Walking a supramolecular tightrope: A self-assembled dodecamer from an 8-aryl-2′-deoxyguanosine derivative

Article abstract of DOI:10.1021/ja9040384

(Chemical Equation Presented) Controlling the properties of self-assembled supramolecules via intrinsic parameters (i.e., structural information in the subunits) enables the reliable construction of assemblies of well-defined size and composition. Here we

Full text of DOI:10.1021/ja9040384

Published on Web 07/09/2009
Walking a Supramolecular Tightrope: A Self-Assembled Dodecamer from an
8-Aryl-2′-deoxyguanosine Derivative
Mar´ıa del C. Rivera-Sa´nchez, Ivonne Andu´jar-de-Sanctis, Marilyn Garc´ıa-Arriaga, Vladimir Gubala,
Gerard Hobley, and Jose´ M. Rivera*
Department of Chemistry, UniVersity of Puerto Rico, R´ıo Piedras Campus, R´ıo Piedras, Puerto Rico 00931
Received May 18, 2009; E-mail: jmrivortz@mac.com
Guanosine quadruplexes (GQs) have emerged in recent years as
key players in the development of promising functional nanostruc-
tures.1 GQs are formed by the self-assembly of guanosine subunits
into planar tetramers (G-tetrads) that stack on each other, assisted
by the complexation of a metal cation such as K+ or Na+.
Alternatively, GQs can also form via the folding of G-rich
oligonucleotides (e.g., DNA, RNA) leading to monomeric, dimeric,
and tetrameric structures via the association of one, two, or four
oligonucleotides, respectively.1d,2 In the latter, the number of
G-tetrads is primarily controlled by the sequence (intrinsic param-
eter) of the oligonucleotide, whereas, in the former, such control
can be primarily achieved by adjusting extrinsic parameters (e.g.,
concentration, temperature, solvent,3 the cation template,4 and/or
its counteranion5). Controlling the molecularity via intrinsic
parameters (i.e., structural information in the supramolecular
building blocks6) enables the reliable construction of nanostructures
of well-defined size and composition. In recent years we have
developed 8-aryl-2′-deoxyguanosine (8ArG) derivatives as versatile
recognition motifs for the construction of supramolecular nano-
structures in organic and aqueous media (Figure 1).3a,7 For example,
we have reported the reliable formation of a hexadecamer as the
basis for constructing discrete self-assembled dendrimers.7b So far
we have managed to adjust the intrinsic parameters of 8ArG’s to
program the preferential formation of octamers (O) or hexadecamers
(H) (Figure 1b). However, using the same strategy to program the
formation of the elusive intermediate dodecamer has proven more
difficult.7 Here we report on a lipophilic 8-(3-pyridyl)-2′-deox-
yguanosine derivative that forms a discrete dodecamer with high
fidelity8 and enhanced stability relative to the octamer formed by
an isosteric 8-phenyl-2′-deoxyguanosine derivative.
8ArG derivatives adopt a syn conformation around the glycosidic
bond.9 This limits the number of ways in which the resulting
G-tetrads can orient themselves within the GQ (i.e., higher
preorganization10). In most cases, the steric repulsion imposed by
the groups attached to the ribose moiety in 8ArG’s prevent the
assembly beyond an octamer. However, this steric repulsion can
be compensated by increasing the number of attractive noncovalent
interactions. We have shown that carbonyl groups in the meta
position of a phenyl moiety can engage in additional hydrogen
bonds, among other noncovalent interactions, which enable the
formation of a hexadecamer.7 We hypothesized that derivatives with
heteroaryl groups attached to C8 could engage in additional
dipole-dipole interactions.11 We expected these interactions to be
strong enough to overcome the steric repulsion, enabling the
formation of a dodecamer, but not too strong to drive the formation
of a hexadecamer (Figure 1b).
1a). Both compounds were prepared as previously described by
us, using a Suzuki-Miyaura cross coupling reaction as the key
synthetic step.7d,12,13
Figure 1. (a) Structure of the tetrad formed by 3PyGi and PhGi. (b)
Schematic representation of the various assemblies formed by 8ArG
derivatives; LBA (Loosely Bound Aggregates), O (Octamer), D (Dodecam-
er), H (Hexadecamer).
1H NMR titration studies of PhGi and 3PyGi in CD3CN, using
K+ as the templating cation, support the formation of an octamer
and a dodecamer, respectively. Addition of increasing amounts of
KI to a solution of PhGi reveals the disappearance of the peaks
corresponding to loosely bound aggregates (LBA, Figures 1b, S6)13
with the concomitant appearance of a new set of peaks that are
consistent with an octamer of D4 symmetry (OD4) (Figure 2a). The
corresponding experiments with 3PyGi show a similar trend to
PhGi with up to 0.06 equiv of KI added (Figure S7), suggesting
the formation of a D4-octamer.13 Beyond this point, a new species
with three sets of signals emerges (Figure S7),13 becoming the main
species (62% D, 34% OD4) after the addition of 0.13 equiv of KI
(Figure 2b). The spectral characteristics are consistent with the
formation of a dodecamer of C4 symmetry, where the highest
fidelity8 (94% D, 4% OD4) was reached at 45 mM and 0.7 equiv of
KI.13 The speciation curves shown in Figure 3 summarize the results
from these titration experiments.
To test this hypothesis, we studied the self-assembly in aceto-
nitrile of 8-(3-pyridyl)-2′-deoxyguanosine esterified at the 3′ and
5′ positions with isobutyryl groups (3PyGi) and compared its
behavior to that of the isosteric phenyl derivative (PhGi) (Figure
Further support for the molecularities of (3PyGi)12 and (PhGi)8
comes from Vapor Pressure Osmometry (VPO) and 2D-DOSY
experiments. VPO measurements provide a molecular weight for
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C O M M U N I C A T I O N S
a solution containing (3PyGi)12 ·2KI of 6216 Da with a calculated
value of 5949 Da (Table S1).13 A discrepancy of 267 Da, which is
within the molecular weight of one subunit of 3PyGi (485 Da), is
very reasonable for this technique. Similar measurements with PhGi
are also consistent with the formation of an octamer (4229 Da,
Table S1).13 DOSY experiments14 provided diffusion coefficients
(D, m2s-1) of (5.69 ( 0.05) × 10-10, (5.5 ( 0.2) × 10-10, and (6.7
( 0.3) × 10-10 for (PhGi)8, (3PyGi)12, and (3PyGi)8, respectively.13
Assuming a spherical shape, the Stokes-Einstein equation enables
the following hydrodynamic radii (r, Å): 11.3 ( 0.1, 11.6 ( 0.2,
and 9.5 ( 0.5, for (PhGi)8, (3PyGi)12, and (3PyGi)8, respectively
(Table S2).13,14 The apparent discrepancy in the sizes for (PhGi)8
and (3PyGi)8 may result from a smaller amplitude in the “breathing”
motions for the latter, due to its enhanced noncovalent interactions.
and that significant amounts of assemblies are still detected upon
dilution to 0.5 mM (Table S3, Figures S8-9).13 However, diluting
a solution of (3PyGi)12 shifts the equilibrium toward (3PyGi)8 until
the ratio for both becomes 1:1 at 1 mM. The lower self-assembly
concentration (lsac)15 is 1-2 mM for both (PhGi)8 and (3PyGi)12.
VT experiments reveal the enhanced thermodynamic stability of
(3PyGi)12 over (PhGi)8, giving Tm values of 43.6 and 38.6 °C,
respectively (Figures 3d, S10-11).13 The higher stability of
(3PyGi)12 relative to (PhGi)8 is likely due to the enhanced
noncovalent interactions as discussed below.
Analysis of the titration data using a dose-response curve
supports the notion that the assembly of both (PhGi)8 and (3PyGi)12
are cooperative processes. The calculated Hill coefficients (nH) are
1.6 ( 0.5 and 4.8 ( 0.9, respectively (Figure 3c, Table S4).13 The
comparison and interpretation of such coefficients must be done
with care, since the mechanism of formation of the octamer is likely
to be different than that of the dodecamer.16 Nonetheless, it is
reasonable to state that the extent of cooperativity in the formation
of (3PyGi)12 is higher than that of (PhGi)8.17
Computer modeling, supported by 2D NMR experiments,
provides a compelling representation for the structure of (3PyGi)12
and hints to the rationale for its formation. The information in the
guanine moiety dictates how four 8ArG subunits organize them-
selves in a planar tetrad. Considering that the 8-aryl group
preorganizes the subunits into the syn conformation, only one type
of tetrad is possible. Since both octamers formed by PhGi and
3PyGi are D4-symmetric (as shown by NMR13), the interphases
between the two tetrads must be homogeneous (i.e., head-to-head,
hh, or tail-to-tail, tt; Figure 4b).18 Intertetrad H2′(T1)-H2′(T2) NOE
correlations support an hh interphase between those tetrads (Figures
4b, S18).13 With this arrangement, the octamers formed by 8ArG’s
are stabilized by eight CH-π contacts between the C2′H2 and the
aryl rings in both T1 and T2 (Figure 4c). This is one of the reasons
why quadruplexes made by 8ArG’s are more stable, and self-
assemble with greater fidelity, than those formed by the parent G.7d
The formation of (3PyGi)12 requires four additional subunits to
assemble onto the existing (3PyGi)8. The newly added tetrad (T3)
orients itself with its “head” facing the “tail” of T2. This
arrangementissupportedbymultipleNOEsincludingH1′(T2)-H1′(T3)
and H11(T2)-H1′(T3) (Figure 4b), which would not be present
with a tt interphase.13
1
Figure 2. Partial H NMR spectra (500 MHz, 298.2 K) of (a) PhGi and
(b) 3PyGi in CD3CN with 0.7 equiv of KI. The peaks on the left (11.4-12.6
ppm) correspond to the N1H’s, and those on the right (5.7-6.5 ppm)
correspond to the CH1’s.
In essence, the formation of (3PyGi)12 is enabled by an optimum
balance between repulsive and attractive noncovalent interactions,
specifically, (i) cation-dipole interactions (somewhat offset by the
electrostatic repulsion between the two templating cations); (ii) π-π
interactions, between T2 and T3; and (iii) dipole-dipole interactions
between the pyridyl moieties (Figure 4d). Besides the dipole created
by the nitrogen in the pyridyl ring, electron density surface maps
(Figure 4e,f) show that opposite sides of such rings have slightly
different electron densities that reinforce π-π interactions. The
formation of the dodecamer (PhGi)12 is energetically unfavorable
because the enhanced steric repulsion between the ester groups
cannot be compensated by attractive dipole-dipole interactions like
in (3PyGi)12. Likewise, formation of the hexadecamer (3PyGi)16
is prevented by a similar steric repulsion between the ester groups.
At this stage, additional dipole-dipole interactions are not enough
to stabilize such a hexadecamer.3,7
Figure 3. Speciation curves for (a) PhGi and (b) 3PyGi. (c) Dose-response
curves for (PhGi)8 (blue) and (3PyGi)12 (green) at 30 mM in CD3CN at
298.2 K (Table S4).13 (d) Melting profiles as determined by VT-NMR for
(PhGi)8 and (3PyGi)12 (dashed blue and green lines, respectively) and the
corresponding first derivative plots (solid lines). UA refers to unidentified
assemblies.
Guanine is an excellent information-rich recognition motif,1 but
there is significant ambiguity in that information,6 leading to context
dependent self-assembly.3 This is not necessarily a disadvantage,
and it is in fact often convenient for the elaboration of responsive
systems. However, certain applications require a more reliable self-
assembly, less ambiguity, which can only be achieved by modulat-
The stability of both (PhGi)8 and (3PyGi)12 was assessed by
variable concentration (VC) and variable temperature (VT) NMR
experiments. VC studies in CD3CN reveal that both (PhGi)8 and
(3PyGi)12 compose greater than 90% of the mixtures above 20 mM
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why 8ArG derivatives are excellent platforms for the development
of well-defined, predictable, and reliable supramolecular structures
that form with high fidelity.7
We are currently studying the scope and limitations of other
8ArG derivatives that also show a propensity to form dodecamers.
Adding these to the existing octamer- and hexadecamer-forming
8ArG derivatives will enable the preparation of a wide variety of
self-assembled dendrimers and other nanostructures where the size
and the number of functional elements can be fine-tuned for specific
applications. The results of such studies will be reported in due
course.
Acknowledgment. We thank NIH-SCoRE (2506GM08102) for
financial support. M.C.R.S. thanks NASA-PR and PRIDCO, and
M.G.A. thanks NIH-RISE for graduate fellowships. I.A.D. thanks
the MARC program for an undergraduate fellowship.
Supporting Information Available: Detailed synthetic procedures,
characterization for all new compounds, experimental protocols and
NMR data. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Figure 4. (a) Top and (b) side views for a model of (3PyGi)12 where some
atoms are omitted for clarity. (a) The top tetrad (T3) has its “tail” (i.e.,
rotation in the direction of the magenta arrows is counterclockwise, Figure
S18)13,18 facing up and its “head” in contact with the “tail” of the middle
tetrad (T2). (b) T1 and T2 correspond to the initially formed octamer, and
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black double-headed arrows show selected NOEs indicating intratetrad (T3)
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ing intrinsic parameters. The contrasting behavior of PhGi and
3PyGi illustrates how specific information can be programmed6
into 8ArG subunits by increasing their preorganization10 and/or the
number of contacts that engage in attractive interactions. This is
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