serine
tetrapeptidew
Giovanni N. Roviello,*z Domenica Musumeci,z Enrico M. Bucci and Carlo Pedone
Received 29th September 2010, Accepted 6th December 2010
DOI: 10.1039/c0mb00214c
This work concerns a dithymine tetrapeptide, which can be seen as a new analogue of a
dinucleoside monophosphate, made of both unfunctionalized and thymine-containing L-serine
units alternated in the sequence. The new nucleopeptide was obtained on the solid phase by two
different synthetic strategies. The first one is suitable to easily realize nucleopeptides with
homonucleobase sequences, obtained by assembling an oligoserine backbone and then
simultaneously coupling the free serine hydroxyl groups with the carboxymethylated nucleobase.
The other strategy, which makes use of a Fmoc-protected nucleo-L-serine monomer, allows for
the obtainment of nucleopeptides with mixed nucleobase sequences. CD spectroscopic studies and
laser light scattering experiments, performed on solutions of the novel nucleopeptide, suggested
the formation of supramolecular networks based on the self-assembly of the dithymine
tetrapeptide molecules. Furthermore, CD binding studies with natural nucleic acids revealed a
very weak interaction between the nucleopeptide and DNA (but not RNA). Molecular networks
based on this biodegradable and water-soluble nucleopeptide, which is more resistant in plasma
than standard tetrapeptides (and oligopeptides), contain a hydrophobic core which could provide
the necessary environment to incorporate poorly water-soluble drugs, as evidenced by
fluorescence spectroscopy. Furthermore, our studies evidenced that the structure of the
tetrapeptide-based supramolecular assembly can be modified by metal ions as evidenced by
UV interaction studies with Cu2+
.
In the last decades, a great relevance has been attributed to
hydrogels as well as water-soluble macromolecular networks
useful for the incorporation and the release of genes and
drugs.11 These polymeric systems are governed by non-
covalent bonds occurring between the subunits and can be
based on one or more types of monomeric units. Among the
different classes of molecules able to form supramolecular
architectures it is worth to mention systems characterized by
polynucleobase-molecules such as nucleic acids, which are
particularly important in nanomedicine,12 and peptide nucleic
acids,13 as well as systems characterized by monomeric units
bringing a single nucleobase which can form gels based on the
cooperative effects of the complementary nucleobases.14
Taking into account all these considerations we designed
and realized a novel analogue (1) of TpT DNA dinucleoside
monophosphate (2, Fig. S1, ESIw), characterized by a peptide
backbone comprising both nucleobase-functionalized and free
L-serine moieties alternated in the sequence. In this oligomer
the atoms of the backbone which bring the DNA bases are
separated by the same number of bonds (i.e. 6, Fig. S1, ESIw)
found in DNA, and the nucleobases are anchored to the
hydroxyl groups of the L-serine units by means of ester bonds.
Introduction
Many investigations regarding novel antigene or antisense
strategies have shown that the ribose phosphodiester linkage
can be replaced in novel oligonucleotide mimics by several
modifications.1 For example, several attempts to use a polyamide
backbone as an alternative oligonucleotide linkage have been
reported, allowing for the obtainment of several DNA analogues
with interesting properties.2–9 As a general rule, in a nucleo-
peptide backbone two a-aminoacid units are isomorphous in
terms of their length (six bonds) to one sugar-phosphate residue
of the DNA and RNA backbone. Therefore, the repeating unit of
a potential nucleic acid-binding nucleopeptide could be built of
two amino acid moieties, one of which derivatized with the
nucleobase, directly joined to one another by a peptide bond.10
Istituto di Biostrutture e Bioimmagini – CNR, Via Mezzocannone 16,
80134 Napoli, Italy. E-mail: giroviel@unina.it;
Fax: +39 (0)81-2534574; Tel: +39 (0)81-2534585
w Electronic supplementary information (ESI) available: Figure S1,
LC-ESIMS profiles, CD DNA binding study and HPLC profiles
relative to the crude mixtures obtained after the nucleopeptide
syntheses. See DOI: 10.1039/c0mb00214c
z These authors equally contributed to this work.
c
This journal is The Royal Society of Chemistry 2011
Mol. BioSyst., 2011, 7, 1073–1080 1073

View Article Online
Underivatized L-serines were introduced as spacers between
the nucleobase-carrying aminoacids in order to achieve the
proper distance between two consecutive nucleobases, as well
as to improve the water-solubility of the resulting nucleo-
peptide, as expected for the presence of the hydrophilic
hydroxyl moieties. This TpT DNA dinucleoside mono-
phosphate analogue is expected to be more resistant to
enzymatic degradation than DNA but also biodegradable
due to its real peptide nature. The self-assembly properties
as well as the ability to interact with nucleic acids of the novel
alternate L-serine-based nucleopeptide were investigated, as
described in the present work.
diethyl ether and purified by RP-HPLC on a C18 column.
ESI-MS (positive ions) confirmed the identity of the
dinucleotide analogue (Fig. S2, ESIw).
Synthesis of the Fmoc-protected monomer 5 for the solid phase
assembly of nucleopeptide 1
We realized a convenient and fast synthetic route to chiral
Fmoc nucleo-L-serine monomer, in which the (S)-2-amino-
3-hydroxypropanoic moiety is connected to the DNA
nucleobase by an ester bond, suitable for the solid phase
synthesis of nucleopeptides. The nucleobase-containing
monomer was synthesized starting from the commercially
available Fmoc-L-Ser(tBu)-OH (3, Fig. 2). In the first synthetic
step the tert-butyl group was selectively removed with trifluoro-
acetic acid to give the free hydroxyl group in b position
(compound 4, Fig. 2). ESI-MS (positive ions) confirmed the
identity of this intermediate (Fig. S3, ESIw).
Subsequently, Fmoc-L-Ser-OH 4 was coupled with the
nucleobase acetic acid under different synthetic conditions
with the best results coming from the use of DIPC/DMAP
in DMF as solvent (Fig. 2). After RP-HPLC purification the
desired product 5 was obtained in 30% yield and characterized
by 13C/1H NMR and ESIMS (Fig. S4, ESIw). Subsequently,
the nucleo-tetrapeptide 1 was assembled in the solid phase
using an alternative synthetic strategy employing the coupling
of both monomer 5 and the commercial Fmoc-L-Ser(tBu)-OH
achieved with PyBop/DIPEA as the activating system (Fig. 3).
The yield for the incorporation of the novel nucleoaminoacid 5
was about 80%, while the overall yield for the obtainment of
1, calculated on the basis of the UV Fmoc test, was about
50%. ESI-MS (positive ions) confirmed the identity of the
oligomer.
Results and discussion
Solid phase synthesis of homonucleobase–oligoserines
We realized an easy synthetic route to homonucleobase–
oligoserines (Fig. 1). Briefly, a tetrapeptide was assembled in
the solid phase by using both commercial Fmoc-L-Ser(tBu)-OH
and Fmoc-L-Ser-OH (the latter was obtained as described below
in this work), and PyBOP/DIPEA as the activating system. By
previous experiments we did not find any reactivity of the free
hydroxyl groups under the coupling conditions used (data not
shown). No capping step was performed to avoid the acetylation
of the free hydroxyl groups, which were essential for the
subsequent functionalization with the nucleobases. Then, the
free hydroxyl groups were esterified with the nucleobase acetic
acid (TCH2COOH) by using DCC/DMAP as the activating
system. After final Fmoc removal a UV Fmoc test revealed a
67% overall yield for the tetraserine backbone synthesis.
Subsequently, the crude oligomer was detached from the
resin by acidic treatment (95% TFA), precipitated with cold
Fig. 1 Synthesis of the L-serine-based nucleo-tetrapeptide.
c
This journal is The Royal Society of Chemistry 2011
1074 Mol. BioSyst., 2011, 7, 1073–1080

View Article Online
Supramolecular self-assembly of nucleopeptides—CD
spectroscopy
Molecular self-assembly of 1 was investigated using CD by
monitoring the value of a CD signal at 200 nm at different
nucleopeptide concentrations (Fig. 4a). An intense negative
peak at 200 nm appeared upon increasing the concentration of
nucleopeptide from 10 to 78 mM. By plotting the CD signal at
200 nm as a function of the nucleopeptide concentration, we
observed two main slope changes (Fig. 4b), corresponding
probably to two tridimensional states of the supramolecular
network. The CD spectra of the peptide did not present any
significant change other than a decrease in molar ellipticity
indicating that there was no substantial variation in the
nucleopeptide conformation. Hence, it can be concluded that
the conformational preference of dithyminyl tetraserine is not
changed during the self-assembly.
By examining the CD spectra at higher concentration levels
(275–550 mM ), it was possible to appreciate not only a further
reduction in CD intensity recorded at 200 nm (Fig. 4d), but
also an induced CD contribution of the nucleobases consisting
of a positive band at about 270 nm and a negative band at
about 295 nm (Fig. 4c).
Fig. 2 Synthesis of monomer 5.
Interestingly, the route to the nucleopeptide 1 which
makes use of monomer 5 provided an overall yield higher
than that achieved by the strategy involving the esterification
on the solid phase with the nucleobase–acetic acid
(Fig. 2). However, the latter approach minimizes the number
of synthetic and purification steps required and results,
thus convenient for the synthesis of homonucleobase–
oligoserines. On the other hand, the strategy described in
Fig. 3, which can be extended also to the other nucleobases
(i.e. A, G and C) by using the appropriate nucleobase–acetic
acids, allows for the synthesis of nucleopeptides with mixed
sequence.
The CD spectra relative to two nucleopeptide solutions at
different values of pH and temperatures were also recorded
(Fig. 5). A major degree of supramolecular organization of the
dithymine nucleopeptide was detected at pH 7.5 and at
temperatures lower than 30 1C.
Static laser light scattering
Static laser light scattering experiments were performed on
nucleopeptide solutions at different concentrations. The data
collected were in accord with the formation of supramolecular
networks whose size did not vary significantly with the
Fig. 3 Synthesis of nucleo-tetrapeptide 1 by using monomer 5.
c
This journal is The Royal Society of Chemistry 2011
Mol. BioSyst., 2011, 7, 1073–1080 1075

View Article Online
Fig. 4 (a) CD spectra of nucleopeptide 1 in phosphate buffer (pH = 7.5) at various concentrations; (b) CD value at 200 nm as a function of the
nucleopeptide 1 concentration (10–78 mM); (c) CD spectra of nucleopeptide 1 in phosphate buffer (pH = 7.5): 10 (black line); 54 (dashed black),
275 (cyan) and 550 mM (purple); (d) CD values at 220 nm as a function of the nucleopeptide 1 concentration (10, 19, 46, 63, 78, 275 and 550 mM).
Fig. 6 Hydrodynamic radius vs. nucleopeptide concentration.
and presence of various concentrations of nucleopeptide 1
are shown in Fig. 7a. For each concentration value, ANS
fluorescence stabilized after about 90 min. The changes in
emission were tracked until the system reached equilibrium.
Fluorescence intensity of ANS at 530 nm was plotted against
peptide 1 concentrations (inset of Fig. 7a). The intensity
increased with increasing the peptide concentration, indicating
Fig. 5 CD spectra of nucleopeptide 1 (a) 550 mM at pH 6 (black) and
pH 7.5 (red); (b) 30 mM at pH 7.5 at 20 (red), 30 (black), 40 (pink) and
50 (blue) 1C.
that the ANS binding site became more nonpolar on aggregation.
From the plot of ANS fluorescence intensity vs. nucleopeptide
concentration (Fig. 7a, inset), a change in the slope at about
60 mM concentration can be detected, corresponding to the
critical aggregate concentration of the nucleopeptide.
Furthermore, we evaluated the ability of nucleopeptide 1 to
form supramolecular networks also by kinetic FITC fluorescence
experiments. We observed a time-dependent effect of the nucleo-
peptide on FITC fluorescence (Fig. 7b). In particular, the
increase of FITC fluorescence intensity in time after addition
of 1 sustains the hypothesis of molecular networking and the
formation of a hydrophobic core which provides the necessary
environment to incorporate poorly water-soluble molecules, such
as FITC (see Fig. 7b). The changes in emission were tracked until
the system reached equilibrium (2 h).
concentration in the 25–280 mM range (with a mean hydro-
dynamic radius of about 45 nm estimated in this range) but
significantly increased after the 300 mM concentration level
(Fig. 6).
Fluorescence spectroscopy
Fluorescence aggregation studies on 1 were also carried out
using 8-anilino-naphthalene sulfonate (ANS) as an external
probe. Indeed, ANS can be used as a sensitive probe
to monitor the assembly of oligopeptides. Representative
fluorescence spectra of ANS in phosphate buffer in the absence
c
1076 Mol. BioSyst., 2011, 7, 1073–1080
This journal is The Royal Society of Chemistry 2011

View Article Online
Fig. 7 (a) Fluorescence titration of ANS (30 mM) with nucleopeptide 1 (0–90 mM) in phosphate buffer (pH = 7.5). ANS alone (grey line); 50
(magenta), 70 (dashed black), 80 (red) and 90 mM (black) of 1 added. Fluorescence spectra relative to the 30, 40 and 60 mM concentration of 1 were
omitted for clarity; inset of Fig. 7a: ANS fluorescence intensity at 530 nm vs. peptide 1 concentrations; (b) kinetic experiment on nucleopeptide 1
aggregation: fluorescence spectra of FITC (121 mM in H2O; pH = 7.0), 5 (black line), 60 (red) and 120 (grey) min after the addition of 6.5 mM
nucleopeptide 1; inset of Fig. 7b: time dependence of FITC fluorescence intensity after addition of nucleopeptide 1.
Metal ion binding studies
decreases in the presence of copper(II) cations, confirming also
in this case an interaction of the metal with the nucleobases
which causes a clear stacking effect. In other words the L-serine
nucleopeptide, which is intrinsically able to form supra-
molecular networks, shows also a nucleobase stacking effect
in the presence of copper(II) which is an interesting feature
which may open up new possibilities for the fabrication of
nucleopeptide–metal hybrid materials governed by noncovalent
nucleopeptide–metal ion interactions.
It is known that nucleobase stacking can occur in the
presence of different metal cations such as copper(II) and
palladium(II).15 This interaction is at the basis of supra-
molecular architectures assembled by the copper(II) binding
both in nucleopeptide molecules and in nucleosides or
nucleotides.16ꢀ18 Interestingly, copper(II)–peptide complexes
can also possess biological activity as reported for the natural
tripeptide glycyl-L-histidyl-L-lysine (GHK) which acts as a cell
growth factor.19 In order to investigate the influence of
copper(II) ions on the supramolecular networks based on the
dithymine tetraserine, we performed UV experiments in which
the absorbance of a 10 mM solution of nucleopeptide, in the
presence of phosphate buffer at pH 7.5, was recorded after
adding increasing amounts of a CuCl2 solution. As can be seen
in Fig. 8 the UV absorbance due to the thymine moieties
Biological studies—DNA binding evaluation
DNA binding ability of the L-serine-based nucleopeptide was
evaluated by CD spectroscopy. Firstly, the CD profile of the
single strand nucleopeptide 1 in 10 mM phosphate buffer
(pH 7.5) was analysed in order to evaluate any pre-organization
of the molecule. By examining the CD behaviour of 1 in the
range 190–240 nm at different temperatures (5, 10 and 25 1C)
no significant a-helical contribution was detected (data not
shown).
Regarding the binding with DNA, CD experiments were
performed at 5 1C in a tandem cell recording the ‘‘sum’’
spectrum of the separated components and the ‘‘mix’’
spectrum, recorded after mixing the two ligand solutions,
relative to 1 equivalent in nucleobase of dA12 with respect to
nucleopeptide 1. The slight difference observed between the
‘‘sum’’ (green line) and ‘‘mix’’ (blue) CD spectra suggested a
very weak interaction between the nucleopeptide and DNA
(Fig. S5, ESIw). On the other hand, no interaction with RNA
(A12, polyA and polyU) was revealed by analogous experiments
in the same experimental conditions (data not shown).
Human plasma stability assay
The enzymatic resistance of the novel nucleopeptide was
investigated by incubating the oligomer in 40% human plasma
at 37 1C and analyzing, by RP-HPLC, samples withdrawn
from the reaction mixture at various times (Fig. 9). By
analyzing the results of the stability assay, it can be deduced
that the nucleopeptide is biodegradable and is more resistant
than a natural oligonucleotide, presenting a t1/2 of about
3 hours.
Fig. 8 (a) UV absorbance of a 10 mM solution of nucleopeptide 1,
(10 mM phosphate buffer at pH 7.5, 25 1C) in the presence of 0, 5, 10, 15,
20, 25 and 30 equivalents of Cu(II), (b) plot of UV absorbance at 205
(pink) and 267 (blue) nm vs. Cu(II)/nucleopeptide concentration ratio.
c
This journal is The Royal Society of Chemistry 2011
Mol. BioSyst., 2011, 7, 1073–1080 1077

View Article Online
(monitoring at 260 nm) by building up a gradient starting
with buffer A (0.05% TFA in water) and applying buffer B
(0.05% TFA in acetonitrile) with a flow rate of 0.8 ml minꢀ1
.
Semi-preparative purifications were performed on a Hewlett
Packard/Agilent 1100 series HPLC, equipped with a diode
array detector, by using a Phenomenex Juppiter C18 300 A
(10 mm, 10 ꢁ 250 mm) column. Gradient elution was
performed at 25 1C (monitoring at 260 nm) by building up a
gradient starting with buffer A0 (0.1% TFA in water) and
applying buffer B0 (0.1% TFA in acetonitrile) with a flow rate
of 4 ml minꢀ1. Samples were lyophilized in a FD4 Freeze
Dryer (Heto Lab Equipment) for 16 h.
Fig. 9 Area vs. time profile relative to the human serum stability
assay. In 40% human plasma (T = 37 1C) the dithymine tetrapeptide
presents a t1/2 = 3.02 h.
Circular dichroism (CD) spectra were obtained on a Jasco
J-810 spectropolarimeter, while, ultraviolet (UV) spectra were
recorded on a UV-Vis Jasco model V-550 spectrophotometer,
both equipped with
a Peltier ETC-505T temperature
Experimental section
controller, using an absorption Hellma quartz cell with light
paths of 1 cm and 1 mm, and a Hellma-238-QS tandem quartz
cell (2 ꢁ 0.4375 cm). Quantification of the purified oligomer
was performed by UV measurements (T = 80 1C, absorbance
value at l = 260 nm) of 1 dissolved in MilliQ water: the
epsilon value used (17.2 mMꢀ1) was calculated using the molar
extinction coefficient of the thymine PNA monomer
(8.6 mMꢀ1). Fluorescence experiments were performed on a
JASCO model FP-750 spectrofluorimeter equipped with a
Peltier temperature controller, using a micro fluorescence cell
(Hellma, Suprasil quartz, 10 ꢁ 2 mm light path, 0.5 ml
volume). For light scattering a MiniDAWN Treos spectro-
meter (Wyatt Instrument Technology Corp.) equipped with a
laser operating at 658 nm was used in a batch mode (off-line).
Abbreviations
The following abbreviations are used: ANS (8-anilino-1-
naphthalenesulfonic acid); DCC (1,3-dicyclohexylcarbodiimide);
DCM (dichloromethane); DCU (1,3-dicyclohexylurea); DIPC
(N,N0-diisopropylcarbodiimide); DIPEA (N,N0-diisopropyl-
ethylamine); DMAP (4-dimethylaminopyridine); DMF
(N,N-dimethylformamide); DMSO-d6 (deuterated dimethyl
sulfoxide); FITC (fluorescein isothiocyanate); Fmoc (9-fluorenyl-
methoxycarbonyl); MBHA (4-methylbenzhydrylamine); NMP
(4-methylpyrrolidone); PyBop (benzotriazole-1-yl-oxy-tris-
pyrrolidino-phosphonium hexafluorophosphate); TCH2COOH
(thymin-1-yl acetic acid); TFA (trifluoroacetic acid); TIS
(triisopropyl silane).
Chemicals
Solid phase synthesis of nucleopeptide 1
Fmoc-L-Ser(tBu)-OH and PyBop were purchased from
Novabiochem. Anhydroscan DMF and NMP were from
LabScan. Piperidine was from Biosolve. CH3CN for HPLC
chromatography and acetic anhydride were from Reidel-de
The synthesis was carried out in short PP columns (4 ml)
equipped with a PTFE filter, a stopcock and a cap on a
Rink-amide resin using the peptide Fmoc chemistry. The
tetrapeptide H-[Ser(T)-Ser]2-NH2 (1, Fig. 1) was assembled
on Rink-amide–NH2 resin (0.5 mmol gꢀ1, 40 mg, 20 mmol) by
alternatively coupling Fmoc-L-Ser-OH (0.52 M in DMF dry,
9 eq., 381 ml) and the commercial Fmoc-L-Ser(tBu)-OH
(0.52 M in DMF dry, 9 eq., 381 ml), and using PyBOP
(0.52 M in NMP dry, 9 eq., 381 ml)/DIPEA (18 eq., 61 ml) as
the activating system (762 ml DMF/NMP, 1 : 1 v/v; for
20 min). Fmoc deprotection of the amino groups with 20%
piperidine in DMF for 15 min followed each coupling step,
and by the UV Fmoc test (e301 = 7800) it was possible to
evaluate the incorporation yields of each serine unit. No
capping step was performed to avoid the acetylation of the
free serine hydroxyl groups. Before the final Fmoc removal,
the free hydroxyl groups were esterified with the nucleobase
acetic acid: to a stirred solution of TCH2COOH (400 mmol,
10 eq. per OH, 74 mg) in NMP (600 ml) at 0 1C, a solution of
DCC (440 mmol, 11 eq. per OH, 91 mg)/DMAP (20 mmol,
0.5 eq. per OH ) in NMP (700 ml) was added under Ar
atmosphere. After 1 h, the white precipitate of DCU was
removed by filtration in a dry-box, and the yellow filtrate
solution containing the activated nucleobase acetic acid was
added to the pre-swelled resin and allowed to react for 3 h. The
UV test of the final Fmoc revealed an overall yield for the
Haen. TCH COOH, TFA, Rink-MBHA-amide resin were
¨
2
from Fluka. DCM and TFA (for HPLC) were from Romil.
ANS, deuterated DMSO, DCC, DIPC, DIPEA, DMAP,
FITC and TIS were from Sigma-Aldrich. Diethyl ether was
from Carlo Erba. dA12 was from Biomers.
Apparatus
1H NMR and 13C NMR spectra were recorded at 25 1C on a
Varian unity 400 MHz spectrometer. Chemical shifts (d) are
given in parts per million (ppm). Proton chemical shifts were
referenced to residual CHD2SOCD3 (d = 2.49, quin) signals.
13C NMR chemical shifts were referenced to the solvent
(CD3SOCD3: d = 39.5, sept).
Centrifugations were performed on a Z 200 A Hermle
centrifuge (4 min at 4000 rpm).
Products were analysed and characterized by LC-MS on an
MSQ mass spectrometer (ThermoElectron, Milan, Italy)
equipped with an ESI source operating at 3 kV needle voltage
and 320 1C, and with a complete Surveyor HPLC system,
comprising an MS pump, an autosampler, and a PDA
detector, by using a Phenomenex Jupiter C18 300 A (5 mm,
4.6 ꢁ 150 mm) column. Gradient elution was performed
c
1078 Mol. BioSyst., 2011, 7, 1073–1080
This journal is The Royal Society of Chemistry 2011

View Article Online
tetrapeptide backbone of about 67%. Subsequently, oligomer 1
was detached from the resin by TFA/TIS/H2O (95 : 2.5 : 2.5)
treatment and recovered by precipitation with cold diethyl ether,
centrifugation and lyophilization. The crude material was
purified by semipreparative HPLC on a C18 column using a
linear gradient of 5% (for 5 min) to 30% B0 in A0 over 30 min:
tR = 10.6 min; UV quantification of the purified product gave
4.2 mmol of 1 (2.9 mg; 21% yield). LC-ESIMS characterization
(Fig. S2, ESIw). Method: 5% (5 min) to 40% B in A over 10 min,
tR = 9.32 min; m/z: 1397.18 (found), 1396.25 (expected for
[2 ꢁ (C26H35N9O14) + H]+); 720.61 (found), 722.59 (expected
for [C26H35N9O14 + Na]+); 699.27 (found), 698.63 (expected for
[C26H35N9O14 + H]+).
(3H, s, CH3 thymine); dC (100 MHz, DMSO-d6): 174.6,
172.12, 168.25, 160.0, 154.9, 147.7, 145.4, 144.7, 131.6, 131.1,
129.3, 124.1, 112.6, 69.9, 68.17, 56.8, 52.1, 15.9.
Solid phase synthesis of nucleopeptide 1 by using monomer 5
The tetrapeptide H-[Ser(T)-Ser]2-NH2 (1, Fig. 3) was
assembled on Rink-amide–NH2 resin (0.5 mmol gꢀ1, 20 mg,
10 mmol) by alternatively coupling Fmoc-L-Ser(T)-OH (0.2 M
in NMP, 5 eq., 250 ml) and the commercial Fmoc-L-Ser(tBu)-
OH (5 eq., 19 mg), and using PyBOP (5 eq., 26 mg)/DIPEA
(10 eq., 17 ml) as the activating system in NMP (about 400 ml
for 20 min). Capping was performed with 20% (Ac)2O/5%
DIPEA for 10 min, while Fmoc deprotection of the amino
groups was obtained with 20% piperidine in DMF for 15 min.
The incorporation yields of each Fmoc-L-Ser(T)-OH
monomer (5) were of about 80%. The overall oligomer yield,
calculated on the basis of the UV Fmoc test, was of about
50%. Cleavage from the solid support and purification of
oligomer 1 were performed as described before. ESI-MS
characterization confirmed the identity of the nucleopeptide.
UV quantification of the purified product gave 5 mmol of 1
(40% yield).
Synthesis of the Fmoc-L-Ser(T)-OH monomer (5)
The nucleobase-containing monomer was synthesized starting
from the commercially available Fmoc-L-Ser(tBu)-OH (3,
Fig. 2). About 100 mg of 3 (0.261 mmol) were treated with
TFA/DCM 1 : 1 (3 ml, RT, 2 h) to selectively remove the
tert-butyl group. After removal of TFA and DCM under
nitrogen stream, the residue was resuspended in 6 ml of cold
diethyl ether by the aid of sonication, and Fmoc-L-Ser-OH (4)
was recovered by centrifugation as a white precipitate (84 mg,
0.257 mmol, almost quantitative yield). Characterization of
Fmoc-L-Ser-OH 4. LC-ESIMS-method: 15% (5 min) to 95% B
in A over 15 min, tR = 12.50 min; m/z: 983.82 (found), 983.03
(expected for [3 ꢁ (C18H17NO5) + H]+); 678.29 (found),
677.67 (expected for [2 ꢁ (C18H17NO5) + Na]+); 656.59
(found), 655.69 (expected for [2 ꢁ (C18H17NO5) + H]+);
350.83 (found), 350.33 (expected for [C18H17NO5 + Na]+);
329.55 (found), 328.35 (expected for [C18H17NO5 + H]+)
(Fig. S3, ESIw). NMR-dH (400 MHz, CDCl3) 7.72–7.24 (8H,
m, aromatic CH Fmoc), 6.25 (1H, d, 3JH,H = 7.2 Hz, CONH),
4.51–4.12 (4H, m, FmocCH-CH2, FmocCH-CH2, CHalpha),
4.10–3.89 (2H, bdd, CH2CHalpha); dC (100 MHz, CDCl3)
173.6, 157.0, 144.1, 141.6, 128.1, 127.5, 125.6, 120.3, 67.6,
CD studies
All the CD spectra were recorded using the following
parameters: scanning speed, 50 nm minꢀ1; data pitch, 2 nm;
band width, 2 nm; response, 4 s; 7 accumulations.
Nucleic acids binding studies were performed at 5 1C in
10 mM phosphate buffer (pH 7.5), using a tandem cell. CD
spectra relative to the self-assembly studies of 1 were
performed at 10 1C in 10 mM phosphate buffer (pH 7.5),
using a 1 mm quartz cell. For each nucleopeptide concentration,
various CD spectra were collected until the system reached
equilibrium.
63.3, 56.4, 47.4. Subsequently, compound
4 (84 mg,
Human plasma stability assay
0.257 mmol) was dissolved in 1 ml DMF and coupled with
thymine acetic acid (1.5 eq., 71 mg), which was previously
preactivated with DIPC (1.5 eq., 60 ml)/DMAP (cat., ca. 1 mg)
in DMF (1 ml), at room temperature (Fig. 2). After 21 h,
the reaction was quenched by adding water, freezing and
lyophilizing the mixture. The crude material was resuspended
in 20% aqueous CH3CN and purified by semipreparative
HPLC on a C18 column using a linear gradient of 20%
(5 min) to 80% B in A over 20 min; tR = 19.5 min. The
desired product 5 was obtained in 30% yield (39 mg,
0.079 mmol). Characterization of Fmoc-L-Ser(T)-OH 5.
LC-ESIMS-method: 15% (5 min) to 95% B0 in A0 over
15 min; tR = 12.82 min; m/z: 987.83 (found), 987.97 (expected
for [2 ꢁ (C25H23NO8) + H]+); 532.46 (found), 532.58
(expected for [C25H23NO8 + K]+); 517.71 (found), 516.47
180 ml of nucleopeptide 1 solution (92 mM) was added to 120 ml
of 100% Human Plasma (HP) in a micro-vial, and the mixture
was incubated at 37 1C, resulting in a final 55 mM concentration
of 1 and 40% HP. Aliquots (30 ml) of the reaction mixture
were withdrawn at various times (0, 15, 30 min, and 1, 2, 3, 4,
5, 6, 15 h), added to a solution of 20% CH3CN in H2O (0.1%
TFA), left 2 min at 85 1C, and analyzed by RP-HPLC using a
linear gradient of 5 (3 min) to 21% CH3CN (0.1%TFA) in
H2O (0.1%TFA) over 9 min (1.0 mL minꢀ1, at 45 1C).
Static light scattering
A stock solution of nucleopeptide was filtered through a 0.02
mm Millex syringe driven filter unit (Millipore, Bedford, MA).
After dilution samples containing the nucleopeptide were
prepared in 10 mM phosphate buffer, pH 7.5. All measurements
were registered in triplicates for 2 min acquisition time. The
hydrodynamic radius (Rh) of the scattering molecules was
derived, using the ASTRA software, from the diffusion
coefficient using the Einstein–Stokes equation.
(expected for [C25H23NO8
+
Na]+); 494.82 (found),
495.49 (expected for [C25H23NO8 + H]+) (Fig. S4, ESIw).
NMR-dH (400 MHz, DMSO-d6): 11.39 (1H, s, NH thymine),
7.90–7.32 (10H, m, aromatic CH Fmoc, Fmoc-NH, CH
thymine), 4.49 (2H, d, FmocCH-CH2), 4.48–4.01 (6H, m,
NCH2CO, FmocCH-CH2, CHalpha, and CH2CHalpha), 1.73
c
This journal is The Royal Society of Chemistry 2011
Mol. BioSyst., 2011, 7, 1073–1080 1079

View Article Online
Fluorescence
References
Fluorescence aggregation studies with ANS were carried out
using the following instrument parameters: scan type, fluorescence
emission; excitation wavelength, 380 nm; emission wavelength,
400 nm; excitation and emission slits, 5 nm; recording speed
125 nm minꢀ1. Emission spectra of a 430 ml solution ANS
alone (30 mM in 10 mM phosphate buffer, pH = 7.5), and
after successive additions (7.8 ml each) of nucleopeptide 1
(550 mM), were collected. The changes in emission were
tracked until the system reached equilibrium (about 90 min).
Experiments were performed at both 25 and 10 1C giving the
same results reported in Fig. 7a. The kinetic experiments on
nucleopeptide 1 aggregation were performed using the following
instrument parameters: scan type, fluorescence emission;
excitation wavelength, 485 nm; emission wavelength,
490 nm; excitation and emission slits, 2.5 nm; recording speed
125 nm minꢀ1. To a 121 mM FITC solution in H2O
(pH = 7.0), 5 ml of 1 (550 mM) were added, obtaining a
nucleopeptide concentration of 6.5 mM. Fluorescence spectra
were collected at the following times: 0, 5, 20, 60, 90, 120,
180 min. Experiments were performed at both 25 and 10 1C
giving the same results reported in Fig. 7b.
1 N. M. Bell and J. Micklefield, Chemical Modification of
Oligonucleotides for Therapeutic, Bioanalytical and Other
Applications, ChemBioChem, 2009, 17, 2691–2703.
2 A. J. H. Nollet, C. M. Hutino and U. K. Pandit, Unconventional
Nucleotide Analogues—I: N 9-Purinyl Alpha-Amino Acids,
Tetrahedron, 1969, 25, 5971–5981.
3 J. D. Buttrey, A. S. Jones and R. T. Walker, Synthetic analogues of
polynucleotides—XII: The resolution of DL-b-(thymin-1-yl)alanine
and polymerisation of the b-(thymin-1-yl)alanines, Tetrahedron,
1975, 31, 73–75.
4 E. Lioy and H. Kessler, Synthesis of a new chiral peptide analogue
of DNA using ornithine subunits and solid-phase peptide synthesis
methodologies, Liebigs Ann., 1996, 2, 201–204.
5 P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Sequence-
selective recognition of DNA by strand displacement with a
thymine-substituted polyamide, Science, 1991, 6, 1497–1500.
6 S. Matsumara, T. Takahashi, A. Ueno and H. Mihara,
Complementary nucleobase interaction enhances peptide–peptide
recognition and self-replicating catalysis, Chem.–Eur. J., 2003, 9,
4829–4837.
7 S. Pensato, M. Saviano, N. Bianchi, M. Borgatti, E. Fabbri,
R. Gambari and A. Romanelli, Gamma-Hydroxymethyl PNAs:
Synthesis, interaction with DNA and inhibition of protein/DNA
interactions, Bioorg. Chem., 2010, 38, 196–201.
8 G. N. Roviello, D. Musumeci, A. De Cristofaro, D. Capasso, S. Di
Gaetano, E. M. Bucci and C. Pedone, Alternate dab-aegPNAs:
synthesis, nucleic acid binding studies and biological activity, Mol.
BioSyst., 2010, 6, 189–195.
9 G. N. Roviello, E. Benedetti, C. Pedone and E. M. Bucci,
Nucleobase-containing peptides: an overview of their characteristic
features and applications, Amino Acids, 2010, 39, 45–57.
10 U. Diederichsen, Pairing Properties of Alanyl Peptide Nucleic
Acids Containing an Amino Acid Backbone with Alternating
Configuration, Angew. Chem., Int. Ed. Engl., 1996, 35, 445–448.
11 X. Shuai, T. Merdan, F. Unger and T. Kissel, Supramolecular gene
delivery vectors showing enhanced transgene expression and good
biocompatibility, Bioconjugate Chem., 2005, 16, 322–329.
12 R. Chhabra, J. Sharma, Y. Liu, S. Rinker and H. Yan, DNA
Self-assembly for Nanomedicine, Adv. Drug Delivery Rev., 2010,
62, 617–625.
13 M. Moccia, D. Musumeci, G. N. Roviello, S. Fusco, M. Valente,
E. M. Bucci, R. Sapio, C. Pedone and P. A. Netti, Preliminary
studies on noncovalent hyperbranched polymers based on PNA
and DNA building blocks, J. Pept. Sci., 2009, 15, 647–653.
14 E. Snip, K. Koumoto and S. Shinkai, Gel formation properties of a
uracil-appended cholesterol gelator and cooperative effects of the
complementary nucleobases, Tetrahedron, 2002, 58, 8863–8873.
15 M. Mizutani, I. Kubo, K. Jitsukawa, H. Masuda and H. Einaga,
Nucleobase stacking evidenced on ternary metal (palladium(II),
copper(II)) complexes with nucleobase amino acids and aromatic
diimines, Inorg. Chem., 1999, 38, 420–421.
Conclusions
In conclusion, we realized by two different synthetic strategies
a dithymine tetrapeptide (1, Fig. 1), made of both thymine-
containing and unfunctionalized L-serine units alternated in
the sequence. Our novel analogue showed a good solubility in
water, a desirable property for a peptide-like oligonucleotide
mimetic. We demonstrated by the above described studies that
this novel analogue of TpT DNA dinucleoside mono-
phosphate can form supramolecular architectures, containing
a hydrophobic core able to incorporate poorly water-soluble
molecules, which are modified by metal ion interaction.
Furthermore, the nucleopeptide is biodegradable and is able
to interact very weakly with complementary DNA. The
preliminary findings of the present work encourage us to
further study the alternated L-serine/L-nucleoserine nucleo-
peptides, obtainable in the solid phase with an appropriate
sequence and number of bases, as novel materials that could
be beneficial in the biomedical research as drug delivery
agents.
16 A. Kuesel, J. Zhang, M. Alvarino Gil, C. A. Stueckl, W.
Meyer-Klaucke, F. Meyer and U. Diederichsen, Metal binding
within a peptide based nucleobase stack with tuneable double
strand topology, Eur. J. Inorg. Chem., 2005, 4317–4324.
17 D. Armentano, T. F. Mastropietro, M. Julve, R. Rossi, P. Rossi
and G. De Munno, A New Octanuclear Copper(II)–Nucleoside
Wheel, J. Am. Chem. Soc., 2007, 129, 2740–2741.
Acknowledgements
18 M. G. Santangelo, A. Medina-Molner, A. Schweiger, G. Mitrikas
and B. Spingler, Structural analysis of Cu(II) ligation to the
50-GMP nucleotide by pulse EPR spectroscopy, JBIC, J. Biol.
Inorg. Chem., 2007, 12, 767–775.
19 C. Conato, R. Gavioli, R. Guerrini, H. Kozlowski, P. Mlynarz,
C. Pasti, F. Pulidori and M. Remelli, Copper complexes of
glycyl-histidyl-lysine and two of its synthetic analogues: chemical
behaviour and biological activity, Biochim. Biophys. Acta, 2001,
1526, 199–210.
We thank Prof. Antonio Roviello, Dr Mariangela Castiglione,
Dr Valentina Roviello, Dr Giuseppe Perretta, Mrs Angela
Galeotafiore and Mr Leopoldo Zona for their precious
suggestions and invaluable assistance. We are also grateful
to the institutions that supported our laboratory (Consiglio
`
Nazionale delle Ricerche and Universita degli Studi di Napoli
‘Federico II’).
c
1080 Mol. BioSyst., 2011, 7, 1073–1080
This journal is The Royal Society of Chemistry 2011