Expanding the hoogsteen edge of 2′-deoxyguanosine: Consequences for G-quadruplex formation

Article abstract of DOI:10.1021/ol048013v

(Chemical Equation Presented) The synthesis and self-assembling properties of 8-aryl-2′-deoxyguanosine derivatives are described. Our studies suggest that a properly placed acetyl group can increase the stability and specificity of the resulting G-quadrup

Full text of DOI:10.1021/ol048013v

ORGANIC
LETTERS
2004
Vol. 6, No. 25
4735-4738
Expanding the Hoogsteen Edge of
-Deoxyguanosine: Consequences for
2′
G-Quadruplex Formation
Vladimir Gubala, Jose´ E. Betancourt, and Jose´ M. Rivera*
Department of Chemistry, UniVersity of Puerto Rico, R´ıo Piedras Campus,
R´ıo Piedras, Puerto Rico 00931
jriVera@cnnet.upr.edu
Received September 29, 2004
ABSTRACT
The synthesis and self-assembling properties of 8-aryl-2
placed acetyl group can increase the stability and specificity of the resulting G-quadruplex supramolecules by enhancing noncovalent interactions
such as hydrogen bonds and -stacking.
′-deoxyguanosine derivatives are described. Our studies suggest that a properly
π
Advances in supramolecular chemistry and related fields rely
on the availability of recognition motifs with increased
selectivity, specificity, and ease of synthesis. Nucleosides
in general, and guanosine (G) in particular, stand out as
excellent candidates for the elaboration of a variety of
supramolecular structures. Guanosine can form tetrameric
structures (G-tetrads) that self-assemble in the presence of a
variety of cations to form higher ordered structures known
as G-quadruplexes. We describe the self-assembling proper-
ties of lipophilic 8-aryl-dG analogues (8ArdG); in particular,
we demonstrate that such properties can be modulated by
the presence of an aryl group attached to C8 at the guanine
base, as well as the nature and stereochemical arrangement
of functional groups capable of increasing the number of
hydrogen bonds within a tetrad. We show that, in CDCl3 or
CD3CN and in the presence of KI, 8-(3-acetylphenyl)-dG
forms assemblies of increased thermal stability and specificity
when compared with the parent dG or a related compound
that lack an expanded Hoogsteen edge.
G-Quadruplexes are relevant to areas as diverse as cancer
research and nanotechnology.1a The G-rich sequence of
telomeres, ends of chromosomes, have a propensity to form
(3) Two notable examples for the modulation of the supramolecular
behavior of G-analogues is illustrated by the work of the Gottarelli and
Davis groups. The former evaluated the impact of long alkyl chains attached
to the sugar ring of dG as a means of controlling the formation of liquid
crystalline phases, and the latter demonstrated how to control the kinetics
of self-assembly by changing the anion bound at the surface of the
quadruplex (See refs 2d and 2a, respectively).
(4) Kettani, A.; Gorin, A.; Majumdar, A.; Hermann, T.; Skripkin, E.;
Zhao, H.; Jones, R. J. Mol. Biol. 2000, 297, 627-644.
(5) Molecular modeling was performed using HyperChem 7.5 for
Windows (from Hypercube, Inc.) with the Amber 99 force field.
(6) Western, E. C.; Daft, J. R.; Johnson, E. M.; Gannett, P. M.;
Shaughnessy, K. H. J. Org. Chem. 2003, 68, 6767-6774.
(7) Sessler’s group reported the formation of G-tetrads in the absence
of a templating metal cation, both in the solid state and in solution: (a)
Sessler, J. L.; Sathiosatham, M.; Doerr, K.; Lynch, V.; Abboud, K. A.
Angew. Chem., Int. Ed. 2000, 39, 1300-1303. For a recent related study,
see: (b) Giorgi, T.; Lena, S.; Mariani, P.; Cremonini, M. A.; Masiero, S.;
Pieraccini, S.; Rabe, J. P.; Samori, P.; Spada, G. P.; Gottarelli, G. J. Am.
Chem. Soc. 2003, 125, 14741-14749.
(8) (a) Borzo, M.; Detellier, C.; Laszlo, P.; Paris, A. J. Am. Chem. Soc.
1980, 102, 1124-1134. (b) Bouhoutsos-Brown, E.; Marshall, C. L.;
Pinnavaia, T. J. J. Am. Chem. Soc. 1982, 104, 6576-6584. (c) Fisk, C. L.;
Becker, E. D.; Miles, H. T.; Pinnavaia, T. J. J. Am. Chem. Soc. 1982, 104,
3307-3314.
(1) (a) Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668-698. (b)
Mergny, J.-L.; Riou, J.-F.; Mailliet, P.; Teulade-Fichou, M.-P.; Gilson, E.
Nucl. Acids Res. 2002, 30, 839-865.
(2) (a) Shi, X.; Mullaugh, K. M.; Fettinger, J. C.; Hofstadler, S. A.; Davis,
J. T. J. Am. Chem. Soc. 2003, 125, 10830-10841. (b) Rinaldi, R.; Maruccio,
G.; Biasco, A.; Arima, V.; Cingolani, R.; Giorgi, T.; Masiero, S.; Spada,
G. P.; Gottarelli, G. Nanotechnology 2002, 13, 398-403. (c) Shi, X.;
Fettinger, J. C.; Davis, J. T. Angew. Chem., Int. Ed. 2001, 40, 2827-2831.
(d) Mezzina, E.; Mariani, P.; Itri, R.; Masiero, S.; Pieraccini, S.; Spada, G.
P.; Spinozzi, F.; Davis, J. T.; Gottarelli, G. Chem. Eur. J. 2001, 7, 388-
395.
10.1021/ol048013v CCC: $27.50
© 2004 American Chemical Society
Published on Web 11/10/2004

G-quadruplex structures in vitro. Molecules capable of
binding to and stabilizing G-quadruplexes have shown prom-
ising anticancer activity because of their inhibitory effect
on telomerase.1b In the areas of supramolecular chemistry
and nanotechnology, the Davis and Gottarelli groups have
made various lipophilic G-analogues for the construction of
self-assembled ionophores, self-assembled liquid crystals,
and other molecular devices.1a,2 The stated importance of
G-quadruplexes makes it appropriate to determine structure-
function relationships with G-analogues. In particular,
G-analogues that are modified at the guanine base could be
used to modulate the properties of oligonucleotides or other
self-assembled structures containing them.3
grooves of a G-quadruplex, it is solvent-exposed, and in
aqueous solutions it is most likely H-bound to water
molecules. In related structures such as the DNA-hexad, two
of the four H*s are hydrogen bound to adenine bases.4
Molecular modeling shows that a G-analogue with a
hydrogen bond acceptor located near H* could make an
additional H-bond.5 A specific example of a G-analogue with
an expanded Hoogsteen edge is shown in Figure 1b. The
oxygen, shown in red, in the 8-(3-acetylphenyl) group could
hydrogen bond to N2H*, forming a tetrad with up to four
extra hydrogen bonds. The resulting G-quadruplexes formed
from the stacking of such tetrads are expected to have
additional noncovalent interactions (π-stacking, CH-π) and
also to be more specific, i.e., forming mostly one type of
supramolecular structure, and more stable than the naturally
occurring G-quadruplexes.
Each guanine base in a G-tetrad has an unpaired hydrogen
bond donor (H*) at N2 that is not hydrogen bound within
the structure (Figure 1a). Because H* is located at the
The key step in the synthesis of 8ArdG analogues is a
Suzuki-Miyaura cross-coupling reaction using the method-
ology recently published by the Shaughnessy group (Scheme
1).6 Esterification of the 5′- and 3′-hydroxyl groups in 1, 3,
Scheme 1. Synthesis of dG Analogues
and 4 affords analogues 1a, 3a, and 4a, respectively, which
are soluble in organic solvents.
Compounds 1a, 3a, and 4a show sharp and well-defined
signals in DMSO-d6 that are indicative of the presence of
mostly monomeric species (Figure 2a). In CDCl3, the peaks
are also sharp and well-resolved with the exception of the
N1H peaks, which are slightly exchange-broadened, indicat-
ing the formation of loosely bound aggregates (Figure 2b,
Scheme 2, M).7 The spectra of 1a, 3a, and 4a in CDCl3
changes significantly upon the addition of KI. Metal cations
such as K+ promote the formation of octamers, hexadecam-
ers, and higher ordered aggregates by lipophilic guanosine
analogues in organic solvents2 and by guanosine nucleotides
and oligonucleotides in aqueous environments.8
Titration experiments of 1a, 3a, and 4a in CDCl3 with KI
reveal the ratio of monomers to metal cation in the quadru-
plex and the specificity for such quadruplexes as a function
of [K+] (Figure 2). Below a 1:8 ratio of K+ to monomer, 1a
shows a preference for the formation of a head-to-tail (ht)9
octamer (O), but beyond a 1:8 ratio, the spectrum becomes
more complex with the emergence of several species in
Figure 1. (a) Top view of a G-tetrad showing the directions of
the hydrogen bond donors (green arrows) on the Watson-Crick
edge. (b) Top view of the 3b-tetrad in which the oxygen from the
3-acetyl group expands the Hoogsteen edge of the guanine base,
allowing the formation of up to four additional hydrogen bonds
per tetrad. The double-headed arrows indicate selected two-
dimensional NOESY cross-peaks. (c) Top view and side view of a
molecular model of the 3b-tetrad and a cartoon representation of
it. The former shows the direction of the hydrogen bond donors on
the Watson-Crick edge that define the head (h) of the tetrad when
the rotation is clockwise, as shown, and the tail (t) when the
direction is counterclockwise.
4736
Org. Lett., Vol. 6, No. 25, 2004

ments. VT experiments over a 112 K temperature range
reveal the increased thermal stability of (3a)16‚3K+ when
compared with (1a)n‚mK+ and (4a)8‚K+ (Figure 3a-c). At
Figure 2. Titration experiments (295 K, 500 MHz) for compounds
1a, 3a, and 4a in (a) DMSO-d6 (36, 32, 35 mM) and (b-f) CDCl3
(58, 57, 52 mM) with the following equivalents of KI relative to
monomer: (b) 0, (c) 1/32, (d) 1/8, (e) 1/6, (f) 1/2. The portion of
Figure 3. Variable-temperature experiments for compounds 1a,
3a, and 4a in CDCl3 with 1/6 equiv of KI. The temperatures were
(a) 221, (b) 288, and (c) 333 K.
1
the H NMR spectra shows the region of the N1-H peaks. Key:
M (loosely organized monomers), O (octamer), H (hexadecamers),
P (stacked polymers), as shown in Scheme 2.
221 and 333 K, the main species is still (3a)16‚3K+, whose
1H NMR peaks broaden at 221 K, but remain sharp and show
all the multiplicities at 333 K. The spectra of (4a)8‚K+, in
which the 4-acetyl group cannot form additional hydrogen
bonds, shows the collapse of the main species at 221 K; at
333 K, the peaks are broad and show no multiplicities, both
of which are indicative of a more dynamic system (Figure
3a-c: 4a).
Scheme 2. Hierarchical Self-Assembly for G-Quadruplexes
The dilution experiments in CD3CN allowed us to evaluate
the ratio (f) of ordered species formed by 3a, 4a, and 1a,
which is useful to make comparisons between the stabilities
of (3a)16‚3K+, (4a)8‚K+, and (1a)n‚mK+ (Table 1). In the
different proportions (Figure 2d: 1a). This behavior is
consistent with a shift in the equilibrium from an octamer
toward stacked polymers (Scheme 2, P), which agrees with
the behavior of related dG analogues reported by the
Gottarelli group.2d
Table 1. Fraction of Ordered Species (f)a as a Function of
Total Concentration (GT) of Nucleosides 1a, 3a, or 4a in
CD3CNb at 295 K
Upon titration of 3a with KI, two new species appear, a
species with one set of peaks and a second species with two
sets of peaks (Figure 2: 3a). When [KI] g 3/16 equiv, the
main species (>95%) is the one with two sets of peaks. This
behavior is consistent with the formation of a hexadecamer
(H) with a formula (3a)16‚3K+ and an octamer (O) with a
formula (3a)8‚K+. On the other hand, as we increase the
concentration of KI, 4a shows the formation of primarily a
head-to-head (hh) octamer (O) (4a)8‚K+ with a small amount
of a head-to-tail (ht) octamer (O′). Molecular modeling and
two-dimensional NOESY experiments of (3a)16‚3K+ and
(4a)8‚K+ reveal that the head (h) of the tetrad is less crowded
than its tail (t) because the monomers 3a and 4a stay in the
syn conformation around the glycosidic bond (Scheme 2).7,10
Those experiments also indicate that the interfaces between
the tetrads are hh-tt-hh for (3a)16‚3K+ and hh for (4a)8‚
K+ as shown in Scheme 2.
GT
f 1a
f 3a
f 4a
44
0.84
0.84
0.81
0.81
0.63
0.50
0.15
0
0.96
0.95
0.95
0.94
0.92
0.87
0.73
0.50
0.24
0.94
0.92
0.90
0.88
0.88
0.79
0.56
0.31
0.06
25
20
15
10
5
2
1
0.5
0
a Ratio f ) [XO]/[XT], where [XO] and [XT] represent the concentration
in ordered form (i.e., quadruplexes) and the total concentration of 1a, 3a,
or 4a, respectively. b In CD3CN, both monomeric and assembled species
1
can be clearly distinguished in the H NMR spectra.
The increased stability of the quadruplex formed by 3a
over those formed by 1a and 4a was determined using H
NMR through variable-temperature (VT) and dilution experi-
concentration range between 44 and 10 mM, f remains
effectively constant for both 3a and 4a, but below 10 mM it
is significantly larger for 3a. In contrast, f is lower for 1a
throughout the entire concentration range examined. These
results indicate that an aryl group at C8 and a properly placed
functional group (capable of increasing the number of
hydrogen bonds within a tetrad) impart stability to the
resulting quadruplexes when compared to the parent com-
pound.
1
(9) See Figure 1c for the definition of the head and the tail of a tetrad.
Also, see: Smith, F. W.; Lau, F. W.; Feignon, J. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 10546-10550.
(10) (a) Michelson, A. M.; Monny, C.; Kapuler, A. M. Biochim. Biophys.
Acta 1970, 217, 7. (b) Rao, S. N.; Kollman, P. A. J. Am. Chem. Soc. 1986,
108, 3048-3053. (c) Dias, E.; Battiste, J. L.; Williamson, J. R. J. Am. Chem.
Soc. 1994, 116, 4479-4480.
Org. Lett., Vol. 6, No. 25, 2004
4737

The visibility of separate peaks for N2HWC and N2H* at
temperatures of up to 300 K suggests slower rotation around
the C2-N2 bond caused by the formation of additional
hydrogen bonds.11 Cross-peaks in the two-dimensional
NOESY spectrum of (3a)16‚3K+ measured at 295 K between
H2 and both N2HWC and N2H* and between the acetyl-CH3
and H4 (Figure 1b) confirm that the monomers in the outer
(o) tetrads are in a suitable conformation to make the extra
hydrogen bonds with NH* (Scheme 2, H).12 According to
the same two-dimensional NOESY spectrum, the monomers
in the inner (i) tetrads are prevented from making such
hydrogen bonds because the 8-aryl group is deviated
significantly from the plane of the tetrad (Scheme 2, H).
The increased steric crowding at the tt interface must be
forcing the 8-aryl group to adopt this alternative conformation
as a means of minimizing steric repulsion and maximizing
other noncovalent interactions. The chemical shifts for the
N2H* signals in (3a)16‚3K+ (outer tetrad, inner tetrad) and
(4a)8‚K+ are 7.8, 5.5, and 5.7 ppm, respectively, at 278 K.
The significant downfield chemical shift between the former
(∆δ > 2 ppm) and the latter two is also a strong indicator
for the formation of the putative hydrogen bond.
The 8ArdGs described in this article offer an attractive
way of obtaining easily prepared recognition motifs that can
be used to modulate the supramolecular properties (stoichi-
ometry, specificity, stability) of G-quadruplexes. The evi-
dence presented suggests that such modulation results from
a combination of the following factors: (i) the preorgani-
zation of the 8ArdGs in the syn conformation caused by
substitution at C8; (ii) the larger surface area of the modified
base, which should increase π-stacking interactions; (iii) the
expansion of the Hoogsteen edge obtained by strategically
placing functional groups capable of forming extra hydrogen
bonds. All of these effects combine to increase the stability
and specificity of the resulting quadruplexes. More detailed
studies with other 8ArdGs are underway to determine why
the 3-acetyl group in 3a shifts the equilibrium toward a
hexadecamer as well as the scope and limitations of this
strategy. The results of such studies will be presented soon.
Acknowledgment. We thank Prof. Jeffery T. Davis for
providing us with a preprint copy of reference 1a. V.G.
thanks NSF-EPSCOR, and J.E.B. thanks the UPR-FIPI
program for financial support.
Supporting Information Available: Synthetic procedures
and titration, VT, and two-dimensional NOESY spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(11) Williams, L. D.; Williams, N. G.; Shaw, R. J. Am. Chem. Soc. 1990,
112, 829-833.
(12) Assignment of outer (o) and inner (i) tetrads is based on the large
upfield chemical shift displacement (∆δ up to 1.5 ppm for some peaks,
Figure 2d: 3a) of the latter. This large ∆δ is most likely the result of the
anisotropic shielding created by the aromatic rings of the outer (o) tetrads.
See also Supporting Information, p S9, for more details.
OL048013V
4738
Org. Lett., Vol. 6, No. 25, 2004