Synthesis and biophysical characterization of G-rich oligonucleotides conjugated with sugar-phosphate tails

Article abstract of DOI:10.1080/15257770701528164

A number of 5′-and 3′-glycoconjugates of the oligonucleotide 5′d(TGGGAG)^3′ have been synthesized, exploiting fully automated, online phosphoramidite-based solid phase strategy, as potential anti-HIV-1 agents. The thermodynamic stability of the resulting quadruplexes has been investigated by thermal denaturation studies, via a detailed CD Q1 analysis. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701528164

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Synthesis and Biophysical
Characterization of G-Rich
Oligonucleotides Conjugated with Sugar-
Phosphate Tails
J. D'Onofrio a , E. Erra b , G. Di Fabio a , A. Iadonisi a , L. Petraccone
b , L. De Napoli a , G. Barone b , J. Balzarini c , C. Giancola b & D.
Montesarchio a
a Dipartimento di Chimica Organica e Biochimica , Università degli
Studi di Napoli “Federico II,” , Napoli, Italy
b Dipartimento di Chimica , Universitá degli Studi di Napoli
“Federico II,” , Napoli, Italy
c Rega Institute for Medical Research , Katholieke Universiteit
Leuven , Leuven, Belgium
Published online: 05 Dec 2007.
To cite this article: J. D'Onofrio , E. Erra , G. Di Fabio , A. Iadonisi , L. Petraccone , L. De
Napoli , G. Barone , J. Balzarini , C. Giancola & D. Montesarchio (2007) Synthesis and Biophysical
Characterization of G-Rich Oligonucleotides Conjugated with Sugar-Phosphate Tails, Nucleosides,
Nucleotides and Nucleic Acids, 26:10-12, 1225-1229, DOI: 10.1080/15257770701528164
To link to this article: http://dx.doi.org/10.1080/15257770701528164
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1225–1229, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701528164
SYNTHESIS AND BIOPHYSICAL CHARACTERIZATION
OF G-RICH OLIGONUCLEOTIDES CONJUGATED
WITH SUGAR-PHOSPHATE TAILS
J. D’Onofrio
Dipartimento di Chimica Organica e Biochimica, Universita` degli Studi
2
di Napoli “Federico II,” Napoli, Italy
E. Erra Dipartimento di Chimica, Universita` degli Studi di Napoli “Federico II,”
2
Napoli, Italy
G. Di Fabio and A. Iadonisi
Dipartimento di Chimica Organica e Biochimica,
2
Universita` degli Studi di Napoli “Federico II,” Napoli, Italy
L. Petraccone
Dipartimento di Chimica, Universita` degli Studi di Napoli “Federico
2
II,” Napoli, Italy
L. De Napoli
Dipartimento di Chimica Organica e Biochimica, Universita` degli Studi
2
di Napoli “Federico II,” Napoli, Italy
G. Barone
Dipartimento di Chimica, Universita` degli Studi di Napoli “Federico II,”
2
Napoli, Italy
J. Balzarini
Leuven, Belgium
Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Dipartimento di Chimica, Universita` degli Studi di Napoli “Federico II,”
2
C. Giancola
2
Napoli, Italy
D. Montesarchio
Dipartimento di Chimica Organica e Biochimica, Universita` degli
2
Studi di Napoli “Federico II,” Napoli, Italy
ꢁ
ꢁ
A number of 5ꢁ-and 3ꢁ-glycoconjugates of the oligonucleotide 5 d(TGGGAG)3 have been syn-
2
thesized, exploiting fully automated, online phosphoramidite-based solid phase strategy, as potential
anti-HIV-1 agents. The thermodynamic stability of the resulting quadruplexes has been investigated
by thermal denaturation studies, via a detailed CD analysis.
Keywords Conjugated oligonucleotides; G-quadruplex structures sugar-phosphate
tails; anti-HIV-1 agents
Address correspondence to D. Montesarchio, Dipartimento di Chimica Organica e Biochimica,
Universita` degli Studi di Napoli “Federico II,” Via Cintia 4, I-80126 Napoli, Italy. E-mail: daniela.
montesarchio@unina.it
1225

1226
J. D’Onofrio et al.
Recently, Hotoda and coworkers demonstrated that a number of G-
rich oligonucleotides, decorated with large aromatic groups at their 5ꢁ-
end, are potent anti-HIV active compounds.[1] Interestingly, the lead
ꢁ
ꢁ
sequence5 d(TGGGAG)3 was found to be inactive against HIV-1 without
end modifications, and active at µM concentrations when 5ꢁ-conjugated with
aromatic groups.[2,3] This antiviral activity was correlated with the ability of
G-rich sequences to form stable quadruplex structures,[4,5] postulated to be
the species directly interacting with the in vivo targets.
Among the various modifications proposed to improve the pharma-
cological profile of oligonucleotides, end-modification with carbohydrates
may generate useful hybrids for targeting cells with DNA fragments. Glyco-
conjugation increases nucleases resistance and cell uptake and can also re-
duce unwanted aggregation. Extensive research has been carried out to
conjugate peptides to carbohydrate-based ligands; on the contrary, only a
few examples of DNA glyco-conjugates are reported in the literature. In
this frame, we[6] recently described a straightforward protocol based on the
usage of appropriate glucose and sucrose phosphoramidite derivatives, as
model compounds for mono- and disaccharide building blocks, which can
be easily incorporated in the solid phase in a standard automated oligonu-
cleotide synthesis to generate stable phosphodiester linkages connecting
the saccharide residues to one or both OH termini of the oligonucleotide
chain.
Here we present the design and synthesis of a set of model glyco-
ꢁ
ꢁ
conjugated oligonucleotides carrying Hotoda’s sequence d(5 TGGGAG3 ),
together with preliminary results on their biophysical characterization. We
have synthesized 5ꢁ- and 3ꢁ-conjugated 6-mers b-g (Scheme 1 and Table 1)
and compared their quadruplex formation abilities with unmodified
6-mer a. Glucose and sucrose-containing oligomers b-e were prepared
essentially following previously reported procedures.[6] This synthetic
strategy has been here extended to mannose-linked oligomers f and g,
as examples of glyco-conjugation which can find direct application for
TABLE 1 Synthesized oligonucleotides and Tm values of
the resulting quadruplexes, obtained by CD-monitored
(at 264 nm) thermal denaturation curves
Sequence (5ꢁ-3ꢁ)
Tm (◦C)
ꢁ
ꢁ
5 TGGGAG3 (a)
41
26
44
35
36
31
45
1
1
1
1
1
1
1
ꢁ
ꢁ
glucose-5 TGGGAG3 (b)
ꢁ
ꢁ
5 TGGGAG3 -glucose (c)
ꢁ
ꢁ
sucrose-5 TGGGAG3 (d)
ꢁ
ꢁ
5 TGGGAG3 -sucrose (e)
ꢁ
ꢁ
mannose-5 TGGGAG3 (f)
ꢁ
ꢁ
5 TGGGAG3 -mannose (g)

G-Rich Olligonucleotides
1227
SCHEME 1
lectine recognition. In all cases, starting from 100 mg of commercially
available guanosine-functionalized CPG support (0.1 meq/g, used in
connection with phosphoramidite 5 for the synthesis of 5ꢁ-conjugated
oligomers) or with the first sugar residue (7, 0.06-0.08 meq/g, used for the
3ꢁ-conjugation7), on average 80-110 OD units of pure a-g could be isolated,
after HPLC purification, then characterized by MALDI-TOF MS.
CD studies for all the investigated oligomers showed spectra with a typ-
ical negative band at ca. 240 nm and a large positive band at 264 nm,
diagnostic of tetramolecular, parallel G-quadruplex structures. To deter-
mine the thermodynamic stability of the resulting quadruplexes, thermal

1228
J. D’Onofrio et al.
ꢁ
ꢁ
FIGURE 1 Superimposed CD spectra of 5 TGGGAG3 (a) taken in the temperature range 10–75◦C.
denaturation studies were carried out via detailed CD analyses. Data con-
cerning the unmodified sequence are here reported in Figures 1 and 2. The
measured Tm data for all the studied quadruplexes, listed in Table 1, show
that the saccharide-phosphate tails in G-rich oligomers do influence the sta-
bility of the resulting complexes. In the case of the disaccharide moiety, a
destabilization occurred both in the 5ꢁ- and 3ꢁ-conjugation (d and e, respec-
tively). The insertion of a monosaccharide-phosphate residue at the 5ꢁ end
ꢁ
ꢁ
FIGURE 2 Plot of the molar ellipticities of 5 TGGGAG3 (a) at 264 nm as a function of the temperature.

G-Rich Olligonucleotides
1229
(b and f) was detrimental to quadruplex formation, while a ꢀTm = + 3–4◦C
was obtained for the 3ꢁ-conjugation (c and g).
The complete definition of the thermodynamic parameters for the
quadruplex complexes, as well as studies on the in vitro antiviral activity
of the novel glyco-conjugated oligonucleotides, are currently underway and
will be presented in due course. These data can be useful to develop novel
non-antisense anti-HIV active oligonucleotide-based lead compounds.
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logically active oligodeoxyribonucleotides. 5. 5ꢁ-End-substituted d(TGGGAG) possesses anti-human
immunodeficiency virus type 1 activity by forming a G-quadruplex structure. J. Med. Chem. 1998, 41,
3655–3663.
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7. Oligonucleotides c, e, and g could have alternatively synthesized using commercially available univer-
sal solid supports and 2ꢁ-deoxyribonucleoside 5ꢁ-phosphoramidites, in connection with sugar phos-
phoramidite building block 5. This strategy was here not adopted on the basis of the overall higher
costs and lower expected yields associated with the usage of nucleoside 5ꢁ-phosphoramidites.