Nonpolymeric thermosensitive supramolecules

Article abstract of DOI:10.1021/ja9070927

(Figure Presented) Here we show 2′-deoxyguanosine derivatives that self-assemble in aqueous media into discrete supramolecular hexadecamers and exhibit the lower critical solution temperature (LCST) phenomenon. Spectroscopic, calorimetric, and electron mi

Full text of DOI:10.1021/ja9070927

Published on Web 11/02/2009
Nonpolymeric Thermosensitive Supramolecules
Jose´ E. Betancourt and Jose´ M. Rivera*
Department of Chemistry, UniVersity of Puerto Rico, R´ıo Piedras Campus, R´ıo Piedras, Puerto Rico 00931
Received August 21, 2009; E-mail: jmrivortz@mac.com
Naturally occurring molecular machines self-assemble into a wide
variety of nanostructures that are able to sense environmental
changes and respond to them accordingly. Artificial stimuli
responsive (i.e., smart) materials have found numerous applications
in biomedicine and nanotechnology.1 This is due to their ability to
undergo large changes in a property triggered by a relatively small
stimulus. In thermosensitive materials, when a water-soluble
substance is made increasingly hydrophobic, it will reach a range
of compositions upon which it will show a lower critical solution
temperature (LCST) before becoming completely insoluble.2 Poly-
mers represent the overwhelming majority of known substances
that show the LCST phenomenon by undergoing a coil to globule
transition and/or self-assembling into aggregates upon reaching a
transition temperature (Tt).1c,3,4 Thermally responsive polymers,
most notably, elastin-like polypeptides (ELPs)5 and poly(N-
isopropylacrylamide) (pNIPAAm),6 are attractive due to their
suitability for fundamental studies as well as their practical uses in
a plethora of applications.7-11
Although, in principle, nonpolymeric and well-defined supramo-
lecular assemblies could be developed to show this property, to
the best of our knowledge, there are still no known examples of
such substances. Here we show 2′-deoxyguanosine derivatives that
self-assemble in aqueous media into discrete supramolecular
hexadecamers and exhibit the LCST phenomenon. Furthermore,
the Tt can be tuned to higher values by the addition of a more
hydrophilic derivative. These findings uncover a new paradigm in
the development of smart thermosensitive materials with properties
and applications complementary to those of polymers.4
Supramolecular self-assembly offers a complementary biomi-
metic strategy to the use of polymers for the development of
functional nanostructures.1,12,13 Attractive features of supramo-
lecular self-assembly include synthetic economy and the dynamic
exchange of subunits. Our efforts in this field have been aimed at
studying the self-assembly of 2′-deoxyguanosine derivatives, in
particular, modulation of the resulting supramolecular structures
by replacing H8 in the guanine moiety with a functionalized aryl
group (Figure 1).14 We have shown that the nature and substitution
pattern of the aryl group can dictate the molecularity and stability
(thermal and kinetic) of the resulting supramolecules as well as
their selectivity for binding metal cations.14c Furthermore, we have
shown the reversible high fidelity switching between a hexadecamer
and an octamer by simply changing the solvent.14d
The 8-(meta-acetylphenyl)-2′-deoxyguanosine (mAG) moiety is
an attractive recognition motif that enables the construction of well-
defined and discrete supramolecular nanostructures.15 We demon-
strated this by using it to construct lipophilic hexadecameric self-
assembled dendrimers (SADs).16 Our recent discovery that a
positively charged hydrophilic mAG derivative self-assembles
isostructurally into hexadecamers in aqueous media15 prompted us
to explore the construction of hydrophilic SADs. During such
studies, we discovered that, upon assembly, derivative 2 (Figure
1a) exhibited the LCST phenomenon (Vide infra).
Figure 1. (a) Chemical structure of the tetrad formed by 1 and 2. (b) Top
view of the tetrad shown in a. (c) Side view of the hexadecamer 216•3KI
(abbreviated throughout the article as 216), formed by four coaxially stacked
tetrads. In b and c the ester side chains attached to the 2′-deoxyribose rings
are omitted for clarity. (d) Same as b but with the ester groups highlighted
with a dotted surface. (e) Similar to d in CPK representation and with the
ester groups of all the subunits shown. (f) Side view of e. In all the models
the carbon atoms are magenta, the oxygen atoms are red, the nitrogen atoms
are blue, the potassium cations are yellow, and the hydrogen atoms are
white.
Both 1 and 2 (Figure 1a,b) were synthesized using a methodology
similar to that previously reported by us16 (Figure S1).17 At room
temperature, both derivatives are insoluble in an aqueous phosphate-
buffered solution (pH 7.1), but upon addition of KI (2 M), they
1
become soluble. H NMR experiments of soluble solutions of 1
and 2 (both at 20 mM, pH 7.1, 2 M KI) in H2O-D2O (9:1,
potassium buffer) reveal the signatures for the self-assembly in
1
aqueous media. The H NMR spectrum shows the characteristic
double set of signals corresponding to two pairs of tetrads (N1H)
in different chemical environments (Figure 2a,b). The 2D NOESY
spectrum supports the formation of 116 and 216, by showing the
signature cross peaks characteristic of a hexadecamer in water15
(Figure S7).17 The enhanced solubility is not a simple salting-in
process, but it also results from the self-assembly of 16 subunits in
9
16666 J. AM. CHEM. SOC. 2009, 131, 16666–16668
10.1021/ja9070927 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
which the more hydrophobic aryl components are clustered together
in a central, relatively hydrophobic, core (Figure 1c), surrounded
by the more hydrophilic groups (e.g., triazole rings, hydroxyl
groups) facing the solvent (Figure 1d-f).
which upon deconvolution reveal the overlap of two different
processes with temperatures of 58.7 and 72.7 °C. The excellent
overlap of the first peak with the transmittance curve indicates a
direct correlation to the Tt. The close match between the endother-
mic DSC peak corresponding to the melting temperature of 116 (Tm
) 77.2 °C, Figure S12)17 and the second peak from the sample of
216 strongly suggests that it belongs to the melting of the
hexadecamers within the suspended particles. The results from VT
NMR experiments with 216 (Tm ) 76.7 °C, Figure S11)17 correlate
well with those from the DSC. Furthermore, the signature peaks
for the hexadecamer are observed at temperatures above the Tt,
which is a strong indication that the hexadecamers undergo the
phase transition intact (i.e., as well-defined supramolecules). This
discards the possibility that the phase transition occurs because of
a temperature induced disassembly followed by the precipitation
of the monomeric subunits.
Modulating the Tt for any system that exhibits the LCST
phenomenon can be achieved by adjusting the balance between
hydrophobic and hydrophilic groups.5,8,19 In polymeric systems,
Tt is usually modulated by changing the composition at the synthesis
level, but multiple other strategies such as changes in pH, ionic
strength, and the presence of cosolutes, among others, are usually
employed.20 Simple blending procedures21 and supramolecular
modification of polymeric systems have also been used to achieve
such modulation.3a,22 This led us to hypothesize that adding the
more hydrophilic 1 to a solution of 216 would lead to an increase
in the Tt. A solution containing 1 to 2 (1:15, 20 mM combined)
shows a 1H NMR indicative of a fully mixed hexadecamer (Figure
S8).17 The Tt of this solution is 64.9 °C as determined by
transmittance experiments (Figure 2c) and confirmed by DSC
(Figure S13). Similar experiments using a greater proportion of 1
to 2 (1:1) also show the formation of a mixed hexadecamer, but
this time no Tt was observed in the temperature range 25-90 °C
(data not shown). At this point the system has overcome the
threshold of the hydrophilic/hydrophobic balance required to show
the LCST phenomenon.
Figure 2. 1H NMR spectra (500 MHz, 10% D2O in H2O), DSC, and
turbidity data. (a) NMR spectrum of 216. (b) NMR spectrum of 116. The
red and blue cartoons in a and b represent 216 and 116, respectively. (c)
Turbidity curves (measured at 500 nm) for 216 (red circles), 116 (blue
triangles), and the mixed 1•215 (hollow circles). The measurements were
performed in aqueous phosphate-buffered solution (pH 7.1, 2 M KI). (d)
DSC endotherm with the two resulting deconvolution peaks representing
the Tt, left, and the Tm, right. The overlaid turbidity curve (red circle) shows
an excellent correlation with the DSC peak that corresponds to the Tt. Insets,
vials with a solution of 216 below, left, and above, right, the Tt.
Dynamic light scattering (DLS) studies provide critical informa-
tion regarding the sizes of the various supramolecules as a function
of temperature (24-74 °C). Below the Tt the average hydrodynamic
diameter (DH) for 216 is (4.6 ( 0.2) nm (Figure 3a), which is in
Figure 3. (a) Average hydrodynamic diameters (DH) of 216 (orange squares)
as a function of temperature measured by DLS. The measurements were
made at intervals of 2 °C, allowing the temperature to stabilize before each
measurement. (b) Size distributions for selected temperatures from the DLS
curve shown on a (i-iV). (c) TEM image of a sample of 216 deposited
above the Tt (60 °C). Scale bar, 200 nm. The apparent discrepancy between
the DH determined by DLS and TEM is likely due to the dehydration
of the globules upon drying the sample and the high vacuum conditions of
the TEM measurements.
1
very good agreement to the value obtained from H DOSY NMR
experiments of (4.40 ( 0.12) nm (Table S1).17,23 Upon reaching
Tt, the DH of the sizes of the species in solution increased by 3
orders of magnitude to above 1 µm. Continued heating of the
solution from 58.0 to 66.0 °C lead to a sharp decrease of the DH
until the values stabilized at ∼400 nm. Transmission electron
microscopy (TEM) measurements of these aggregates showed that
they are nonhollow, discrete, and globular nanoparticles of relatively
low polydispersity (Figure 3c). Heating the sample beyond 70 °C
leads to a sudden increase in the DH values with a concomitant
increase in the polydispersity of the resulting aggregates (Figure
3b, 74 °C).
The supramolecular state also leads to the emergence of the
thermosensitive property for 216. Aqueous phosphate-buffered
solutions (pH 7.1, 2 M KI) of both 116 and 216 were completely
homogeneous (i.e., highly soluble) at room temperature. And while
the turbidity for the solution of 116 does not change upon heating
to 90 °C (Figure 2c, blue curve), the solution of 216 suddenly
becomes cloudy at 59 °C. Transmittance measurements at 500 nm
confirm these observations (Figure 2c, red curve). Cooling the
solution back to 25 °C restores the initial conditions (yellowish
transparent solution), and this process can be repeated at least ten
times (Figure S9)17 with no signs of fatigue (e.g., decomposition).
Differential scanning calorimetry (DSC) studies support the
results obtained from the transmittance measurements and provide
further insights into the energetics of the LCST phenomenon.18
DSC measurements reveal a double-peak endotherm transition,
Furthermore, the DLS studies allow us to postulate a mechanism
for this thermally induced assembly (Figure 4). At 25-58 °C the
solution is populated with monodisperse, discrete self-assembled
hexadecamers (Figure 4b,i). Raising the temperature above the Tt
(58 °C) induces the formation of large hydrated globules of
intermediate polydispersity (Figure 4b,ii). A continued heating of
the sample (60.0-70.0 °C) leads to a decrease in the average size
of the globules (Figure 4b,iii). This is likely due to the ejection of
water molecules initially trapped throughout the interior of the
aggregates. Further heating of the solution (>70.0 °C) induces a
9
J. AM. CHEM. SOC. VOL. 131, NO. 46, 2009 16667

C O M M U N I C A T I O N S
to open the door to the development of novel smart materials for
biomedical and nanotechnological applications.1,4
Acknowledgment. We thank NIH-SCoRE (Grant No.
2506GM08102) and NCRR-NIH (Grant No. P20 RR016470) for
financial support. J.E.B. thanks the Alfred P. Sloan Foundation
and NSF-IFN-EPSCoR (01A-0701525) for graduate fellowships.
We also thank Adriana Herrera (UPRM) for help with DLS
measurements; Dr. Maxime Guinel (UPRRP) for TEM images;
´
and Amalia Avila, Aura Collazo, and Luis Oquendo (UPRRP)
for technical assistance during the initial stages of this project.
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.
References
(1) (a) Yoshida, M.; Lahann, J. ACS Nano 2008, 2, 1101–1107. (b) Rodriguez-
Hernandez, J.; Checot, F.; Gnanou, Y.; Lecommandoux, S. Prog. Polym.
Sci. 2005, 30, 691–724. (c) de Las Heras Alarcon, C.; Pennadam, S.;
Alexander, C. Chem. Soc. ReV. 2005, 34, 276–285.
(2) Simmons, D. S.; Sanchez, I. C. Macromolecules 2008, 41, 5885–5889.
(3) (a) Kumar, A.; Srivastava, A.; Galaev, I.; Mattiasson, B. Prog. Polym. Sci.
2007, 32, 1205–1237. (b) Chen, G.; Hoffman, A. S. Nature 1995, 373,
49–52.
(4) Dai, S.; Ravi, P.; Tam, K. C. Soft Matter 2009, 5, 2513–2533.
(5) Urry, D. J. Phys. Chem. B 1997, 101, 11007–11028.
(6) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163–249.
(7) Gil, E. S.; Hudson, S. A. Prog. Polym. Sci. 2004, 29, 1173–1222.
(8) Urry, D. W.; Gowda, D. C.; Parker, T. M.; Luan, C. H.; Reid, M. C.; Harris,
C. M.; Pattanaik; Harris, R. D. Biopolymers 1992, 32, 1243–1250.
(9) Chilkoti, A.; Dreher, M. R.; Meyer, D. E.; Raucher, D. AdV. Drug DeliVery
ReV. 2002, 54, 613–630.
Figure 4. Proposed mechanism of hierarchical assembly. (a) Isothermal
self-assembly of 2 to form 216. (b) Thermal induced assembly of 216. Upon
reaching the Tt, 216 forms a hydrated globule (ii) with a hydrodynamic
diameter of ∼1 µm. (iii) The entropically favorable dehydration of the
globule leads to a more compact nanoparticle with a diameter of ∼325
nm. (iV) Beyond 74 °C the assemblies melt, inducing the formation of ill-
defined aggregates of large polydispersity.
(10) Lao, U.; Kostal, J.; Mulchandani, A.; Chen, W. Nat. Protocols 2007, 2,
1263–1268.
(11) Rzaev, Z.; Dinc¸er, S. Prog. Polym. Sci. 2007, 32, 534–595.
(12) Lehn, J. M. Science 2002, 295, 2400–2403.
(13) Whitesides, G. M. Small 2005, 1, 172–179.
(14) (a) Gubala, V.; Betancourt, J. E.; Rivera, J. M. Org. Lett. 2004, 6, 4735–
4738. (b) Rivera-Sa´nchez, M. d. C.; Andu´jar-de-Sanctis, I.; Gar´ıa-Arriaga,
M.; Gubala, V.; Hobley, G.; Rivera, J. M. J. Am. Chem. Soc. 2009, 131,
10403–10405. (c) Gubala, V.; De Jesus, D.; Rivera, J. M. Tetrahedron
Lett. 2006, 47, 1413–1416. (d) Betancourt, J. E.; Mart´ın-Hidalgo, M.;
Gubala, V.; Rivera, J. M. J. Am. Chem. Soc. 2009, 131, 3186–3188.
(15) Garc´ıa-Arriaga, M.; Hobley, G.; Rivera, J. M. J. Am. Chem. Soc. 2008,
130, 10492–10493.
melting of the hexadecamers, which promotes the formation of
larger polydisperse aggregates (Figure 4b,iV).
The results presented in this article provide compelling evidence
that the self-assembly of nonpolymeric small molecules can lead
to the development of smart, discrete, and well-defined supramol-
ecules. Furthermore, reactions at the supramolecular level, in which
two similar subunits with different hydrophobicities are mixed,
enables the modulation of the LCST phenomenon. We are currently
evaluating the scope of this strategy by synthesizing a series of
derivatives of different hydrophobicities to fine-tune the Tt over a
wider range. Studies aimed at exploring the possibility of using
other stimuli, such as pH, to trigger the LCST phenomenon are
underway. The extension of the LCST phenomenon from the
macromolecular to the nonpolymeric supramolecular realm is likely
(16) Betancourt, J. E.; Rivera, J. M. Org. Lett. 2008, 10, 2287–2290.
(17) See Supporting Information.
(18) Schild, H. G.; Tirrell, D. A. J. Phys. Chem. 1990, 17, 163–249.
(19) Kisselev, A.; Manias, E. Fluid Phase Equilib. 2007, 261, 69–78.
(20) Urry, D. Biopolymers 1998, 47, 167–178.
(21) (a) Ferna´ndez-Trillo, F.; van Hest, J. C. M.; Thies, J. C.; Michon, T.;
Weberskirch, R.; Cameron, N. R. Chem. Commun. 2008, 2230–2232. (b)
Yu, Y.; Nakamura, D.; DeBoyace, K.; Neisius, A. W.; McGown, L. B. J.
Phys. Chem. B 2008, 112, 1130–1134.
(22) Schmitz, S.; Ritter, H. Angew. Chem., Int. Ed. 2005, 44, 5658–5661.
(23) Cohen, Y.; Avram, L.; Frish, L. Angew. Chem., Int. Ed. 2005, 44, 520–554.
JA9070927
9
16668 J. AM. CHEM. SOC. VOL. 131, NO. 46, 2009