G.N. Roviello et al. / International Journal of Pharmaceutics 415 (2011) 206–210
209
(positive ions) which confirmed the identity of the oligomer. The
nucleopeptide showed a good water solubility and a good chemical
stability in the aqueous solutions.
4. Conclusion
In conclusion, a Fmoc-protected thymine nucleoamino acid (3,
Fig. 2), useful for the solid phase assembly of aromatic nucleopep-
tides, was prepared for the first time by a convenient synthetic
route and fully characterized by NMR and ESI-MS. Furthermore,
we realized by a synthetic strategy employing the novel Fmoc-
protected nucleoamino acid, a dithymine tetrapeptide (4, Fig. 4),
made of both thymine-containing and unfunctionalized l-tyrosine
units alternated in the sequence. The synthetic procedure designed
and realized in this work is flexible and should allow for the
properties of both the nucleoamino acid and the nucleopeptide
here described in analogy to the reports on single-nucleobase-
bearing and nucleopeptide-based networks (Snip et al., 2002;
Moccia et al., 2009; Roviello et al., 2011a,b) in view of their
employment for the realization of supramolecular structures ben-
eficial in the biomedical research as drug and gene delivery
tools.
Fig. 5. LC–ESI-MS (positive ions) of nucleopeptide 4; tR = 8.77 min; method: 15%
(5 min) to 95% B in A over 15 min (A = 0.05% TFA in H2O, B = 0.05% TFA in CH3CN).
recovered by precipitation with cold diethyl ether, centrifu-
gation and lyophilization. The crude material was purified by
semipreparative HPLC on a C18 column using a linear gradient
of 25% (for 5 min) to 70% Bꢀ in Aꢀ over 25 min: tR = 12.3 min;
The purified oligomer 4 was dissolved in MilliQ water and
quantified by UV measurements (T = 80 ◦C, absorbance value at
ꢀ = 260 nm). The epsilon value used for the quantification of the
oligomer (17.2 m M−1) was calculated using the molar extinction
coefficient of thymine PNA monomer (8.6 m M−1); UV quantifi-
cation of the purified product gave 3.2 mol of 1 (3.2 mg; 32%
yield). LC–ESI-MS characterization of nucleopeptide 4. Method: 15%
(5 min) to 95% Bꢀ in Aꢀ over 15 min; tR = 8.77 min. ESI-MS (Fig. 5)
m/z: 1041.51 (found), 1041.12 (expected for [C50H51N9O14 + K]+);
1024.14 (found), 1025.01 (expected for [C50H51N9O14 + Na]+);
1002.27 (found), 1003.03 (expected for [C50H51N9O14 + H]+).
Acknowledgments
We thank Dr. Mariangela Castiglione, Dr. Annalisa Cesarani
and Dr. Valentina Roviello for their precious suggestions, and Dr.
Giusepppe Perretta and Mr. Leopoldo Zona for their invaluable
technical assistance. We are also grateful to the institutions that
supported our laboratory (Consiglio Nazionale delle Ricerche and
Università degli Studi di Napoli ‘Federico II’).
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We realized a convenient and fast synthetic route to a chiral
Fmoc nucleo-l-tyrosine monomer, in which the (S)-2-amino-3-
(4ꢀ-hydroxyphenyl)-propanoic moiety was connected to the DNA
nucleobase by an ester bond, suitable for the solid phase synthesis
of aromatic nucleopeptides. The nucleobase-containing monomer
was synthesized starting from the commercially available Fmoc-
l-Tyr(tBu)-OH (1, Fig. 2). In the first synthetic step the tert-butyl
group was selectively removed with trifluoroacetic acid to give the
intermediate 2 with the free phenolic hydroxyl group (Fig. 2).
Subsequently, Fmoc-l-Tyr-OH 2 was coupled with the commer-
conditions with the best results coming from the use of DIPC/DMAP
in DMF as solvent in the presence of TMP (Fig. 2). After RP-HPLC
purification the desired product 3 was obtained in 34% yield and
characterized by 13C/1H NMR and ESI-MS (Fig. 3).
Roviello, G.N., Gröschel, S., Pedone, C., Diederichsen, U., 2009. Synthesis of
novel MMT/acyl-protected nucleo alanine monomers for the preparation of
DNA/alanyl-PNA chimeras. Amino Acids 38, 1301–1309.
3.2. Synthesis of the aromatic nucleopeptide 4
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Subsequently, the nucleo-tetrapeptide 4 was assembled in solid
phase using a synthetic strategy employing the coupling of both
monomer 3 and the commercial Fmoc-l-Tyr(tBu)-OH achieved by
using PyBop/DIPEA as activating system (Fig. 4). Nucleopeptide was
purified by RP HPLC, quantified by UV and characterized by ESI-MS