Design, synthesis and characterisation of guanosine-based amphiphiles

Article abstract of DOI:10.1002/chem.201101827

A small library of sugar-modified guanosine derivatives has been prepared, starting from a common intermediate, fully protected on the nucleobase. Insertion of myristoyl chains and of diverse hydrophilic groups, such as an oligoethylene glycol, an amino acid or a disaccharide chain, connected through in vivo reversible ester linkages, or of a charged functional group provided different examples of amphiphilic guanosine analogues, named G1-G7 herein. All of the sugar-modified derivatives were positive in the potassium picrate test, showing an ability to form G-tetrads. CD spectra demonstrated that, as dilute solutions in CHCl₃, distinctive G-quadruplex systems may be formed, with spatial organisations dependent upon the structural modifications. Two compounds, G1 and G2, proved to be good low-molecular-weight organogelators in polar organic solvents, such as methanol, ethanol and acetonitrile. Ion transportation experiments through phospholipid bilayers were carried out to evaluate their ability to mediate H⁺ transportation, with G5 showing the highest activity within the investigated series. Moreover, G3 and G5 exhibited a significant cytotoxic profile against human MCF-7 cancer cells in in vitro bioassays.

Full text of DOI:10.1002/chem.201101827

DOI: 10.1002/chem.201101827
Design, Synthesis and Characterisation of Guanosine-Based Amphiphiles
Luca Simeone,[a] Domenico Milano,[a] Lorenzo De Napoli,[a] Carlo Irace,[b]
Antonio Di Pascale,[b] Mariangela Boccalon,[c] Paolo Tecilla,[c] and
Daniela Montesarchio*[a]
Abstract: A small library of sugar-
modified guanosine derivatives has
modified derivatives were positive in
the potassium picrate test, showing an
ability to form G-tetrads. CD spectra
demonstrated that, as dilute solutions
in CHCl3, distinctive G-quadruplex sys-
tems may be formed, with spatial or-
ganisations dependent upon the struc-
tural modifications. Two compounds,
G1 and G2, proved to be good low-mo-
lecular-weight organogelators in polar
organic solvents, such as methanol, eth-
anol and acetonitrile. Ion transporta-
tion experiments through phospholipid
bilayers were carried out to evaluate
their ability to mediate H+ transporta-
tion, with G5 showing the highest ac-
tivity within the investigated series.
Moreover, G3 and G5 exhibited a sig-
nificant cytotoxic profile against
human MCF-7 cancer cells in in vitro
bioassays.
been prepared, starting from
a
common intermediate, fully protected
on the nucleobase. Insertion of myris-
toyl chains and of diverse hydrophilic
groups, such as an oligoethylene glycol,
an amino acid or a disaccharide chain,
connected through in vivo reversible
ester linkages, or of a charged function-
al group provided different examples
of amphiphilic guanosine analogues,
named G1–G7 herein. All of the sugar-
Keywords: amphiphiles · antiprolif-
eration · G-quadruplexes · iono-
phores · nucleosides
Introduction
lar devices and in pursuing more efficient therapeutic strat-
egies.[1,2] In this area, nucleolipids derived from biologically
active nucleosides/nucleotides can be very promising pro-
drugs with unique structural and biophysical properties.
These features can be ascribed to their ease of self-assembly
into stable liposomes in aqueous media, thus providing effi-
cient uptake through the phospholipid bilayers by cells, and
conferring protection to the drug from extracellular enzy-
matic degradation.[3] From an alternative perspective, in-
creasing interest is also devoted to these compounds as gene
transfection agents, particularly for DNA and siRNA in vivo
delivery.[4]
Nucleolipids are hybrid molecules consisting of a lipid core
covalently linked to a nucleobase, nucleoside or nucleotide.
Hybrid lipid–nucleoside structures occur in eukaryotic and
prokaryotic cells, showing antimicrobial, antifungal, antiviral
or anti-tumour properties. Over the last two decades, several
novel nucleolipids have been synthesised that combine the
pharmacological potential of nucleosides or nucleotides with
the aggregation properties of vesicle-forming lipids. Recent-
ly, these compounds have attracted significant attention as
useful synthetic platforms in the design of artificial molecu-
Although several amphiphilic nucleolipids based on ade-
nosine, cytosine, uridine or thymidine are described in the
literature, very few examples are known for guanosine[1,5]
and the potential applications of guanosine-based amphi-
philes in biomedical applications and/or as self-assembling
materials are almost completely unexplored. Furthermore,
guanosine-based systems deserve special interest in supra-
molecular chemistry due to the known self-aggregation
properties of guanine and guanosine derivatives. Several
supramolecular architectures generated by lipophilic guano-
sines based on unusual structural motifs, such as G-tetrads
stabilised by metal cations or, in the absence of cations, G-
ribbon structures that in some cases are also organised in
sheet-like two dimensional assemblies, have been described
in the literature.[6–9]
[a] Dr. L. Simeone, Dr. D. Milano, Prof. L. De Napoli,
Prof. D. Montesarchio
Department of Organic Chemistry and Biochemistry
University “Federico II” of Napoli
Via Cintia, 4, 80126 Napoli (Italy)
Fax : (+39) 081-674393
E-mail: daniela.montesarchio@unina.it
[b] Dr. C. Irace, Dr. A. Di Pascale
Department of Experimental Pharmacology
University “Federico II” of Napoli
Via D. Montesano, 48, 80131 Napoli (Italy)
[c] Dr. M. Boccalon, Prof. P. Tecilla
Department of Chemical Sciences, University of Trieste
Via L. Giorgieri, 34127 Trieste (Italy)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101827. It contains general
synthetic methods, detailed molecular characterisation (1H and
13C NMR and MS data) and the synthesis of compounds 1–7a–d and
8–16 and general procedures for the ionophoric activity experiments.
Herein, the preparation of a set of amphiphilic guanosine-
based derivatives by simple synthetic protocols combining
different bio-inspired building blocks is described. Their
structural and biological properties are investigated in pre-
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FULL PAPER
liminary studies aimed at identifying new tools with the po-
tential to profitably complement the lipophilic congeners re-
cently studied by the research groups of G. P. Spada,[6–9] J. T.
Davis[9–12] and K. Araki[13–15], among others, further expand-
ing the chemical diversity offered by nucleosides.
the nucleoside moiety and are rapidly cleaved by cell ester-
ases, liberating the “free” nucleosides within the cells, once
transportation through the lipid bilayers has been achieved.
For the preparation of a library of amphiphilic, sugar-
modified guanosine derivatives, the general design presented
herein is based on the insertion of a saturated fatty acid resi-
due, that is myristic acid, as the lipophilic chain. In the syn-
thesised compounds, named G1–G7, this group was attached
to the secondary hydroxyl groups of ribose to give bi-tailed
compounds G1–G5, or to the 5’-OH group, to give mono-
tailed derivative G7. Higher molecular diversity was intro-
duced through the use of different hydrophilic groups, with
polyethers (introduced in G1, G6 and G7), charged func-
tional groups (in G3), natural a-amino acids (in G2 and G4)
and carbohydrates (in G5) represented. Aiming for a
simple, versatile and finely tunable synthetic method, the li-
brary of sugar-modified amphiphilic analogues was thus pre-
pared via a common intermediate (3, Scheme 1) that is ex-
haustively protected on the nucleobase[16] and containing
Results and Discussion
Synthesis of compounds G1–G7: A variety of synthetic strat-
egies have been used to prepare nucleolipids. In most cases,
the design is based on the chemical modification of the
sugar functionality, thus minimising the perturbation of the
nucleobase moiety and keeping their critical recognition
sites unaltered. Ester functionalities are typically considered
to be ideal chemical connections for nucleosidic ribose deri-
vatisations, offering several advantages. These linkages can
be obtained through straightforward and high yielding con-
densation reactions that do not require prior modification of
Scheme 1. Synthesis of intermediate 3: i) Boc2O (Boc=tert-butoxycarbonyl), triethylamine (TEA), 4-dimethylaminopyridine (DMAP), CH3CN, RT, 72 h,
57%; ii) NH3, CH3OH, 12 h, RT, quantitative.
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D. Montesarchio et al.
three ribosidic OH groups available for condensation reac-
tions with various appendages.
chromatography. This derivative was then exhaustively de-
acetylated by treatment with methanolic ammonia, giving
target compound 3 in 56% overall yield over the three
steps.
For the G1–G5 series, following the approach of Grinstaff,
Berthꢁlꢁmy and co-workers,[1] the secondary ribose OH
functionalities were exploited to insert fatty acid residues,
whereas the 5’-OH was derivatised with different hydrophil-
ic groups, introduced to balance the hydrophobic contribu-
tion of the lipid chains. Compound G7 was designed as an
analogue of G1 containing the same lipophilic and hydro-
philic tails but with an inverted ratio. Uniquely, compound
G6 only contains polyether groups. In this manner, the
series of compounds with 2, 1 or 0 myristic acid tails and, in
turn, 1, 2 or 3 triethylene glycol groups (G1, G7, and G6, re-
spectively) was generated.
The synthetic route to the target sugar-modified guano-
sine analogues was thus realised by starting from compound
3, obtained in three straightforward steps from guanosine
(Scheme 1). The Boc group, though seldom used for nucleo-
base protection in nucleoside chemistry,[17] was selected to
protect the guanine moiety due to the easy installation pro-
cedure, its lipophilic character, which facilitates guanosine
manipulation in organic solvents, and above all the conven-
ient removal conditions. As a matter of fact, Boc deprotec-
tion can be achieved by mild acidic treatment (dilute tri-
fluoroacetic acid (TFA) in anhydrous organic solvents),
which is fully compatible with ester linkages and produces
only volatile side products, thereby not requiring column
chromatography to give the
The synthesis of compounds G1–G5 was accomplished
through two simple manipulations starting from derivative
6, in turn obtained in three steps from intermediate 3
(Scheme 2). Reaction of 3 with TBDPSCl and imidazole in
DMF provided 5’-protected nucleoside 4, which was then
condensed with myristic acid in the presence of DCC, clean-
ly giving 2’,3’-di-O-myristoyl derivative 5. TBDPS-group re-
moval was achieved by treatment of 5 with the Et3N·3HF in
THF, leading to target compound 6 in 70% overall yield
from 3. G1 was then obtained in almost quantitative yields
in two steps, involving first the coupling of 5’-OH-deprotect-
ed nucleoside
6 with monomethoxy(triethylene glycol)
acetic acid, previously prepared in our laboratory,[19] by
using DCC as the condensing agent, followed by an acidic
treatment with TFA (10%) in CH2Cl2 for 2 h at RT, to ach-
ieve full nucleobase deprotection.
Hybrid nucleosides G2 and G4 were obtained by using a
similar procedure based on the coupling of derivative 6 with
commercially available Fmoc-protected a-amino acids
(Fmoc-GluACTHNUTRGENN(GU OtBu)-OH and Fmoc-SerACHTUTGNREN(NUGN OTrt)-OH, respective-
ly), and also mediated by DCC activation. The choice of
Fmoc-protection in lieu of Boc-protection could, in princi-
ple, allow further derivatisation of the final compounds, for
pure, deprotected products.
To obtain compound 3, gua-
nosine was first regioselectively
protected[18] by treatment with
acetic anhydride in CH3CN and
TEA. Tri-O-acetylated deriva-
tive 1 was then reacted with an
excess of di-tert-butyl dicarbon-
ate (Boc2O) in CH3CN in the
presence of TEA and DMAP,
giving fully protected nucleo-
side 2. Interestingly, this com-
pound, obtained in more ac-
ceptable yields (57%) than pre-
viously reported procedures,[17]
showed a tert-butyl group at the
O6 position. This, in analogy
with a previous report on gua-
nine protection,[16] may be at-
tributed to spontaneous loss of
CO2 from the original N1 Boc
carbamate, which has under-
gone an internal transposition
to the O6 tert-butyl ether. In
contrast to the previously re-
ported case,[16] we could not iso-
late the tris-Boc-protected in-
termediate and only compound
Scheme 2. Synthesis of G1–G4: i) tert-butyldiphenylsilyl chloride (TBDPSCl), imidazole, DMF, RT, 2 h, 92%;
ii) CH
48 h, RT, 80%; iv) coupling with: for 7a: CH3O
Fmoc-Glu(OtBu)OH (Fmoc=fluorenylmethoxycarbonyl) in CH2Cl2, DCC, RT, 1.5 h; for 7d: Fmoc-Ser-
(OTrt)OH (Trt=trityl) in CH2Cl2, DCC, RT, 1.5 h; for 7c: SO3·Et3N, DMF, 45 min, RT, 70%; v) only for 7b
and 7d: Fmoc removal with piperidine (10%) in DMF, RT, 20–40 min; vi) trifluoroacetic acid (TFA; 10%) in
(CH2)12COOH, N,N’-dicyclohexylcarbodiimide (DCC), DMAP, 12 h, RT, 95%; iii) Et3N·3HF, THF,
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
2 was recovered after column CH2Cl2, RT, 2 h, quantitative.
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Guanosine-Based Amphiphiles
FULL PAPER
instance, by peptide elongation or attachment of additional
functional groups. For both compounds, Fmoc removal,
cleanly achieved by reaction with piperidine (10%) in DMF,
was followed by treatment with TFA (10%) in CH2Cl2 for
2 h at RT, ensuring an effective one-pot deprotection of the
guanine and of the side-chains of glutamic acid and serine
residues, masked as tert-butyl ester and trityl ether groups,
respectively.
Compound G3 was prepared by reacting 6 with the com-
mercially available complex SO3·Et3N in DMF, followed by
TFA deprotection, carried out as described above. This gave
the desired 5’-O-sulfate derivative in 70% yield over the
two steps.
Preparation of glyco–nucleolipid hybrid G5, incorporating
a disaccharide residue chosen to confer increased hydrophil-
ic character to the final derivative compared to the G1–G4
series, required prior synthetic elaboration of the disacchar-
ide building block. To this end, a,a’-d-trehalose was selected
on the basis of its favourable properties: this is, in fact, a
readily available, symmetrical, non-reducing disaccharide
that does not undergo mutarotation and shows higher chem-
ical stability compared with other common disaccharides.
The last issue is of utmost importance from a synthetic point
of view considering that the overall strategy to obtain the
described guanosine-containing nucleolipids is based on a
final acidic treatment. The succinic group was chosen as the
linker providing covalent attachment of the trehalose resi-
due to the 5’-OH end of the guanosine scaffold. This was in-
troduced on one primary hydroxyl of the disaccharide after
it had been subjected to the following derivatisations (de-
picted in Scheme 3): 1) mono-silylation of the original disac-
charide, carried out with TBDPSCl and imidazole in DMF
at 08C, giving 8 in 86% yield; 2) exhaustive Boc protection
of the remaining seven hydroxyl groups by treatment with
Boc2O in CH3CN in the presence of TEA and DMAP, lead-
ing to 9 in 58% yield; 3) removal of the tert-butyldiphenyl-
silyl protecting group by reaction with Et3N·3HF in THF,
providing 10 in 80% yield; 4) reaction with succinic anhy-
dride and catalytic DMAP in THF, giving the target succiny-
lated trehalose derivative 11 in 78% yield. This building
block was finally used in the DCC-promoted coupling with
nucleoside 6, yielding target molecule 12 in 86% yield. As
in the case of G1–G4, target compound G5 was then ob-
tained upon treatment of 12 with TFA, quantitatively giving
complete sugar and nucleobase deprotection in a single step.
Derivative G6 was obtained in two steps starting from 3.
As depicted in Scheme 4, these consisted of exhaustive deri-
vatisation with monomethoxy(triethylene glycol) acetic
acid[19] in the presence of DCC, followed by treatment of 13
with TFA.
Finally, derivative G7 was prepared from nucleoside 4
upon esterification of both secondary hydroxyl groups with
monomethoxy(triethylene glycol) acetic acid (Scheme 5).[19]
This lead to compound 14, which was desilylated, coupled
Scheme 3. Synthesis of G5: i) TBDPSCl, imidazole, DMF, 08C, 50 min, 86%; ii) Boc2O, TEA, DMAP, CH3CN, 48 h, RT, 58%; iii) Et3N·3HF, THF, RT,
72 h, 80%; iv) succinic anhydride, DMAP, THF, RT, 24 h, 78%; v) coupling with 6, DCC, CH2Cl2, RT, 3 h, 86%; vi) TFA (10%) in CH2Cl2, RT, 3 h,
quantitative.
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D. Montesarchio et al.
All the intermediate products synthesised were purified
by silica gel column chromatography and then fully charac-
terised by 1H and 13C NMR spectroscopy and ESI mass
spectrometry. Final compounds G1–G7, obtained from the
corresponding nucleobase-protected precursors by a simple
treatment with TFA (10%) in CH2Cl2, followed by repeated
co-evaporations from isopropanol until the excess TFA had
disappeared completely, were identified on the basis of their
NMR and ESI-MS spectra.
Characterisation of G1–G7
Gelling ability: Preliminary structural investigations have
been attempted on G1–G7 by means of NMR spectroscopy.
In all cases, NMR analysis of the compounds dissolved in
CDCl3 showed dramatically broadened and very badly re-
solved signals in the 1H and 13C NMR spectra that prevented
useful structural information from being obtained. With
time, the signal broadening became more and more noticea-
1
ble, particularly in the imino-proton region of the H NMR
spectra, suggesting the presence of growing superstructures
undergoing dynamic equilibria. Upon changing the solvent
from CDCl3 to the less structure-inducing CD3OD and/or
varying the temperature in the range 288–328 K, only minor
benefits in terms of resolution and line sharpness were ob-
served. These results suggested
Scheme 4. Synthesis of G6: i) CH3OACHTUNGTRNEUNG(CH2CH2O)3CH2COOH, DCC,
DMAP, CH2Cl2, RT, 48 h, 42%; ii) TFA (10%) in CH2Cl2, RT, 3 h, quan-
titative.
a
strong tendency for these
compounds to form highly ag-
gregated systems.
As a first step to determining
the properties of the newly syn-
thesised amphiphilic deriva-
tives, we investigated their solu-
bility and gelling ability (select-
ed data are given in Table 1; for
full
characterisation,
see
Table S1 in the Supporting In-
formation). A strong tendency
to aggregate, attributable to the
well-known ability of guanine-
based systems to form a rigid
network of intermolecular hy-
drogen bonds, has been con-
firmed for all of the synthesised
samples, and their capacity for
being organogelators evaluated
by the inversion method.[20] In
all cases, analysis of these sys-
tems at concentrations lower
than 20 mm did not indicate any
tendency to form stable gels,
and higher concentrations were
therefore tested.
Scheme 5. Synthesis of G7: i) CH3O
CATHGNURTENN(UNG CH2CH2O)3CH2COOH, DCC, DMAP, CH2Cl2, RT, 1 h; ii) Et3N·3HF,
THF, RT, 18 h; iii) CH3A(CH2)12COOH, DCC, DMAP, CH2Cl2, RT, 2 h; iv) TFA (10%) in CH2Cl2, RT, 2.5 h.
CHTUNGTRENNUNG
G1 and G2, examined in the
with myristic acid at the 5’-OH group, and then deprotected
at the guanine moiety.
range 3–7% w/w (corresponding to ca. 25–70 mm), gave
stable gels in polar solvents, such as methanol, ethanol and
CH3CN (pictures of the gels obtained from G1 and G2 are
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Guanosine-Based Amphiphiles
FULL PAPER
Table 1. Solubility properties of derivatives G1–G7 in selected solvents.[a]
CHCl3, but no relevant contribution to the self-as-
sembling capability of the nucleoside; it can there-
fore be assumed that its overall structuring process
is only guided by the guanosine moiety and not in-
fluenced by the sugar decorations. This nucleoside,
found to be positive to the potassium picrate test,
showed a CD spectrum with a single negative band
at 259 nm, very close to the maximum exhibited in
the absorbance spectrum registered in the same sol-
vent (262 nm). This correspondence between the
G1
G2
G3
G5
G6
G7
Solvent
26 mm
57 mm 57 mm
67 mm
57 mm 57 mm 57 mm 57 mm
CH3OH G[b] 3% G 6%
S
G[b] 7%
S
S
S
S
I
S
I
I
S
S
S
S
S
I
ethanol
CH3CN
S
S
S
G
G[b] 6%
S
[a] G=gel; S=homogeneous solution; I=insoluble. [b] Rapid gelification was ach-
ieved by leaving the compound at +48C.
shown in Figure S1 in the Supporting Information). In com-
parable concentration conditions (5% w/w), lipophilic gua-
nosine derivatives based on 2’,3’-O-isopropylidenguanosine,
carrying different n-alkylsilyl ethers at the 5’-position, syn-
thesised by Araki and co-workers,[14] were found to gelate
from n-decane, cyclohexane and n-hexane. Thus, the mole-
cules we synthesised offer new examples of low-molecular-
weight organogelators that may profitably complement both
the known lipophilic guanosines and the hydrophilic, natu-
rally occurring guanosine mono- and polyphosphates, given
that they cover a completely different solvent polarity range
that falls between highly apolar solvents and water. Analy-
sing the obtained results from a qualitative point of view,
comparison of the G1, G7 and G6 systems indicates that
two long aliphatic chains are essential for gelation; on the
other hand, the presence of charged (as in G3) or markedly
hydrophilic groups (as in G5) appears to negatively affect
the formation of stable gels. G4 is apparently a unique case,
being essentially insoluble in all of the most commonly used
organic solvents, with the exception of DMSO.
CD and UV spectra (see Figures S3 and S4 in the Support-
ing Information) points to a random distribution of the G-
tetrads generated by this nucleoside in solution and thus not
producing ordered three-dimensional structures with charac-
teristic orientations of the chiral centres. Of the investigated
amphiphilic derivatives, only G3 and G5 showed CD spectra
comparable, apart from the sign, to their UV/Vis ones, in
analogy with 2’,3’,5’-tri-O-acetylguanosine (see Figures S5
and S6 in the Supporting Information). On the contrary, G1,
G2, G6 and G7, all having UV/Vis profiles very similar to
the reference compound (data not shown), exhibited distinc-
tive CD spectra with two intense bands of opposite sign, di-
agnostic of regular G-tetrad stacking in organised and well-
defined octameric complexes with C4 or D4 symmetry (Fig-
ures 1–4, respectively).
CD studies: To gain useful information on the conformation-
al behaviour of the synthesised amphiphilic compounds in
organic solvents, CD spectra were acquired in the non-struc-
turing solvent CHCl3. For G4, which is only soluble in
DMSO and therefore not directly comparable with the
other systems under investigation, CD data were not ac-
quired.
To preliminarily assess the ability of these systems to
form G-tetrads, qualitative potassium picrate tests were car-
ried out by following the procedure described for lipophilic
guanosine derivatives.[21] In all cases, these colorimetric tests
confirmed the ability of these compounds, dissolved in
CHCl3, to extract K+ ions from aqueous solutions contain-
ing the yellow salt potassium picrate and transfer them to
the organic phase. The yellow colour of the organic phase
after this treatment is indicative of G-tetrad formation by
the guanosine derivatives, which attract K+ ions from aque-
ous solution; these cations in turn carry with them picrate
anions, otherwise highly insoluble in organic solvents, to
ensure electroneutrality (see Figure S2 in the Supporting In-
formation).
Figure 1. CD spectra of compound G1 at 2.7 mm in CHCl3; a) before ad-
dition of potassium picrate, lmax =256, 283 nm; b) after addition of potas-
sium picrate, lmax =262, 285 nm.
Following the rationalisation introduced by A. Randazzo,
G. P. Spada and co-workers,[22] the position of the opposite
sign bands in the CD spectra of guanosine-based systems
can provide information on the G-tetrad stacking not only
in G-rich oligonucleotides, that is, in systems in which the
stacked guanines within each strand are covalently linked by
the sugar–phosphate oligonucleotide backbone, but also in
non-covalently linked guanosine monomers. So the presence
of a negative band centred at about 240 nm and a positive
band at 260 nm is diagnostic of G-tetrads organised in ho-
mopolar stacking (also referred to as “H-to-T”, with H and
T representing the “head” and “tail” surfaces, respectively,
of a G-tetrad[23] facing each other). A blueshift of 20–30 nm
Next, compounds G1–G3 and G5–G7 were analysed by
CD spectroscopy as dilute solutions in CHCl3, before and
after the addition of potassium picrate. 2’,3’,5’-Tri-O-acetyl-
guanosine was studied in parallel as a reference compound,
having sugar modifications conferring high solubility in
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D. Montesarchio et al.
G7 tend to generate octamers containing only “H-to-H” or
“T-to-T” G-tetrad stacking. Interestingly, G1, G6 and G7
have spectra diagnostic of G-tetrad structuring even before
addition of K+ ions. Since their G-tetrad-forming ability has
been previously proven by positive potassium picrate tests,
these results can only be explained by assuming that these
systems display an extraordinary affinity for K+, which
could be extracted from the traces present in solvents, glass-
ware, and so forth and thus incorporated into stable com-
plexes even before direct contact with potassium solutions.
To confirm this hypothesis, after potassium picrate addition,
G1, G6 and G7 were treated with an excess of the cryptand
4,7,13,16,21,24-hexaoxa-1,10-diazabicycloACHTNUTRGNEUNG[8.8.8]hexacosane,
which sequesters K+ ions, and then analysed by CD spec-
troscopy, giving only residual CD signals (data not shown).
CD-monitored melting experiments were carried out on
G1, G2, G6 and G7 to obtain information on the thermal
stability of the stacked G-tetrad systems. For all of these
compounds, under the studied conditions, a significant, non-
linear decrease in the CD signal intensity was observed on
increasing the temperature, even if not in the form of well-
defined S-shaped curves that refer to a single transition with
a clearly identifiable melting temperature. This would be ex-
pected in the case of a single, columnar G-quadruplex struc-
ture involving all of the G-tetrads stacked in a highly hier-
archical order. In all cases, curves attributable to the super-
imposition of multiple transitions were registered suggesting
the coexistence in solution of non-homogeneous dissociating
systems, probably containing a variable number of stacked
G-tetrads, although essentially stable at room temperature
(see Figure S7 in the Supporting Information).
Figure 2. CD spectra of compound G2 at 2.7 mm in CHCl3; a) before ad-
dition of potassium picrate; b) after addition of potassium picrate, lmax
=
257, 293 nm.
Figure 3. CD spectra of compound G6 at 2.7 mm in CHCl3; a) before ad-
Taken together, these results show that for guanosine-
based self-assembly of compounds G1–G3 and G5–G7 in
CHCl3 a non-negligible role is played by the sugar function-
alisation. If the ribose decorations are not able to elicit in-
termolecular interactions, as is the case of 2’,3’,5’-tri-O-ace-
tylguanosine, no specific three-dimensional structure is ob-
served. For G1, G2, G6 and G7, the CD spectra showed the
presence of regular G-tetrad stacking. However, the CD-
monitored thermal denaturation curves do not support the
existence of single, well-defined G-quadruplex complexes.
Interestingly, of these four nucleosides, only G6, which ex-
clusively carries polyether chains on the ribose moiety, is
able to give homopolar stacking. On the contrary, the asym-
metrically substituted guanosine derivatives carrying both
lipophilic and hydrophilic groups tend to form heteropolar
G-tetrad stacks. This difference in behaviour can be ex-
plained in terms of relevant contributions to the overall self-
assembly given by alkyl and polyether chains, which, in con-
tributing to self-assembly in solution, may favour a particu-
lar three-dimensional arrangement of the stacked G-tetrads.
Following this interpretation, G3 and G5 do not show a reg-
ular G-tetrad stacking pattern in CHCl3, probably because
of steric hindrance of the substituents in G5 and the pres-
ence of net negative charges in G3 that prevent the forma-
tion of ordered aggregates.
dition of potassium picrate; b) after addition of potassium picrate; lmax
249, 265 nm.
=
Figure 4. CD spectra of compound G7 at 2.7 mm in CHCl3; a) before ad-
dition of potassium picrate; b) after addition of potassium picrate; lmax
264, 295 nm.
=
of both bands is diagnostic of heteropolar, “H-to-H” or “T-
to-T”, stacking.
Inspection of the spectra allows us to conclude that G6
exhibits homopolar G-tetrad stacking, whereas G1, G2 and
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Guanosine-Based Amphiphiles
FULL PAPER
Detailed microstructural characterisation studies on G1–
G7 are currently underway to determine the stability, size
and shape of the corresponding superstructures, and will be
reported in due course. The studies will involve a combined
experimental approach by using dynamic light scattering
(DLS) measurements to reveal the formation and size distri-
bution of the aggregates, small-angle neutron scattering
(SANS) measurements to provide an estimation of the
thickness of the aggregates and microscope images.
Ionophoric activity: The group of J. T. Davis has recently
demonstrated that potassium-promoted self-assembly of lip-
ophilic guanosine derivatives may contribute to stabilising
ion channels active in phospholipid membranes.[10–12] We
therefore investigated the ionophoric activity of compounds
G1–G7 by using a standard base–pulse assay that reports
H+/OHꢀ transportation directly and cation/anion transpor-
tation indirectly. Liposomes (100 nm diameter) containing
the pH-sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid
trisodium salt (HPTS) were prepared in a 4-(2-hydroxyeth-
Figure 5. Normalised fluorescence change in HPTS emission (FI, lex
=
403 and 460 nm, lem =510 nm) as a function of time after addition of the
base NaOH (50 mL, 0.5m) to EYPC/EYPG liposomes (95:5, 100 nm di-
ameter) loaded with HPTS (0.1 mm), total lipid concentration=0.17 mm,
HEPES (25 mm), NaCl (100 mm), pH 7.0, total volume=3 mL, 258C) in
the presence of G1–G7 derivatives (2%) and Amphotericin B (AmB;
1%). The concentration of the ionophores is given in percent with re-
spect to the total concentration of the lipids. The control trace has been
recorded in the absence of the ionophore. EYPC=egg yolk phosphati-
dylcholine; EYPG=egg yolk phosphatidylglycerol;
yl)-1-piperazineethanesulfonic
acid
(HEPES)
buffer
(pH 7.0) containing NaCl (or MCl and NaX in the experi-
ments for cation and anion selectivity, respectively; 100 mm)
and, once the ionophore was added, the external pH was
suddenly brought to 7.6 by addition of NaOH (or MOH in
experiments for cation selectivity) and the fluorescence
emission of the dye recorded. The emission maximum for
HPTS is about 510 nm in both the acidic and conjugate-base
forms. However, the excitation wavelength of the acidic
form (403 nm) is significantly different from that of the con-
jugate-base form (460 nm) so that the acid/base ratio is di-
rectly reflected in the emission intensity modulation pro-
duced by alternating excitation at the two wavelengths.[24] In
essence, the modulated emission signal, obtained by cycling
the excitation wavelength and recording the emission at
510 nm, reports the effective pH within the liposome. An in-
crease in HPTS fluorescence emission indicates basification
of the liposome inner water pools that may be correlated to
H+/OHꢀ transportation and to the associated cation/anion
symport or antiport.[25] The results obtained for compounds
G1–G7 are reported in Figure 5. Amphotericin B (AmB), a
naturally occurring ionophore, was used as a positive control
in these experiments.
Amphiphilic guanosine derivatives G1–G4 are mostly in-
active, whereas an increasing ability to discharge the pH
gradient is observed for compounds G7, G6 and G5. The
most active derivative is G5 followed by G6, both character-
ised by a large hydrophilic portion, that is, the disaccharide
residue in G5 and three monomethoxy(triethylene glycol)
chains in G6. The activity/hydrophilicity relationship is fur-
ther confirmed by the trend observed for G1, G6 and G7,
which differ in number of monomethoxy(triethylene glycol)
residues: on increasing the number of polar chains, the ac-
tivity increases. Similar behaviour was previously observed
by us for some structurally related CyPLOS derivatives,[26]
for which the activity was also strongly dependent on the
number of polar ethylene glycol chains appended to a rigid
macrocyclic scaffold and the activity was attributed to a de-
stabilisation of the phospholipid bilayer caused by the inser-
tion of polar chains.
The ionophoric activity of compounds G1–G7 is not influ-
enced by the presence of K+ ions. Experiments performed
in the conditions utilised to produce Figure 5 but by using
KCl instead of NaCl and KOH instead of NaOH, and there-
fore in the presence of a high concentration of potassium
ions as the only externally added cation, show kinetic pro-
files for the pH discharge that are virtually superimposable
on those obtained in the presence of Na+ ions (Figure S8 in
the Supporting Information). This suggests that in the phos-
pholipid membranes the potassium ion is not able to pro-
mote the assembly of the guanosine derivatives as is ob-
served for more concentrated solutions in chloroform. The
low tendency of simple guanosine derivatives to form G-
quartets in phospholipid membranes was not unexpected
and parallels the findings reported by the group of J. T.
Davis. Stable G-quartets have indeed only been reported for
systems in which the guanosine moieties are covalently
linked to a suitable scaffold[10] or inserted into derivatives in
which self-assembly is mainly driven by the formation of hy-
drogen bonds between bis-urea[11] or bis-carbamate subu-
nits.[12] It appears that the single interaction between potassi-
um ions and guanosine is not strong enough to promote the
self-assembling process in membranes, possibly due to the
localisation of the guanosine moieties close to the surface of
the bilayer where water may compete efficiently with the
formation of the G-tetrads.
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D. Montesarchio et al.
The hypothesis that, under the conditions of the kinetic
experiments, the amphiphilic guanosine derivatives are
mainly monomeric is supported by the study of the depend-
ence of the ionophoric activity on the ionophore concentra-
tion. The kinetic profiles obtained at different concentra-
tions of G5 and G6 derivatives are reported in Figure S9 in
the Supporting Information. The data fit well with a first-
order kinetic process and the apparent first-order rate con-
stants were obtained by non-linear regression analysis of the
fluorescence data versus time. The observed rate constants
for the transportation process are reported in Figure 6. The
linear trend observed is a clear indication that the active
species responsible for the transportation process is mono-
meric.
Figure 6. Dependence of kobs [sꢀ1] on the concentration of ionophores G5
and G6. The original kinetic profiles are reported in Figure S9 in the Sup-
porting Information. For conditions see Figure 5.
Figure 7. a) Cation selectivity for ionophore G5 (3% concentration) by
using the HPTS assay (MCl (100 mm), pH 7.0, base pulse by addition of
MOH (50 mL, 0.5m)). b) Anion selectivity for ionophore G5 (2% concen-
tration) by using the HPTS assay (NaX (100 mm), pH 7.0, base pulse by
addition of NaOH (50 mL, 0.5m)). The kinetics were corrected for sponta-
neous permeation of the different anions. Other conditions as specified
in Figure 5.
To better characterise the ionophoric activity of the am-
phiphilic guanosine derivatives, we investigated the influ-
ence of the nature of the cation or anion present in solution
on the transportation process in the case of G5, which is the
most active derivative. The HPTS assay may indeed give in-
direct information on the cation and/or anion selectivity of
the transportation process. The transmembrane discharge of
the externally applied pH gradient, which is signalled by an
increase in HPTS fluorescence emission, may derive by H+
efflux from the liposome inner water pool or by OHꢀ influx
from the bulk water to the inner water pool of the liposome.
In any case, this ion traffic must be counterbalanced. This
occurs through four processes overall: H+/Na+ antiport,
OHꢀ/Clꢀ antiport, H+/Clꢀ symport and Na+/OHꢀ symport
processes. Therefore, from a comparison of kinetic experi-
ments performed in the presence of different cations and
anions, it is possible to gain indirect evidence on their effect
on the transportation process. The results obtained for the
cation and anion selectivity experiments by using the
group I alkali metals and the halogen anions as representa-
tive examples are reported in Figure 7a and 7b, respective-
ly.
Inspection of Figure 7 shows that the rate of transporta-
tion does not depend a great deal on the cation present
whereas it is strongly influenced by the anion and increases
on going from fluoride to iodide, following the lyotropic se-
quence. This suggests that the transportation process is gov-
erned by the translocation of the anion and in particular
limited by the cost of its dehydration.
As a final control experiment, we verified the ability of
guanosine derivative G5 to promote the leakage of calcein
dye trapped inside the inner water pool of liposomes.[27]
Even at a concentration of 5%, G5 is unable to promote
the leakage of calcein (Figure S10 in the Supporting Infor-
mation), suggesting that the pore formed is relatively small
and not large enough to allow the transit of this large anion-
ic dye.
Taken together, the obtained results indicate that the am-
phiphilic guanosines G1–G7 probably partition in the mem-
brane by positioning the guanine moiety and the sugar ring
close to the surface of the liposome and the appended
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Chem. Eur. J. 2011, 17, 13854 – 13865

Guanosine-Based Amphiphiles
FULL PAPER
chains within the membrane. If the ribose decorations have
a balanced amphiphilic character, as in the case of G5 and
G6, they destabilise the membrane, allowing the transit of
small ions in a non-selective process mainly governed by the
cost of dehydration of the anion. The ionophore acts in a
monomeric form even in the presence of potassium ions,
which appear, under the studied conditions, to be unable to
promote the aggregation of the guanosine derivatives. The
ionophore is too short to span the membrane and the iono-
phoric activity is probably related to the formation of a dis-
ordered zone in the membrane characterised by an in-
creased permeability. Ions are therefore able to cross this
disordered zone in a process that is intrinsically poorly se-
lective and is strongly correlated to the lipophilicity of the
anion.
several anti-proliferative drugs, these compounds did not
show unspecific in vitro cytotoxicity towards tumour and
non-tumour cell lines. This might suggest specific interac-
tions with biological targets. Detailed studies of the struc-
ture–activity relationship will be carried out in an extensive
investigation aimed at developing nucleolipids with selective
antineoplastic activity.
Conclusion
In this study, a general and versatile synthetic strategy has
been developed to obtain a library of amphiphilic, sugar-
modified analogues of guanosine, generated by very simple
and high yielding manipulations starting from a common
precursor, which had been fully protected on the nucleo-
base. Molecular diversity is ensured by insertion of different
hydrophilic groups, such as amino acids, carbohydrates, oli-
goethers and lipophilic residues, as well as fatty acid chains.
In all cases, these appendages were attached to the ribose
moiety through ester linkages, obtained under mild and very
effective coupling conditions and, in principle, easily cleaved
inside cells by esterases.
For all synthesised compounds, qualitative potassium pic-
rate tests confirmed the ability to extract potassium ions
from aqueous solutions into CHCl3 and thus to form G-tet-
rads. Analysis of CD spectra of dilute solutions of these de-
rivatives in CHCl3 has revealed distinctive patterns of differ-
ent G-tetrad self-assemblies for G1, G2, G6 and G7; for G6,
the CD spectrum was diagnostic of homopolar stacking of
the G-quartets, whereas for the other three compounds the
CD bands supported the presence of G-quartets with heter-
opolar stacking. Two derivatives, G1 and G2, show unusual
gelling abilities in polar solvents, such as methanol, ethanol
and acetonitrile, complementing those of lipophilic guano-
sine derivatives that are known to form stable organogels in
highly apolar solvents. When analysed for their ion transpor-
tation abilities, G5 was shown to be the most active com-
pound and, as a general rule, the activity was strongly corre-
lated with the presence of a large hydrophilic portion in the
molecule. An interesting antiproliferative activity was found
for G3 and G5 when tested on MCF-7 cancer cells, with IC50
values in the vicinity of 20 mm, while no cytotoxicity
emerged for normal, control cells. Taken together, these
data show that guanosine-based amphiphiles display a varie-
ty of unusual properties, which are largely and finely tuna-
ble, as a function of the nature and number of the ribose
substituents. This renders this class of compounds of great
interest for both their biological/biomedical potential and
innovative applications related to the development of novel
self-assembling materials.
The in vitro screening of their antiproliferative activity: Anti-
viral[28] and anti-cancer[29] activity has been discovered for
several G-rich oligonucleotides and associated to the unusu-
al ability of these molecules to self-assemble into G-quartet-
based superstructures, which are recognised in vivo by spe-
cific proteins. On the other hand, for almost half a century,
modified nucleosides with biological activities have been
searched for and a great deal of biomedical interest is cur-
rently associated with nano-aggregated systems.[30] Since
novel guanosine derivatives G1–G7 seem to combine both
the ability to form G-tetrads with the ability to generate
large nano-aggregates, in vitro experiments have been car-
ried out on a panel of cancer and non-cancer cell lines to in-
vestigate their bioactivity in a preliminary screening.
To this end, cell lines were treated for 48 h with G1–G7,
at various concentrations, in a growth inhibition assay and
the cytotoxicity was determined in terms of IC50 value
(Table 2). The results show, for G1–G5, a moderate to weak
selective cytotoxicity against both human MCF-7 breast ade-
nocarcinoma and WiDr epithelial colorectal adenocarcino-
ma cells. In particular, G3 and G5 proved to be the most
active compounds, exhibiting a significant antiproliferative
profile against MCF-7 cells, with IC50 values of 22 and
17 mm, respectively. IC50 values within the micromolar range
are generally consistent with an ability to interfere with cell
viability and/or proliferation. Interestingly, in contrast to
Table 2. Cytotoxicity profile of compounds G1–G6 against cancer and
non-cancer cell lines, IC50 [mM][a]. G7 showed IC50 values ꢁ103 mm in all
cases.
Cell line[b] G1
G2
G3
445ꢂ6
G4
130ꢂ5
G5
436ꢂ9
G6
HeLa
>103 198ꢂ4
>103
>103
>103
WiDr
MCF-7
C6
82ꢂ6
90ꢂ6 135ꢂ10 130ꢂ11 136ꢂ10
185ꢂ12
96ꢂ5
22ꢂ4
616ꢂ5
750ꢂ4
46ꢂ7
152ꢂ8
>103
17ꢂ5
>103 245ꢂ4
302ꢂ5 880ꢂ14
>103 >103
3T3-L1
>103
>103
[a] IC50 values are expressed as the mean ꢂSEM (SEM= standard error
of the mean) (n=24) of three independent experiments. Bold values
show IC50 values <100 mm. [b] HeLa=human cervical cancer cells; MCF-
7=human breast adenocarcinoma cells; WiDr=human epithelial color-
ectal adenocarcinoma cells; C6=rat glioma cells; 3T3L1=murine embry-
onic fibroblasts.
Experimental Section
Ionophoric activity: HPTS assay: A mixture of EYPC in chloroform
(225 mL, 100 mgmLꢀ1
, 30 mmol) and EYPG in chloroform (60 mL,
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13863

D. Montesarchio et al.
20 mgmLꢀ1, 1.5 mmol) was evaporated with Argon flux to form a thin
film and then dried under high vacuum for 3 h. This lipid cake was hy-
drated with HPTS solution (1.5 mL, 0.1 mm; HEPES (25 mm), NaCl
(100 mm, pH 7)) for 30 min at 408C. The lipid suspension was submitted
to five freeze–thaw cycles (ꢀ1968C/408C) by using liquid nitrogen and a
thermostatic bath and then extruded under nitrogen pressure (15 bar) at
room temperature (10 extrusions through a polycarbonate membrane
(0.1 mm)). The LUV suspension was separated from the extravesicular
dye by size exclusion chromatography (SEC; stationary phase: pre-
packed Sephadex G-25 column; mobile phase: HEPES buffer) and dilut-
ed with HEPES buffer to give a stock solution with a lipid concentration
of 5 mm (assuming 100% lipid incorporation into the liposomes). The
lipid suspension (104 mL) was placed in a fluorimetric cell, diluted to
3040 mL with the same buffer solution used for the liposome preparation
and kept under gentle stirring. The total lipid concentration in the fluori-
metric cell was 0.17 mm. An aliquot of the ionophore solution (5–30 mL
Acknowledgements
We thank MIUR (PRIN) for grants in support of this investigation and
Centro di Metodologie Chimico-Fisiche (CIMCF), Universitꢂ di Napoli
“Federico II” for the MS and NMR facilities.
[1] A. Gissot, M. Camplo, M. W. Grinstaff, P. Berthꢁlꢁmy, Org. Biomol.
Chem. 2008, 6, 1324–1333, and references therein.
[2] H. Rosemeyer, Chem. Biodivers. 2005, 2, 977–1062.
[3] A. H. Farji, P. Wipf, Bioorg. Med. Chem. 2009, 17, 2950–2962.
[4] For recent examples, see: a) H. W. Yang, W. Jeong, E.-K. Bang,
E. M. Jeon, B. H. Kim, Org. Biomol. Chem. 2011, 9, 291–296; b) C.
Ceballos, S. Khiati, C. A. H. Prata, X.-X. Zhang, S. Giorgio, P.
Marsal, M. W. Grinstaff, P. Berthꢁlꢁmy, M. Camplo, Bioconjugate
Chem. 2010, 21, 1062–1069; c) S. Khiati, N. Pierre, S. Andriamanari-
vo, M. W. Grinstaff, N. Arazam, F. Nallet, L. Navailles, P. Berthꢁlꢁ-
my, Bioconjugate Chem. 2009, 20, 1765–1772.
[5] An example of guanosine-based amphiphiles is described in: H. R.
Jung, T. K. Vu, S. K. Choi, S. M. Park, R. H. Kim, Bull. Korean.
Chem. Soc. 2010, 31, 549–550. Therein, the authors produced five
nucleobase-protected amphiphilic derivatives of guanosine in 17%
overall yields on average. It is to be noted that the presented syn-
thetic scheme does not allow for final guanine deprotection of the
guanosine derivatives.
[6] S. Lena, S. Masiero, S. Pieraccini, G. P. Spada, Chem. Eur. J. 2009,
15, 7792–7806, and references therein.
[7] S. Lena, S. Masiero, S. Pieraccini, G. P. Spada, Mini-Reviews in Or-
ganic Chemistry 2008, 5, 262–273.
[8] G. Gottarelli, G. P. Spada, Chem. Rec. 2004, 4, 39–49.
[9] J. T. Davis, G. P. Spada, Chem. Soc. Rev. 2007, 36, 296–313.
[10] M. S. Kaucher, W. A. Harrell, J. T. Davis, J. Am. Chem. Soc. 2006,
128, 38–39.
[11] L. Ma, M. Melegari, M. Colombini, J. T. Davis, J. Am. Chem. Soc.
2008, 130, 2938–2939.
[12] L. Ma, W. A. Harrell, J. T. Davis, Org. Lett. 2009, 11, 1599–1602.
[13] K. Araki, I. Yoshikawa, Top. Curr. Chem. 2005, 256, 133–165.
[14] I. Yoshikawa, S. Yanagi, Y. Yamaji, K. Araki, Tetrahedron 2007, 63,
7474–7481.
[15] I. Yoshikawa, J. Sawayama, K. Araki, Angew. Chem. 2008, 120,
1054–1057; Angew. Chem. Int. Ed. 2008, 47, 1038–1041.
[16] R. W. Wang, B. Gold, Org. Lett. 2009, 11, 2465–2468.
[17] S. A. Sikchi, P. G. Hultin, J. Org. Chem. 2006, 71, 5888–5891.
[18] A. Matsuda, M. Shinozaki, M. Suzuki, K. Watanabe, T. Miyasaka,
Synthesis 1986, 385–386.
[19] L. Simeone, G. Mangiapia, C. Irace, A. Di Pascale, A. Colonna, O.
Ortona, L. De Napoli, D. Montesarchio, L. Paduano, Mol. BioSyst.
2011, 7, 3075–3086.
[20] For examples, see: a) R. Iwaura, K. Yoshida, M. Masuda, K. Yase,
T. Shimizu, Chem. Mater. 2002, 14, 3047–3053; b) R. Iwaura, K.
Yoshida, M. Masuda, M. Ohnishi-Kameyama, M. Yoshida, T. Shimi-
zu, Angew. Chem. 2003, 115, 1039–1042; Angew. Chem. Int. Ed.
2003, 42, 1009–1012.
of the appropriate mother solution to obtain the desired molcompound
/
mollipid ratio) was then added to the lipid suspension and the cell was in-
cubated at 258C for 30 min. HPTS emission was monitored at 510 nm
and modulated by alternating excitation at 403 nm and 460 nm on a 1+
1 s cycle. The concentration of the conjugate-base form is related to the
emission intensity at 510 nm during the period when the dye is excited at
460 nm (E460) whereas the concentration of the protonated form is relat-
ed to the emission intensity at 510 nm during the period when the dye is
excited at 403 nm (E403). After incubation, the time course of fluores-
cence was recorded for 200 s and then NaOH (50 mL, 0.5m) was rapidly
added through an injector port and the fluorescence emission was record-
ed for 1200 s. Maximal changes in dye emission were obtained by final
lysis of the liposomes with detergent (40 mL, 5% aqueous Triton X-100
solution). The relative intensity of E460/E403 (F) was calculated. The
extent of transportation was calculated and normalised by using Equa-
tion (1), in which Ft is the relative emission intensity measured at time t,
F0 is the relative emission intensity at ionophore addition before the base
pulse and F1 is the relative emission intensity at saturation after lysis
with Triton. The apparent first-order rate constants for the transportation
process were obtained by non-linear regression analysis of the fluores-
cence data versus time.
À
Á
Ft ꢀ F0
ð1Þ
FI ¼
ꢃ 100
ðF1 ꢀ F0Þ
Determination of cation and anion selectivity with the HPTS assay: The
vesicle suspension (104 mL stock solution, prepared as described above)
was placed in a fluorimetric cell and diluted to 3040 mL with the appro-
priate buffer solution (HEPES (25 mm, pH 7), MCl (M=Li+, Na+, K+,
Rb+ or Cs+; 100 mm) or NaX (X=Fꢀ, Clꢀ, Brꢀ or Iꢀ; 100 mm)). The
total lipid concentration in the fluorimetric cell was 0.17 mm. An aliquot
of the ionophore as a solution in DMSO (5–30 mL of the appropriate
mother solution to obtain the desired molcompound/mollipid ratio) was then
added to the lipid suspension and the cell was incubated at 258C for
30 min. HPTS emission was monitored at 510 nm and excitation wave-
lengths of 403 and 460 nm were used concurrently. After incubation, the
time course of fluorescence was recorded for 200 s and then MOH (M=
Li+, Na+, K+, Rb+ or Cs+ depending on the cation present in the extra-
vesicular buffer solution; 50 mL, 0.5m;) was rapidly added through an in-
jector port and the fluorescence emission was recorded for 1200 s. Maxi-
mal changes in dye emission were obtained by final lysis of the liposomes
with detergent (40 mL, 5% aqueous Triton X-100 solution). The extent of
transportation was calculated and normalised as previously described.
[21] G. Gottarelli, S. Masiero, G. P. Spada, J. Chem. Soc. Chem.
Commun. 1995, 2555–2557.
[22] S. Masiero, R. Trotta, S. Pieraccini, S. De Tito, R. Perone, A. Ran-
dazzo, G. P. Spada, Org. Biomol. Chem. 2010, 8, 2683–2692.
[23] Following the classification used in Ref. [22], a G-tetrad shows the
“head” surface (or H) if it is oriented with the hydrogen-bonding
network going clockwise from the donor to the neighbour acceptor;
“tail” (or T) is defined as the opposite, with the donor-to-acceptor
Hydrogen-bonding system running anticlockwise.
[24] T. M. Fyles, H. Luong, Org. Biomol. Chem. 2009, 7, 733–738.
[25] N. Sakai, S. Matile, J. Phys. Org. Chem. 2006, 19, 452–460.
[26] a) S. Licen, C. Coppola, J. DꢃOnofrio, D. Montesarchio, P. Tecilla,
Org. Biomol. Chem. 2009, 7, 1060–1063; b) C. Coppola, A. Paciello,
G. Mangiapia, S. Licen, L. De Napoli, L. Paduano, P. Tecilla, D.
Montesarchio, Chem. Eur. J. 2010, 16, 13757–13772.
Cell cultures and microculture bioassays for evaluation of cell growth
and proliferation: The bioactivity of compounds G1–G7 was investigated
by evaluation of cell viability and proliferation on a limited panel of cell
lines consisting of tumour C6 rat glioma cells, HeLa human cervical
cancer cells, WiDr human epithelial colorectal adenocarcinoma cells,
MCF-7 human breast adenocarcinoma cells, and non-tumour 3T3L1
murine embryonal fibroblasts. Experimental details of the bioactivity
studies are reported in the Supporting Information.
[27] Y. Tsao, L. Huang, Biochemistry 1985, 24, 1092–1098.
13864
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Guanosine-Based Amphiphiles
FULL PAPER
[28] For a recent review, see: a) B. Gatto, M. Palumbo, C. Sissi, Curr.
Med. Chem. 2009, 16, 1248–1265; for recent examples, see: b) G. Di
Fabio, J. DꢃOnofrio, M. Chiapparelli, B. Hoorelbeke, D. Monte-
sarchio, J. Balzarini, L. De Napoli, Chem. Commun. 2011, 47, 2363–
2365; c) E. Pedersen, J. T. Nielsen, C. Nielsen, V. V. Filichev, Nucleic
Acids Res. 2011, 39, 2470–2481.
Humana Press, Totowa, 2009, pp. 379–392; c) A. C. Girvan, Y. Teng,
L. K. Casson, S. D. Thomas, S. Jueliger, M. W. Ball, J. B. Klein,
W. M. Pierce, Jr., S. S. Barve, P. J. Bates, Mol. Cancer Ther. 2006, 5,
1790–1799.
[30] S. R. Meyers, F. S. Juhn, A. P. Griset, N. R. Luman, M. W. Grinstaff,
J. Am. Chem. Soc. 2008, 130, 14444–14445.
[29] a) S. Balasubramanian, L. H. Hurley, S. Neidle, Nat. Rev. Drug
Discov. 2011, 10, 261–275; b) P. J. Bates, E. W. Choi, L. V. Nayak in
Methods in Molecular Biology, Vol. 542, Gene Therapy of Cancer:
Methods and Protocols, 2nd ed. (Eds.: W. Walther, U. S. Stein),
Received: June 15, 2011
Revised: August 16, 2011
Published online: November 3, 2011
Chem. Eur. J. 2011, 17, 13854 – 13865
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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